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Pregnancy-related mortality in Africa and Asia: evidence from INDEPTH Health and Demographic Surveillance System sites | <sec id="st1"><title>Background</title><p>Women continue to die in unacceptably large numbers around the world as a result of pregnancy, particularly in sub-Saharan Africa and Asia. Part of the problem is a lack of accurate, population-based information characterising the issues and informing solutions. Population surveillance sites, such as those operated within the INDEPTH Network, have the potential to contribute to bridging the information gaps.</p></sec><sec id="st2"><title>
Objective</title><p>To describe patterns of pregnancy-related mortality at INDEPTH Network Health and Demographic Surveillance System sites in sub-Saharan Africa and southeast Asia in terms of maternal mortality ratio (MMR) and cause-specific mortality rates.</p></sec><sec id="st3"><title>Design</title><p>Data on individual deaths among women of reproductive age (WRA) (15–49) resident in INDEPTH sites were collated into a standardised database using the INDEPTH 2013 population standard, the WHO 2012 verbal autopsy (VA) standard, and the InterVA model for assigning cause of death.</p></sec><sec id="st4"><title>Results</title><p>These analyses are based on reports from 14 INDEPTH sites, covering 14,198 deaths among WRA over 2,595,605 person-years observed. MMRs varied between 128 and 461 per 100,000 live births, while maternal mortality rates ranged from 0.11 to 0.74 per 1,000 person-years. Detailed rates per cause are tabulated, including analyses of direct maternal, indirect maternal, and incidental pregnancy-related deaths across the 14 sites.</p></sec><sec id="st5"><title>Conclusions</title><p>As expected, these findings confirmed unacceptably high continuing levels of maternal mortality. However, they also demonstrate the effectiveness of INDEPTH sites and of the VA methods applied to arrive at measurements of maternal mortality that are essential for planning effective solutions and monitoring programmatic impacts.</p></sec> | <contrib contrib-type="author"><name><surname>Streatfield</surname><given-names>P. Kim</given-names></name><xref ref-type="aff" rid="AF0001">1</xref><xref ref-type="aff" rid="AF0002">2</xref><xref ref-type="aff" rid="AF0003">3</xref></contrib><contrib contrib-type="author"><name><surname>Alam</surname><given-names>Nurul</given-names></name><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0004">4</xref><xref ref-type="aff" rid="AF0005">5</xref></contrib><contrib contrib-type="author"><name><surname>Compaoré</surname><given-names>Yacouba</given-names></name><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0006">6</xref><xref ref-type="aff" rid="AF0007">7</xref></contrib><contrib contrib-type="author"><name><surname>Rossier</surname><given-names>Clementine</given-names></name><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0006">6</xref><xref ref-type="aff" rid="AF0007">7</xref><xref ref-type="aff" rid="AF0008">8</xref></contrib><contrib contrib-type="author"><name><surname>Soura</surname><given-names>Abdramane B.</given-names></name><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0006">6</xref><xref ref-type="aff" rid="AF0007">7</xref></contrib><contrib contrib-type="author"><name><surname>Bonfoh</surname><given-names>Bassirou</given-names></name><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0009">9</xref><xref ref-type="aff" rid="AF0010">10</xref></contrib><contrib contrib-type="author"><name><surname>Jaeger</surname><given-names>Fabienne</given-names></name><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0009">9</xref><xref ref-type="aff" rid="AF0011">11</xref></contrib><contrib contrib-type="author"><name><surname>Ngoran</surname><given-names>Eliezer K.</given-names></name><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0009">9</xref><xref ref-type="aff" rid="AF0012">12</xref></contrib><contrib contrib-type="author"><name><surname>Utzinger</surname><given-names>Juerg</given-names></name><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0009">9</xref><xref ref-type="aff" rid="AF0011">11</xref></contrib><contrib contrib-type="author"><name><surname>Gomez</surname><given-names>Pierre</given-names></name><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0013">13</xref><xref ref-type="aff" rid="AF0014">14</xref></contrib><contrib contrib-type="author"><name><surname>Jasseh</surname><given-names>Momodou</given-names></name><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0013">13</xref><xref ref-type="aff" rid="AF0014">14</xref></contrib><contrib contrib-type="author"><name><surname>Ansah</surname><given-names>Akosua</given-names></name><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0015">15</xref><xref ref-type="aff" rid="AF0016">16</xref></contrib><contrib contrib-type="author"><name><surname>Debpuur</surname><given-names>Cornelius</given-names></name><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0015">15</xref><xref ref-type="aff" rid="AF0016">16</xref></contrib><contrib contrib-type="author"><name><surname>Oduro</surname><given-names>Abraham</given-names></name><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0015">15</xref><xref ref-type="aff" rid="AF0016">16</xref></contrib><contrib contrib-type="author"><name><surname>Williams</surname><given-names>John</given-names></name><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0015">15</xref><xref ref-type="aff" rid="AF0016">16</xref></contrib><contrib contrib-type="author"><name><surname>Addei</surname><given-names>Sheila</given-names></name><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0017">17</xref><xref ref-type="aff" rid="AF0018">18</xref></contrib><contrib contrib-type="author"><name><surname>Gyapong</surname><given-names>Margaret</given-names></name><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0017">17</xref><xref ref-type="aff" rid="AF0018">18</xref></contrib><contrib contrib-type="author"><name><surname>Kukula</surname><given-names>Vida A.</given-names></name><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0017">17</xref><xref ref-type="aff" rid="AF0018">18</xref><xref ref-type="aff" rid="AF0019">19</xref></contrib><contrib contrib-type="author"><name><surname>Bauni</surname><given-names>Evasius</given-names></name><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0020">20</xref><xref ref-type="aff" rid="AF0021">21</xref></contrib><contrib contrib-type="author"><name><surname>Mochamah</surname><given-names>George</given-names></name><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0020">20</xref><xref ref-type="aff" rid="AF0021">21</xref></contrib><contrib contrib-type="author"><name><surname>Ndila</surname><given-names>Carolyne</given-names></name><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0020">20</xref><xref ref-type="aff" rid="AF0021">21</xref></contrib><contrib contrib-type="author"><name><surname>Williams</surname><given-names>Thomas N.</given-names></name><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0020">20</xref><xref ref-type="aff" rid="AF0021">21</xref><xref ref-type="aff" rid="AF0022">22</xref></contrib><contrib contrib-type="author"><name><surname>Desai</surname><given-names>Meghna</given-names></name><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0023">23</xref><xref ref-type="aff" rid="AF0024">24</xref></contrib><contrib contrib-type="author"><name><surname>Moige</surname><given-names>Hellen</given-names></name><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0023">23</xref><xref ref-type="aff" rid="AF0024">24</xref></contrib><contrib contrib-type="author"><name><surname>Odhiambo</surname><given-names>Frank O.</given-names></name><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0023">23</xref><xref ref-type="aff" rid="AF0024">24</xref></contrib><contrib contrib-type="author"><name><surname>Ogwang</surname><given-names>Sheila</given-names></name><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0023">23</xref><xref ref-type="aff" rid="AF0024">24</xref></contrib><contrib contrib-type="author"><name><surname>Beguy</surname><given-names>Donatien</given-names></name><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0025">25</xref><xref ref-type="aff" rid="AF0026">26</xref></contrib><contrib contrib-type="author"><name><surname>Ezeh</surname><given-names>Alex</given-names></name><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0025">25</xref><xref ref-type="aff" rid="AF0026">26</xref></contrib><contrib contrib-type="author"><name><surname>Oti</surname><given-names>Samuel</given-names></name><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0025">25</xref><xref ref-type="aff" rid="AF0026">26</xref></contrib><contrib contrib-type="author"><name><surname>Chihana</surname><given-names>Menard</given-names></name><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0027">27</xref><xref ref-type="aff" rid="AF0028">28</xref></contrib><contrib contrib-type="author"><name><surname>Crampin</surname><given-names>Amelia</given-names></name><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0027">27</xref><xref ref-type="aff" rid="AF0028">28</xref><xref ref-type="aff" rid="AF0029">29</xref></contrib><contrib contrib-type="author"><name><surname>Price</surname><given-names>Alison</given-names></name><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0027">27</xref><xref ref-type="aff" rid="AF0028">28</xref><xref ref-type="aff" rid="AF0029">29</xref></contrib><contrib contrib-type="author"><name><surname>Delaunay</surname><given-names>Valérie</given-names></name><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0030">30</xref><xref ref-type="aff" rid="AF0031">31</xref></contrib><contrib contrib-type="author"><name><surname>Diallo</surname><given-names>Aldiouma</given-names></name><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0030">30</xref><xref ref-type="aff" rid="AF0031">31</xref></contrib><contrib contrib-type="author"><name><surname>Douillot</surname><given-names>Laetitia</given-names></name><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0030">30</xref><xref ref-type="aff" rid="AF0031">31</xref></contrib><contrib contrib-type="author"><name><surname>Sokhna</surname><given-names>Cheikh</given-names></name><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0030">30</xref><xref ref-type="aff" rid="AF0031">31</xref></contrib><contrib contrib-type="author"><name><surname>Collinson</surname><given-names>Mark A.</given-names></name><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0032">32</xref><xref ref-type="aff" rid="AF0033">33</xref><xref ref-type="aff" rid="AF0034">34</xref></contrib><contrib contrib-type="author"><name><surname>Kahn</surname><given-names>Kathleen</given-names></name><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0032">32</xref><xref ref-type="aff" rid="AF0033">33</xref><xref ref-type="aff" rid="AF0034">34</xref></contrib><contrib contrib-type="author"><name><surname>Tollman</surname><given-names>Stephen M.</given-names></name><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0032">32</xref><xref ref-type="aff" rid="AF0033">33</xref><xref ref-type="aff" rid="AF0034">34</xref></contrib><contrib contrib-type="author"><name><surname>Herbst</surname><given-names>Kobus</given-names></name><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0035">35</xref><xref ref-type="aff" rid="AF0036">36</xref></contrib><contrib contrib-type="author"><name><surname>Mossong</surname><given-names>Joël</given-names></name><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0035">35</xref><xref ref-type="aff" rid="AF0036">36</xref><xref ref-type="aff" rid="AF0037">37</xref></contrib><contrib contrib-type="author"><name><surname>Emina</surname><given-names>Jacques B.O.</given-names></name><xref ref-type="aff" rid="AF0003">3</xref></contrib><contrib contrib-type="author"><name><surname>Sankoh</surname><given-names>Osman A.</given-names></name><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0038">38</xref><xref ref-type="aff" rid="AF0039">39</xref><xref ref-type="corresp" rid="cor1">*</xref></contrib><contrib contrib-type="author"><name><surname>Byass</surname><given-names>Peter</given-names></name><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0033">33</xref><xref ref-type="aff" rid="AF0040">40</xref></contrib> | Global Health Action | <p>The tragedy of women dying in the context of being pregnant or giving birth continues to be a major, but almost entirely avoidable, problem. A number of countries consistently achieve a maternal mortality ratio (MMR) of less than 10 maternal deaths per 100,000 live births, but the global MMR remains at levels of several hundred mothers dying for every 100,000 births (<xref rid="CIT0001" ref-type="bibr">1</xref>). Most pregnancy-related deaths (PRD) occur in the world's poorer countries, and, irrespective of medical causes of death, a proportion are due to health systems failures such as ineffective referral and transport systems in cases of emergency. In addition, health information systems are generally weakest where the problem of pregnancy-related mortality is the greatest (<xref rid="CIT0002" ref-type="bibr">2</xref>).</p><p>Much information on pregnancy-related mortality comes from maternal death surveys of various kinds, including Demographic and Household Surveys (DHS) (<xref rid="CIT0003" ref-type="bibr">3</xref>), and often involves indirect methods of identifying maternal deaths, such as the sisterhood method (<xref rid="CIT0004" ref-type="bibr">4</xref>). Data from health facilities are also often used, even though 1) many women do not use facilities for their deliveries, and 2) facilities tend to attract complicated cases. Because all these approaches do not directly access the details of deaths as and when they occur in communities, they are subject to a range of biases and uncertainties which have often hindered understanding of pregnancy-related mortality patterns. Depending on methods used, it may also be difficult to separate maternal deaths (direct and indirect maternal causes) from all PRD (any deaths occurring during or within 6 weeks of pregnancy). Global estimates of maternal mortality, made both by the United Nations Maternal Mortality Interagency Estimates Group (MMEIG) (<xref rid="CIT0001" ref-type="bibr">1</xref>) and the Institute of Health Metrics and Evaluation (IHME) (<xref rid="CIT0005" ref-type="bibr">5</xref>) rely heavily on whatever survey and other data happens to be available at the country level, and consequently are influenced by the same uncertainties.</p><p>Because INDEPTH Network Health and Demographic Surveillance sites (HDSS) follow vital events in defined populations on a continuous basis, they are able to document pregnancy-related mortality directly (<xref rid="CIT0006" ref-type="bibr">6</xref>). Furthermore, since all deaths among women of reproductive age (WRA) are followed up and subject to verbal autopsy (VA) interviews, which include questions about the woman's pregnancy status irrespective of her cause of death, it is possible to look at pregnancy-related mortality as cause-specific mortality rates among WRA, and to assess the effect of pregnancy as a risk factor for all causes of death. This also enables estimation of maternal deaths as a subset of all pregnancy-related mortality, which, together with demographic information on births, allows calculation of MMRs.</p><p>Our aim in this paper is to address these issues on the basis of a dataset collected at 22 INDEPTH Network HDSSs across Africa and Asia. Although these sites are not constituted as a representative sample, they provide point estimates over a wide range of settings and time periods.</p><sec sec-type="methods" id="S0002"><title>Methods</title><p>The overall INDEPTH dataset, available from the INDEPTH Data Repository (<xref rid="CIT0007" ref-type="bibr">7</xref>), from which these pregnancy-specific analyses are drawn, is described in detail elsewhere (<xref rid="CIT0008" ref-type="bibr">8</xref>). For WRA, the dataset documents 15,295 deaths in 3,098,718 person-years of observation across 22 sites. The methods involved in compiling the overall dataset are summarised in <xref ref-type="boxed-text" rid="B0001">Box 1</xref>.</p><p>
<italic>Box 1</italic>. Summary of methodology based on the detailed description in the introductory paper (<xref rid="CIT0008" ref-type="bibr">8</xref>).</p><boxed-text id="B0001" position="float"><p>
<bold>Age–sex–time standardisation</bold>
</p><p>To avoid effects of differences and changes in age–sex structures of populations, mortality fractions and rates have been adjusted using the INDEPTH 2013 population standard (<xref rid="CIT0009" ref-type="bibr">9</xref>). A weighting factor was calculated for each site, age group, sex, and year category in relation to the standard for the corresponding age group and sex, and incorporated into the overall dataset. This is referred to in this paper as age–sex–time standardisation in the contexts where it is used.</p><p>
<bold>Cause of death assignment</bold>
</p><p>The InterVA-4 (version 4.02) probabilistic model was
used for all the cause of death assignments in the overall dataset (<xref rid="CIT0010" ref-type="bibr">10</xref>). InterVA-4 is fully compliant with the WHO 2012 Verbal Autopsy standard and generates causes of death categorised by ICD-10 groups (<xref rid="CIT0011" ref-type="bibr">11</xref>). The data reported here were collected before the WHO 2012 VA standard was available, but were transformed into the WHO 2012 and InterVA-4 format to optimise cross-site standardisation in cause of death attribution. For a small proportion of deaths, VA interviews were not successfully completed; a few others contained inadequate information to arrive at a cause of death. InterVA-4 assigns causes of death (maximum 3) with associated likelihoods; thus, cases for which likely causes did not total to 100% were also assigned a residual indeterminate component. This served as a means of encapsulating uncertainty in cause of death at the individual level within the overall dataset, as well as accounting for 100% of every death.</p><p>
<bold>Overall dataset</bold>
</p><p>The overall public domain dataset (<xref rid="CIT0007" ref-type="bibr">7</xref>) thus contains
between one and four records for each death, with the sum of likelihoods for each individual being unity. Each record includes a specific cause of death, its likelihood, and its age–sex–time weighting.</p></boxed-text><p>It is important to be clear about the definitions of pregnancy status in relation to deaths among WRA. WHO (<xref rid="CIT0001" ref-type="bibr">1</xref>) defines a PRD as ‘the death of a women while pregnant or within 42 days of termination of pregnancy, irrespective of the cause of death’. Further, a maternal death is defined as ‘the death of a woman while pregnant or within 42 days of termination of pregnancy, irrespective of the duration and site of the pregnancy, from any cause related to or aggravated by the pregnancy or its management but not from accidental or incidental causes’, which is therefore a subset of PRD. In these analyses, we do not use the concept of late maternal deaths.</p><p>In this dataset, in seven sites there were fewer than 10 PRD reported (mainly due to smaller sites, or shorter reporting periods), and these sites have been excluded from further analyses. One site, Nouna in Burkina Faso, did not record sufficient detail of pregnancy status in the VA data and therefore was also excluded. Thus, these analyses are based on reports from 14 sites, covering 14,198 deaths over 2,595,605 person-years, for which VAs were successfully completed in 91.1% of cases. Further details of the 14 sites included in the analyses here are available separately (<xref rid="CIT0012" ref-type="bibr">12</xref>–<xref rid="CIT0025" ref-type="bibr">25</xref>). As each HDSS covers a total population, rather than a sample, uncertainty intervals are not shown.</p><p>Although the natural way to analyse these longitudinal data was in terms of pregnancy-related mortality rates per 1,000 person-years, because of the widespread use of MMR as a measure of maternal mortality, we also used data from the same populations covered by the HDSSs to generate numbers of live births, based on total person-time relating to the neonatal period. Survivors into infancy each accounted for 28 days of neonatal person-time, with smaller contributions in the case of neonatal deaths. These estimates of live births for each site were only used to produce the MMR estimates shown in <xref ref-type="fig" rid="F0001">Fig. 1</xref>.</p><fig id="F0001" position="float"><label>Fig. 1</label><caption><p>Estimated maternal mortality ratio (MMR) per 100,000 live births and maternal mortality rates per 1,000 person-years observed among women aged 15–49 at 14 INDEPTH sites.</p></caption><graphic xlink:href="GHA-7-25368-g001"/></fig><p>Identifying maternal deaths as a subset of PRD is not entirely straightforward from VA cause of death data. Taking out the accidental causes is simple enough, but a problem remains in determining the proportion of non-obstetric, non-accidental PRD that are ‘related to or aggravated by the pregnancy or its management’, the so-called indirect maternal deaths. In clinical settings, this is a judgement that is made individually on a case-by-case basis. Anaemia as a cause of PRD is always considered to be indirect. In the absence of sufficiently detailed clinical case records to make individual judgements, but having the advantage of cause of death data for all WRA in the populations covered, we were able to estimate, for each participating site, the excess number of deaths associated with pregnancy for each specific non-obstetric, non-accidental cause, based on the proportions of PRD and non-pregnancy deaths (NPRD) from each such cause:<disp-formula id="FD1"><label/><mml:math id="M1"><mml:mrow><mml:mtext>excess PR</mml:mtext><mml:msub><mml:mtext>D</mml:mtext><mml:mrow><mml:mtext>site</mml:mtext><mml:mo>,</mml:mo><mml:mtext>cause</mml:mtext></mml:mrow></mml:msub><mml:mo>=</mml:mo><mml:mtext>PR</mml:mtext><mml:msub><mml:mtext>D</mml:mtext><mml:mrow><mml:mtext>site</mml:mtext><mml:mo>,</mml:mo><mml:mtext>cause</mml:mtext></mml:mrow></mml:msub><mml:mo>-</mml:mo><mml:mo stretchy="false">(</mml:mo><mml:mtext>NPR</mml:mtext><mml:msub><mml:mtext>D</mml:mtext><mml:mrow><mml:mtext>site</mml:mtext><mml:mo>,</mml:mo><mml:mtext>cause</mml:mtext></mml:mrow></mml:msub><mml:mo>×</mml:mo><mml:mo stretchy="false">(</mml:mo><mml:mtext>PR</mml:mtext><mml:msub><mml:mtext>D</mml:mtext><mml:mrow><mml:mtext>site</mml:mtext></mml:mrow></mml:msub><mml:mo>/</mml:mo><mml:mtext>PR</mml:mtext><mml:msub><mml:mtext>D</mml:mtext><mml:mrow><mml:mtext>site</mml:mtext></mml:mrow></mml:msub><mml:mo>+</mml:mo><mml:mtext>NPR</mml:mtext><mml:msub><mml:mtext>D</mml:mtext><mml:mrow><mml:mtext>site</mml:mtext></mml:mrow></mml:msub><mml:mo stretchy="false">)</mml:mo><mml:mo stretchy="false">)</mml:mo></mml:mrow></mml:math></disp-formula>
</p><p>Although for some causes there may be a negative excess (corresponding to a ‘healthy-pregnancy’ effect), that is not relevant to the calculations here, because in standard clinical determination of indirect maternal deaths, no attention is given to enhanced survival from particular causes.</p><p>In this context, all of these data are secondary datasets derived from primary data collected separately by each participating site. In all cases, the primary data collection was covered by site-level ethical approvals relating to on-going health and demographic surveillance in those specific locations. No individual identity or household location data were included in the secondary data and no specific ethical approvals were required for these pooled analyses.</p></sec><sec sec-type="results" id="S0003"><title>Results</title><p>In a total of 14,198 WRA deaths during 2,595,605 person-years of observation, 12,939 had VA interviews successfully completed, of which 1,222 (9.4%) were pregnancy related. Of the overall person-time observed, 67.4% related to the period 2006–2012. Direct obstetric causes were involved in 472.8 (38.7%) of the 1,222 PRD, and a further 177.1 deaths (14.5%) were estimated to be due to indirect maternal causes. Thus, there were 572.1 PRD (46.8%) (so-called ‘incidental’ deaths) which were not ascribed to maternal causes. The numbers of deaths and person-years of exposure for each site are shown in <xref ref-type="table" rid="T0001">Table 1</xref>.</p><table-wrap id="T0001" position="float"><label>Table 1</label><caption><p>Number of deaths by site and pregnancy status, for 12,939 deaths among women aged 15–49 for whom a verbal autopsy interview was successfully completed, with person-time observed</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1"/><th align="center" rowspan="1" colspan="1"/><th align="center" colspan="4" rowspan="1">Pregnancy-related deaths</th><th align="center" rowspan="1" colspan="1"/></tr><tr><th align="left" rowspan="1" colspan="1"/><th align="center" rowspan="1" colspan="1"/><th colspan="4" rowspan="1">
<hr/>
</th></tr><tr><th align="left" rowspan="1" colspan="1">Site</th><th align="center" rowspan="1" colspan="1">Non-pregnancy deaths</th><th align="center" rowspan="1" colspan="1">Total</th><th align="center" rowspan="1" colspan="1">Direct maternal</th><th align="center" rowspan="1" colspan="1">Indirect maternal</th><th align="center" rowspan="1" colspan="1">Incidental</th><th align="center" rowspan="1" colspan="1">Person-years observed</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">Bangladesh: Matlab</td><td align="center" rowspan="1" colspan="1">576</td><td align="center" rowspan="1" colspan="1">77</td><td align="center" rowspan="1" colspan="1">48.5</td><td align="center" rowspan="1" colspan="1">7.8</td><td align="center" rowspan="1" colspan="1">20.7</td><td align="center" rowspan="1" colspan="1">490,544</td></tr><tr><td align="left" rowspan="1" colspan="1">Bangladesh: AMK</td><td align="center" rowspan="1" colspan="1">172</td><td align="center" rowspan="1" colspan="1">43</td><td align="center" rowspan="1" colspan="1">28.1</td><td align="center" rowspan="1" colspan="1">4.4</td><td align="center" rowspan="1" colspan="1">10.5</td><td align="center" rowspan="1" colspan="1">144,278</td></tr><tr><td align="left" rowspan="1" colspan="1">Burkina Faso: Ouagadougou</td><td align="center" rowspan="1" colspan="1">71</td><td align="center" rowspan="1" colspan="1">13</td><td align="center" rowspan="1" colspan="1">5.7</td><td align="center" rowspan="1" colspan="1">3.3</td><td align="center" rowspan="1" colspan="1">4.1</td><td align="center" rowspan="1" colspan="1">58,795</td></tr><tr><td align="left" rowspan="1" colspan="1">Côte d'Ivoire: Taabo</td><td align="center" rowspan="1" colspan="1">63</td><td align="center" rowspan="1" colspan="1">15</td><td align="center" rowspan="1" colspan="1">6.5</td><td align="center" rowspan="1" colspan="1">4.9</td><td align="center" rowspan="1" colspan="1">4.6</td><td align="center" rowspan="1" colspan="1">22,867</td></tr><tr><td align="left" rowspan="1" colspan="1">The Gambia: Farafenni</td><td align="center" rowspan="1" colspan="1">173</td><td align="center" rowspan="1" colspan="1">90</td><td align="center" rowspan="1" colspan="1">48.5</td><td align="center" rowspan="1" colspan="1">9.8</td><td align="center" rowspan="1" colspan="1">31.7</td><td align="center" rowspan="1" colspan="1">78,447</td></tr><tr><td align="left" rowspan="1" colspan="1">Ghana: Navrongo</td><td align="center" rowspan="1" colspan="1">888</td><td align="center" rowspan="1" colspan="1">79</td><td align="center" rowspan="1" colspan="1">43.5</td><td align="center" rowspan="1" colspan="1">6.1</td><td align="center" rowspan="1" colspan="1">29.4</td><td align="center" rowspan="1" colspan="1">279,802</td></tr><tr><td align="left" rowspan="1" colspan="1">Ghana: Dodowa</td><td align="center" rowspan="1" colspan="1">447</td><td align="center" rowspan="1" colspan="1">48</td><td align="center" rowspan="1" colspan="1">25.2</td><td align="center" rowspan="1" colspan="1">2.7</td><td align="center" rowspan="1" colspan="1">20.1</td><td align="center" rowspan="1" colspan="1">140,074</td></tr><tr><td align="left" rowspan="1" colspan="1">Kenya: Kilifi</td><td align="center" rowspan="1" colspan="1">423</td><td align="center" rowspan="1" colspan="1">142</td><td align="center" rowspan="1" colspan="1">53.2</td><td align="center" rowspan="1" colspan="1">17.6</td><td align="center" rowspan="1" colspan="1">71.2</td><td align="center" rowspan="1" colspan="1">234,111</td></tr><tr><td align="left" rowspan="1" colspan="1">Kenya: Kisumu</td><td align="center" rowspan="1" colspan="1">2,801</td><td align="center" rowspan="1" colspan="1">304</td><td align="center" rowspan="1" colspan="1">73.3</td><td align="center" rowspan="1" colspan="1">44.8</td><td align="center" rowspan="1" colspan="1">185.9</td><td align="center" rowspan="1" colspan="1">252,339</td></tr><tr><td align="left" rowspan="1" colspan="1">Kenya: Nairobi</td><td align="center" rowspan="1" colspan="1">693</td><td align="center" rowspan="1" colspan="1">101</td><td align="center" rowspan="1" colspan="1">35.3</td><td align="center" rowspan="1" colspan="1">15.3</td><td align="center" rowspan="1" colspan="1">50.4</td><td align="center" rowspan="1" colspan="1">223,061</td></tr><tr><td align="left" rowspan="1" colspan="1">Malawi: Karonga</td><td align="center" rowspan="1" colspan="1">301</td><td align="center" rowspan="1" colspan="1">41</td><td align="center" rowspan="1" colspan="1">21.9</td><td align="center" rowspan="1" colspan="1">8.3</td><td align="center" rowspan="1" colspan="1">10.8</td><td align="center" rowspan="1" colspan="1">61,411</td></tr><tr><td align="left" rowspan="1" colspan="1">Senegal: Niakhar</td><td align="center" rowspan="1" colspan="1">120</td><td align="center" rowspan="1" colspan="1">18</td><td align="center" rowspan="1" colspan="1">10.9</td><td align="center" rowspan="1" colspan="1">4.3</td><td align="center" rowspan="1" colspan="1">2.8</td><td align="center" rowspan="1" colspan="1">48,089</td></tr><tr><td align="left" rowspan="1" colspan="1">South Africa: Agincourt</td><td align="center" rowspan="1" colspan="1">2,268</td><td align="center" rowspan="1" colspan="1">143</td><td align="center" rowspan="1" colspan="1">37.1</td><td align="center" rowspan="1" colspan="1">22.8</td><td align="center" rowspan="1" colspan="1">83.1</td><td align="center" rowspan="1" colspan="1">350,944</td></tr><tr><td align="left" rowspan="1" colspan="1">South Africa: Africa Centre</td><td align="center" rowspan="1" colspan="1">2,721</td><td align="center" rowspan="1" colspan="1">108</td><td align="center" rowspan="1" colspan="1">35.1</td><td align="center" rowspan="1" colspan="1">26.2</td><td align="center" rowspan="1" colspan="1">46.7</td><td align="center" rowspan="1" colspan="1">210,841</td></tr></tbody></table></table-wrap><p>
<xref ref-type="fig" rid="F0001">Figure 1</xref> shows maternal mortality rates by site together with MMR results based on the total 649.9 maternal deaths (excluding incidental deaths) and corresponding 307,274 live births. MMRs varied between 128 and 461 per 100,000 live births, while maternal mortality rates ranged from 0.11 to 0.74 per 1,000 person-years observed among WRA.</p><p>
<xref ref-type="table" rid="T0002">Table 2</xref> shows mortality rates by major cause of death categories and pregnancy status, by site. There were major variations between sites in non-pregnancy mortality rates, as reflected in other papers in this series (<xref rid="CIT0008" ref-type="bibr">8</xref>, <xref rid="CIT0026" ref-type="bibr">26</xref>, <xref rid="CIT0027" ref-type="bibr">27</xref>). In particular here, there was a 50-fold range in infectious mortality rates unrelated to pregnancy. <xref ref-type="table" rid="T0003">Table 3</xref> shows mortality rates per 1,000 person-years by detailed cause of death and site, together with the overall proportion of deaths for each cause that was pregnancy related.</p><table-wrap id="T0002" position="float"><label>Table 2</label><caption><p>Cause-specific mortality rates per 1,000 person-years for 12,939 deaths among women aged 15–49 years for whom a verbal autopsy interview was successfully completed, from 14 INDEPTH Network HDSS sites, by cause of death categories and pregnancy status</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1"/><th align="center" colspan="5" rowspan="1">Non-pregnancy mortality</th><th align="center" colspan="6" rowspan="1">Pregnancy-related mortality</th><th align="center" colspan="2" rowspan="1">All causes</th></tr><tr><th align="left" rowspan="1" colspan="1"/><th colspan="13" rowspan="1">
<hr/>
</th></tr><tr><th align="left" rowspan="1" colspan="1"/><th align="center" rowspan="1" colspan="1">Infections</th><th align="center" rowspan="1" colspan="1">Neoplasms</th><th align="center" rowspan="1" colspan="1">NCDs</th><th align="center" rowspan="1" colspan="1">External causes</th><th align="center" rowspan="1" colspan="1">Indeterminate</th><th align="center" rowspan="1" colspan="1">Infections</th><th align="center" rowspan="1" colspan="1">Neoplasms</th><th align="center" rowspan="1" colspan="1">NCDs</th><th align="center" rowspan="1" colspan="1">Obstetric causes</th><th align="center" rowspan="1" colspan="1">External causes</th><th align="center" rowspan="1" colspan="1">Indeterminate</th><th align="center" rowspan="1" colspan="1">Non-pregnancy related</th><th align="center" rowspan="1" colspan="1">Pregnancy related</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">Bangladesh: Matlab</td><td align="center" rowspan="1" colspan="1">0.22</td><td align="center" rowspan="1" colspan="1">0.25</td><td align="center" rowspan="1" colspan="1">0.38</td><td align="center" rowspan="1" colspan="1">0.20</td><td align="center" rowspan="1" colspan="1">0.12</td><td align="center" rowspan="1" colspan="1">0.02</td><td align="center" rowspan="1" colspan="1">0.00</td><td align="center" rowspan="1" colspan="1">0.03</td><td align="center" rowspan="1" colspan="1">0.10</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.01</td><td align="center" rowspan="1" colspan="1">1.17</td><td align="center" rowspan="1" colspan="1">0.16</td></tr><tr><td align="left" rowspan="1" colspan="1">Bangladesh: AMK</td><td align="center" rowspan="1" colspan="1">0.21</td><td align="center" rowspan="1" colspan="1">0.26</td><td align="center" rowspan="1" colspan="1">0.36</td><td align="center" rowspan="1" colspan="1">0.28</td><td align="center" rowspan="1" colspan="1">0.08</td><td align="center" rowspan="1" colspan="1">0.03</td><td align="center" rowspan="1" colspan="1">0.01</td><td align="center" rowspan="1" colspan="1">0.05</td><td align="center" rowspan="1" colspan="1">0.20</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.01</td><td align="center" rowspan="1" colspan="1">1.19</td><td align="center" rowspan="1" colspan="1">0.30</td></tr><tr><td align="left" rowspan="1" colspan="1">Burkina Faso: Ouagadougou</td><td align="center" rowspan="1" colspan="1">0.42</td><td align="center" rowspan="1" colspan="1">0.28</td><td align="center" rowspan="1" colspan="1">0.32</td><td align="center" rowspan="1" colspan="1">0.05</td><td align="center" rowspan="1" colspan="1">0.14</td><td align="center" rowspan="1" colspan="1">0.08</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.04</td><td align="center" rowspan="1" colspan="1">0.10</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.01</td><td align="center" rowspan="1" colspan="1">1.21</td><td align="center" rowspan="1" colspan="1">0.23</td></tr><tr><td align="left" rowspan="1" colspan="1">Côte d'Ivoire: Taabo</td><td align="center" rowspan="1" colspan="1">1.85</td><td align="center" rowspan="1" colspan="1">0.11</td><td align="center" rowspan="1" colspan="1">0.54</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.26</td><td align="center" rowspan="1" colspan="1">0.29</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.32</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.05</td><td align="center" rowspan="1" colspan="1">2.76</td><td align="center" rowspan="1" colspan="1">0.66</td></tr><tr><td align="left" rowspan="1" colspan="1">The Gambia: Farafenni</td><td align="center" rowspan="1" colspan="1">1.28</td><td align="center" rowspan="1" colspan="1">0.23</td><td align="center" rowspan="1" colspan="1">0.29</td><td align="center" rowspan="1" colspan="1">0.08</td><td align="center" rowspan="1" colspan="1">0.32</td><td align="center" rowspan="1" colspan="1">0.23</td><td align="center" rowspan="1" colspan="1">0.03</td><td align="center" rowspan="1" colspan="1">0.13</td><td align="center" rowspan="1" colspan="1">0.62</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.15</td><td align="center" rowspan="1" colspan="1">2.20</td><td align="center" rowspan="1" colspan="1">1.16</td></tr><tr><td align="left" rowspan="1" colspan="1">Ghana: Navrongo</td><td align="center" rowspan="1" colspan="1">0.96</td><td align="center" rowspan="1" colspan="1">0.73</td><td align="center" rowspan="1" colspan="1">0.47</td><td align="center" rowspan="1" colspan="1">0.33</td><td align="center" rowspan="1" colspan="1">0.69</td><td align="center" rowspan="1" colspan="1">0.02</td><td align="center" rowspan="1" colspan="1">0.01</td><td align="center" rowspan="1" colspan="1">0.03</td><td align="center" rowspan="1" colspan="1">0.16</td><td align="center" rowspan="1" colspan="1">0.01</td><td align="center" rowspan="1" colspan="1">0.06</td><td align="center" rowspan="1" colspan="1">3.18</td><td align="center" rowspan="1" colspan="1">0.29</td></tr><tr><td align="left" rowspan="1" colspan="1">Ghana: Dodowa</td><td align="center" rowspan="1" colspan="1">1.71</td><td align="center" rowspan="1" colspan="1">0.32</td><td align="center" rowspan="1" colspan="1">0.52</td><td align="center" rowspan="1" colspan="1">0.17</td><td align="center" rowspan="1" colspan="1">0.47</td><td align="center" rowspan="1" colspan="1">0.09</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.03</td><td align="center" rowspan="1" colspan="1">0.19</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.04</td><td align="center" rowspan="1" colspan="1">3.19</td><td align="center" rowspan="1" colspan="1">0.35</td></tr><tr><td align="left" rowspan="1" colspan="1">Kenya: Kilifi</td><td align="center" rowspan="1" colspan="1">1.11</td><td align="center" rowspan="1" colspan="1">0.18</td><td align="center" rowspan="1" colspan="1">0.26</td><td align="center" rowspan="1" colspan="1">0.13</td><td align="center" rowspan="1" colspan="1">0.12</td><td align="center" rowspan="1" colspan="1">0.22</td><td align="center" rowspan="1" colspan="1">0.02</td><td align="center" rowspan="1" colspan="1">0.07</td><td align="center" rowspan="1" colspan="1">0.24</td><td align="center" rowspan="1" colspan="1">0.01</td><td align="center" rowspan="1" colspan="1">0.05</td><td align="center" rowspan="1" colspan="1">1.80</td><td align="center" rowspan="1" colspan="1">0.61</td></tr><tr><td align="left" rowspan="1" colspan="1">Kenya: Kisumu</td><td align="center" rowspan="1" colspan="1">8.61</td><td align="center" rowspan="1" colspan="1">0.48</td><td align="center" rowspan="1" colspan="1">0.96</td><td align="center" rowspan="1" colspan="1">0.17</td><td align="center" rowspan="1" colspan="1">0.87</td><td align="center" rowspan="1" colspan="1">0.63</td><td align="center" rowspan="1" colspan="1">0.04</td><td align="center" rowspan="1" colspan="1">0.08</td><td align="center" rowspan="1" colspan="1">0.33</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.13</td><td align="center" rowspan="1" colspan="1">11.09</td><td align="center" rowspan="1" colspan="1">1.21</td></tr><tr><td align="left" rowspan="1" colspan="1">Kenya: Nairobi</td><td align="center" rowspan="1" colspan="1">2.20</td><td align="center" rowspan="1" colspan="1">0.10</td><td align="center" rowspan="1" colspan="1">0.28</td><td align="center" rowspan="1" colspan="1">0.24</td><td align="center" rowspan="1" colspan="1">0.29</td><td align="center" rowspan="1" colspan="1">0.17</td><td align="center" rowspan="1" colspan="1">0.00</td><td align="center" rowspan="1" colspan="1">0.07</td><td align="center" rowspan="1" colspan="1">0.17</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.05</td><td align="center" rowspan="1" colspan="1">3.11</td><td align="center" rowspan="1" colspan="1">0.46</td></tr><tr><td align="left" rowspan="1" colspan="1">Malawi: Karonga</td><td align="center" rowspan="1" colspan="1">3.57</td><td align="center" rowspan="1" colspan="1">0.39</td><td align="center" rowspan="1" colspan="1">0.46</td><td align="center" rowspan="1" colspan="1">0.14</td><td align="center" rowspan="1" colspan="1">0.34</td><td align="center" rowspan="1" colspan="1">0.16</td><td align="center" rowspan="1" colspan="1">0.05</td><td align="center" rowspan="1" colspan="1">0.05</td><td align="center" rowspan="1" colspan="1">0.36</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.06</td><td align="center" rowspan="1" colspan="1">4.90</td><td align="center" rowspan="1" colspan="1">0.68</td></tr><tr><td align="left" rowspan="1" colspan="1">Senegal: Niakhar</td><td align="center" rowspan="1" colspan="1">1.34</td><td align="center" rowspan="1" colspan="1">0.20</td><td align="center" rowspan="1" colspan="1">0.31</td><td align="center" rowspan="1" colspan="1">0.03</td><td align="center" rowspan="1" colspan="1">0.62</td><td align="center" rowspan="1" colspan="1">0.06</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.29</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.03</td><td align="center" rowspan="1" colspan="1">2.50</td><td align="center" rowspan="1" colspan="1">0.38</td></tr><tr><td align="left" rowspan="1" colspan="1">South Africa: Agincourt</td><td align="center" rowspan="1" colspan="1">4.33</td><td align="center" rowspan="1" colspan="1">0.39</td><td align="center" rowspan="1" colspan="1">0.47</td><td align="center" rowspan="1" colspan="1">0.36</td><td align="center" rowspan="1" colspan="1">0.91</td><td align="center" rowspan="1" colspan="1">0.17</td><td align="center" rowspan="1" colspan="1">0.00</td><td align="center" rowspan="1" colspan="1">0.05</td><td align="center" rowspan="1" colspan="1">0.12</td><td align="center" rowspan="1" colspan="1">0.01</td><td align="center" rowspan="1" colspan="1">0.06</td><td align="center" rowspan="1" colspan="1">6.46</td><td align="center" rowspan="1" colspan="1">0.41</td></tr><tr><td align="left" rowspan="1" colspan="1">South Africa: Africa Centre</td><td align="center" rowspan="1" colspan="1">11.03</td><td align="center" rowspan="1" colspan="1">0.50</td><td align="center" rowspan="1" colspan="1">0.50</td><td align="center" rowspan="1" colspan="1">0.45</td><td align="center" rowspan="1" colspan="1">0.42</td><td align="center" rowspan="1" colspan="1">0.18</td><td align="center" rowspan="1" colspan="1">0.02</td><td align="center" rowspan="1" colspan="1">0.05</td><td align="center" rowspan="1" colspan="1">0.18</td><td align="center" rowspan="1" colspan="1">0.00</td><td align="center" rowspan="1" colspan="1">0.07</td><td align="center" rowspan="1" colspan="1">12.90</td><td align="center" rowspan="1" colspan="1">0.50</td></tr></tbody></table></table-wrap><table-wrap id="T0003" position="float"><label>Table 3</label><caption><p>Cause-specific mortality rates per 1,000 person-years for 12,939 deaths among women aged 15–49 for whom a verbal autopsy interview was successfully completed, from 14 INDEPTH Network HDSS sites, also showing for each cause of death the proportion of pregnancy-related cases</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1">Cause of death</th><th align="center" rowspan="1" colspan="1">Bangladesh: Matlab</th><th align="center" rowspan="1" colspan="1">Bangladesh: AMK</th><th align="center" rowspan="1" colspan="1">Burkina Faso: Ouagadougou</th><th align="center" rowspan="1" colspan="1">Côte d'Ivoire: Taabo</th><th align="center" rowspan="1" colspan="1">Ghana: Navrongo</th><th align="center" rowspan="1" colspan="1">Ghana: Dodwa</th><th align="center" rowspan="1" colspan="1">The Gambia: Farafenni</th><th align="center" rowspan="1" colspan="1">Kenya: Kilifi</th><th align="center" rowspan="1" colspan="1">Kenya: Kisumu</th><th align="center" rowspan="1" colspan="1">Kenya: Nairobi</th><th align="center" rowspan="1" colspan="1">Malawi: Karonga</th><th align="center" rowspan="1" colspan="1">Senegal: Niakhar</th><th align="center" rowspan="1" colspan="1">South Africa: Agincourt</th><th align="center" rowspan="1" colspan="1">South Africa: Africa Centre</th><th align="center" rowspan="1" colspan="1">% Pregnancy related</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">01.01 Sepsis (non-obstetric)</td><td align="center" rowspan="1" colspan="1">0.006</td><td align="center" rowspan="1" colspan="1">0.013</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.030</td><td align="center" rowspan="1" colspan="1">0.021</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.006</td><td align="center" rowspan="1" colspan="1">0.003</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.011</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.00</td></tr><tr><td align="left" rowspan="1" colspan="1">01.02 Acute resp infect, incl pneumonia</td><td align="center" rowspan="1" colspan="1">0.140</td><td align="center" rowspan="1" colspan="1">0.128</td><td align="center" rowspan="1" colspan="1">0.033</td><td align="center" rowspan="1" colspan="1">0.201</td><td align="center" rowspan="1" colspan="1">0.252</td><td align="center" rowspan="1" colspan="1">0.407</td><td align="center" rowspan="1" colspan="1">0.755</td><td align="center" rowspan="1" colspan="1">0.088</td><td align="center" rowspan="1" colspan="1">1.271</td><td align="center" rowspan="1" colspan="1">0.199</td><td align="center" rowspan="1" colspan="1">1.087</td><td align="center" rowspan="1" colspan="1">0.254</td><td align="center" rowspan="1" colspan="1">0.898</td><td align="center" rowspan="1" colspan="1">0.366</td><td align="center" rowspan="1" colspan="1">11.06</td></tr><tr><td align="left" rowspan="1" colspan="1">01.03 HIV/AIDS-related death</td><td align="center" rowspan="1" colspan="1">0.010</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.315</td><td align="center" rowspan="1" colspan="1">0.871</td><td align="center" rowspan="1" colspan="1">1.064</td><td align="center" rowspan="1" colspan="1">0.633</td><td align="center" rowspan="1" colspan="1">0.637</td><td align="center" rowspan="1" colspan="1">0.879</td><td align="center" rowspan="1" colspan="1">9.236</td><td align="center" rowspan="1" colspan="1">2.208</td><td align="center" rowspan="1" colspan="1">5.032</td><td align="center" rowspan="1" colspan="1">1.322</td><td align="center" rowspan="1" colspan="1">7.851</td><td align="center" rowspan="1" colspan="1">7.701</td><td align="center" rowspan="1" colspan="1">4.63</td></tr><tr><td align="left" rowspan="1" colspan="1">01.04 Diarrhoeal diseases</td><td align="center" rowspan="1" colspan="1">0.028</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.016</td><td align="center" rowspan="1" colspan="1">0.138</td><td align="center" rowspan="1" colspan="1">0.125</td><td align="center" rowspan="1" colspan="1">0.128</td><td align="center" rowspan="1" colspan="1">0.071</td><td align="center" rowspan="1" colspan="1">0.024</td><td align="center" rowspan="1" colspan="1">0.101</td><td align="center" rowspan="1" colspan="1">0.010</td><td align="center" rowspan="1" colspan="1">0.038</td><td align="center" rowspan="1" colspan="1">0.409</td><td align="center" rowspan="1" colspan="1">0.112</td><td align="center" rowspan="1" colspan="1">0.037</td><td align="center" rowspan="1" colspan="1">3.98</td></tr><tr><td align="left" rowspan="1" colspan="1">01.05 Malaria</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.034</td><td align="center" rowspan="1" colspan="1">0.587</td><td align="center" rowspan="1" colspan="1">0.057</td><td align="center" rowspan="1" colspan="1">1.471</td><td align="center" rowspan="1" colspan="1">0.854</td><td align="center" rowspan="1" colspan="1">0.045</td><td align="center" rowspan="1" colspan="1">1.611</td><td align="center" rowspan="1" colspan="1">0.044</td><td align="center" rowspan="1" colspan="1">0.615</td><td align="center" rowspan="1" colspan="1">0.412</td><td align="center" rowspan="1" colspan="1">0.252</td><td align="center" rowspan="1" colspan="1">0.054</td><td align="center" rowspan="1" colspan="1">12.47</td></tr><tr><td align="left" rowspan="1" colspan="1">01.07 Meningitis and encephalitis</td><td align="center" rowspan="1" colspan="1">0.017</td><td align="center" rowspan="1" colspan="1">0.022</td><td align="center" rowspan="1" colspan="1">0.063</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.231</td><td align="center" rowspan="1" colspan="1">0.160</td><td align="center" rowspan="1" colspan="1">0.385</td><td align="center" rowspan="1" colspan="1">0.047</td><td align="center" rowspan="1" colspan="1">0.350</td><td align="center" rowspan="1" colspan="1">0.219</td><td align="center" rowspan="1" colspan="1">0.193</td><td align="center" rowspan="1" colspan="1">0.184</td><td align="center" rowspan="1" colspan="1">0.076</td><td align="center" rowspan="1" colspan="1">0.394</td><td align="center" rowspan="1" colspan="1">6.63</td></tr><tr><td align="left" rowspan="1" colspan="1">01.09 Pulmonary tuberculosis</td><td align="center" rowspan="1" colspan="1">0.275</td><td align="center" rowspan="1" colspan="1">0.304</td><td align="center" rowspan="1" colspan="1">0.032</td><td align="center" rowspan="1" colspan="1">0.226</td><td align="center" rowspan="1" colspan="1">0.314</td><td align="center" rowspan="1" colspan="1">0.954</td><td align="center" rowspan="1" colspan="1">1.116</td><td align="center" rowspan="1" colspan="1">0.245</td><td align="center" rowspan="1" colspan="1">5.845</td><td align="center" rowspan="1" colspan="1">2.326</td><td align="center" rowspan="1" colspan="1">1.369</td><td align="center" rowspan="1" colspan="1">0.651</td><td align="center" rowspan="1" colspan="1">4.094</td><td align="center" rowspan="1" colspan="1">12.950</td><td align="center" rowspan="1" colspan="1">3.38</td></tr><tr><td align="left" rowspan="1" colspan="1">01.99 Other and unspecified infect dis</td><td align="center" rowspan="1" colspan="1">0.017</td><td align="center" rowspan="1" colspan="1">0.009</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.084</td><td align="center" rowspan="1" colspan="1">0.019</td><td align="center" rowspan="1" colspan="1">0.205</td><td align="center" rowspan="1" colspan="1">0.032</td><td align="center" rowspan="1" colspan="1">0.002</td><td align="center" rowspan="1" colspan="1">0.174</td><td align="center" rowspan="1" colspan="1">0.032</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.007</td><td align="center" rowspan="1" colspan="1">0.042</td><td align="center" rowspan="1" colspan="1">0.021</td><td align="center" rowspan="1" colspan="1">9.63</td></tr><tr><td align="left" rowspan="1" colspan="1">02.01 Oral neoplasms</td><td align="center" rowspan="1" colspan="1">0.019</td><td align="center" rowspan="1" colspan="1">0.010</td><td align="center" rowspan="1" colspan="1">0.011</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.057</td><td align="center" rowspan="1" colspan="1">0.011</td><td align="center" rowspan="1" colspan="1">0.028</td><td align="center" rowspan="1" colspan="1">0.007</td><td align="center" rowspan="1" colspan="1">0.056</td><td align="center" rowspan="1" colspan="1">0.010</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.020</td><td align="center" rowspan="1" colspan="1">0.032</td><td align="center" rowspan="1" colspan="1">0.038</td><td align="center" rowspan="1" colspan="1">4.22</td></tr><tr><td align="left" rowspan="1" colspan="1">02.02 Digestive neoplasms</td><td align="center" rowspan="1" colspan="1">0.231</td><td align="center" rowspan="1" colspan="1">0.259</td><td align="center" rowspan="1" colspan="1">0.096</td><td align="center" rowspan="1" colspan="1">0.035</td><td align="center" rowspan="1" colspan="1">0.932</td><td align="center" rowspan="1" colspan="1">0.510</td><td align="center" rowspan="1" colspan="1">0.406</td><td align="center" rowspan="1" colspan="1">0.064</td><td align="center" rowspan="1" colspan="1">0.346</td><td align="center" rowspan="1" colspan="1">0.029</td><td align="center" rowspan="1" colspan="1">0.355</td><td align="center" rowspan="1" colspan="1">0.131</td><td align="center" rowspan="1" colspan="1">0.333</td><td align="center" rowspan="1" colspan="1">0.207</td><td align="center" rowspan="1" colspan="1">2.05</td></tr><tr><td align="left" rowspan="1" colspan="1">02.03 Respiratory neoplasms</td><td align="center" rowspan="1" colspan="1">0.074</td><td align="center" rowspan="1" colspan="1">0.116</td><td align="center" rowspan="1" colspan="1">0.067</td><td align="center" rowspan="1" colspan="1">0.038</td><td align="center" rowspan="1" colspan="1">0.067</td><td align="center" rowspan="1" colspan="1">0.083</td><td align="center" rowspan="1" colspan="1">0.016</td><td align="center" rowspan="1" colspan="1">0.057</td><td align="center" rowspan="1" colspan="1">0.253</td><td align="center" rowspan="1" colspan="1">0.069</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.054</td><td align="center" rowspan="1" colspan="1">0.266</td><td align="center" rowspan="1" colspan="1">0.266</td><td align="center" rowspan="1" colspan="1">6.89</td></tr><tr><td align="left" rowspan="1" colspan="1">02.04 Breast neoplasms</td><td align="center" rowspan="1" colspan="1">0.076</td><td align="center" rowspan="1" colspan="1">0.040</td><td align="center" rowspan="1" colspan="1">0.046</td><td align="center" rowspan="1" colspan="1">0.037</td><td align="center" rowspan="1" colspan="1">0.310</td><td align="center" rowspan="1" colspan="1">0.078</td><td align="center" rowspan="1" colspan="1">0.114</td><td align="center" rowspan="1" colspan="1">0.008</td><td align="center" rowspan="1" colspan="1">0.082</td><td align="center" rowspan="1" colspan="1">0.018</td><td align="center" rowspan="1" colspan="1">0.086</td><td align="center" rowspan="1" colspan="1">0.185</td><td align="center" rowspan="1" colspan="1">0.206</td><td align="center" rowspan="1" colspan="1">0.136</td><td align="center" rowspan="1" colspan="1">1.01</td></tr><tr><td align="left" rowspan="1" colspan="1">02.06 Reproductive neoplasms</td><td align="center" rowspan="1" colspan="1">0.063</td><td align="center" rowspan="1" colspan="1">0.057</td><td align="center" rowspan="1" colspan="1">0.059</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.216</td><td align="center" rowspan="1" colspan="1">0.084</td><td align="center" rowspan="1" colspan="1">0.276</td><td align="center" rowspan="1" colspan="1">0.043</td><td align="center" rowspan="1" colspan="1">0.166</td><td align="center" rowspan="1" colspan="1">0.053</td><td align="center" rowspan="1" colspan="1">0.549</td><td align="center" rowspan="1" colspan="1">0.144</td><td align="center" rowspan="1" colspan="1">0.268</td><td align="center" rowspan="1" colspan="1">0.342</td><td align="center" rowspan="1" colspan="1">4.47</td></tr><tr><td align="left" rowspan="1" colspan="1">02.99 Other and unspecified neoplasms</td><td align="center" rowspan="1" colspan="1">0.041</td><td align="center" rowspan="1" colspan="1">0.070</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.023</td><td align="center" rowspan="1" colspan="1">0.014</td><td align="center" rowspan="1" colspan="1">0.025</td><td align="center" rowspan="1" colspan="1">0.136</td><td align="center" rowspan="1" colspan="1">0.021</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.014</td><td align="center" rowspan="1" colspan="1">0.060</td><td align="center" rowspan="1" colspan="1">0.047</td><td align="center" rowspan="1" colspan="1">2.91</td></tr><tr><td align="left" rowspan="1" colspan="1">03.01 Severe anaemia</td><td align="center" rowspan="1" colspan="1">0.024</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.043</td><td align="center" rowspan="1" colspan="1">0.005</td><td align="center" rowspan="1" colspan="1">0.021</td><td align="center" rowspan="1" colspan="1">0.016</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.038</td><td align="center" rowspan="1" colspan="1">0.007</td><td align="center" rowspan="1" colspan="1">0.102</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.020</td><td align="center" rowspan="1" colspan="1">19.30</td></tr><tr><td align="left" rowspan="1" colspan="1">03.02 Severe malnutrition</td><td align="center" rowspan="1" colspan="1">0.003</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.021</td><td align="center" rowspan="1" colspan="1">0.027</td><td align="center" rowspan="1" colspan="1">0.021</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.020</td><td align="center" rowspan="1" colspan="1">0.028</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.062</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.008</td><td align="center" rowspan="1" colspan="1">0.016</td><td align="center" rowspan="1" colspan="1">3.76</td></tr><tr><td align="left" rowspan="1" colspan="1">03.03 Diabetes mellitus</td><td align="center" rowspan="1" colspan="1">0.021</td><td align="center" rowspan="1" colspan="1">0.012</td><td align="center" rowspan="1" colspan="1">0.018</td><td align="center" rowspan="1" colspan="1">0.054</td><td align="center" rowspan="1" colspan="1">0.047</td><td align="center" rowspan="1" colspan="1">0.005</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.011</td><td align="center" rowspan="1" colspan="1">0.025</td><td align="center" rowspan="1" colspan="1">0.009</td><td align="center" rowspan="1" colspan="1">0.040</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.156</td><td align="center" rowspan="1" colspan="1">0.077</td><td align="center" rowspan="1" colspan="1">6.34</td></tr><tr><td align="left" rowspan="1" colspan="1">04.01 Acute cardiac disease</td><td align="center" rowspan="1" colspan="1">0.024</td><td align="center" rowspan="1" colspan="1">0.057</td><td align="center" rowspan="1" colspan="1">0.029</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.062</td><td align="center" rowspan="1" colspan="1">0.261</td><td align="center" rowspan="1" colspan="1">0.021</td><td align="center" rowspan="1" colspan="1">0.011</td><td align="center" rowspan="1" colspan="1">0.062</td><td align="center" rowspan="1" colspan="1">0.057</td><td align="center" rowspan="1" colspan="1">0.074</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.020</td><td align="center" rowspan="1" colspan="1">0.031</td><td align="center" rowspan="1" colspan="1">11.86</td></tr><tr><td align="left" rowspan="1" colspan="1">04.03 Sickle cell with crisis</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.007</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.004</td><td align="center" rowspan="1" colspan="1">0.010</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">100.00</td></tr><tr><td align="left" rowspan="1" colspan="1">04.02 Stroke</td><td align="center" rowspan="1" colspan="1">0.268</td><td align="center" rowspan="1" colspan="1">0.238</td><td align="center" rowspan="1" colspan="1">0.131</td><td align="center" rowspan="1" colspan="1">0.084</td><td align="center" rowspan="1" colspan="1">0.115</td><td align="center" rowspan="1" colspan="1">0.269</td><td align="center" rowspan="1" colspan="1">0.322</td><td align="center" rowspan="1" colspan="1">0.062</td><td align="center" rowspan="1" colspan="1">0.136</td><td align="center" rowspan="1" colspan="1">0.031</td><td align="center" rowspan="1" colspan="1">0.229</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.254</td><td align="center" rowspan="1" colspan="1">0.122</td><td align="center" rowspan="1" colspan="1">6.27</td></tr><tr><td align="left" rowspan="1" colspan="1">04.99 Other and unspecified cardiac dis</td><td align="center" rowspan="1" colspan="1">0.115</td><td align="center" rowspan="1" colspan="1">0.167</td><td align="center" rowspan="1" colspan="1">0.017</td><td align="center" rowspan="1" colspan="1">0.072</td><td align="center" rowspan="1" colspan="1">0.117</td><td align="center" rowspan="1" colspan="1">0.071</td><td align="center" rowspan="1" colspan="1">0.034</td><td align="center" rowspan="1" colspan="1">0.086</td><td align="center" rowspan="1" colspan="1">0.378</td><td align="center" rowspan="1" colspan="1">0.433</td><td align="center" rowspan="1" colspan="1">0.058</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.233</td><td align="center" rowspan="1" colspan="1">0.435</td><td align="center" rowspan="1" colspan="1">13.15</td></tr><tr><td align="left" rowspan="1" colspan="1">05.01 Chronic obstructive pulmonary dis</td><td align="center" rowspan="1" colspan="1">0.007</td><td align="center" rowspan="1" colspan="1">0.049</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.017</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.008</td><td align="center" rowspan="1" colspan="1">0.009</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.027</td><td align="center" rowspan="1" colspan="1">0.159</td><td align="center" rowspan="1" colspan="1">0.028</td><td align="center" rowspan="1" colspan="1">10.69</td></tr><tr><td align="left" rowspan="1" colspan="1">05.02 Asthma</td><td align="center" rowspan="1" colspan="1">0.043</td><td align="center" rowspan="1" colspan="1">0.016</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.008</td><td align="center" rowspan="1" colspan="1">0.071</td><td align="center" rowspan="1" colspan="1">0.006</td><td align="center" rowspan="1" colspan="1">0.033</td><td align="center" rowspan="1" colspan="1">0.060</td><td align="center" rowspan="1" colspan="1">0.020</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.066</td><td align="center" rowspan="1" colspan="1">0.372</td><td align="center" rowspan="1" colspan="1">0.058</td><td align="center" rowspan="1" colspan="1">17.57</td></tr><tr><td align="left" rowspan="1" colspan="1">06.01 Acute abdomen</td><td align="center" rowspan="1" colspan="1">0.194</td><td align="center" rowspan="1" colspan="1">0.162</td><td align="center" rowspan="1" colspan="1">0.144</td><td align="center" rowspan="1" colspan="1">0.162</td><td align="center" rowspan="1" colspan="1">0.451</td><td align="center" rowspan="1" colspan="1">0.487</td><td align="center" rowspan="1" colspan="1">0.408</td><td align="center" rowspan="1" colspan="1">0.047</td><td align="center" rowspan="1" colspan="1">0.751</td><td align="center" rowspan="1" colspan="1">0.104</td><td align="center" rowspan="1" colspan="1">0.467</td><td align="center" rowspan="1" colspan="1">0.434</td><td align="center" rowspan="1" colspan="1">0.178</td><td align="center" rowspan="1" colspan="1">0.172</td><td align="center" rowspan="1" colspan="1">6.55</td></tr><tr><td align="left" rowspan="1" colspan="1">06.02 Liver cirrhosis</td><td align="center" rowspan="1" colspan="1">0.025</td><td align="center" rowspan="1" colspan="1">0.078</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.028</td><td align="center" rowspan="1" colspan="1">0.045</td><td align="center" rowspan="1" colspan="1">0.029</td><td align="center" rowspan="1" colspan="1">0.025</td><td align="center" rowspan="1" colspan="1">0.003</td><td align="center" rowspan="1" colspan="1">0.084</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.009</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.049</td><td align="center" rowspan="1" colspan="1">0.032</td><td align="center" rowspan="1" colspan="1">15.48</td></tr><tr><td align="left" rowspan="1" colspan="1">07.01 Renal failure</td><td align="center" rowspan="1" colspan="1">0.018</td><td align="center" rowspan="1" colspan="1">0.013</td><td align="center" rowspan="1" colspan="1">0.006</td><td align="center" rowspan="1" colspan="1">0.033</td><td align="center" rowspan="1" colspan="1">0.030</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.027</td><td align="center" rowspan="1" colspan="1">0.003</td><td align="center" rowspan="1" colspan="1">0.064</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.008</td><td align="center" rowspan="1" colspan="1">0.081</td><td align="center" rowspan="1" colspan="1">11.54</td></tr><tr><td align="left" rowspan="1" colspan="1">08.01 Epilepsy</td><td align="center" rowspan="1" colspan="1">0.019</td><td align="center" rowspan="1" colspan="1">0.014</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.043</td><td align="center" rowspan="1" colspan="1">0.058</td><td align="center" rowspan="1" colspan="1">0.050</td><td align="center" rowspan="1" colspan="1">0.119</td><td align="center" rowspan="1" colspan="1">0.014</td><td align="center" rowspan="1" colspan="1">0.051</td><td align="center" rowspan="1" colspan="1">0.016</td><td align="center" rowspan="1" colspan="1">0.023</td><td align="center" rowspan="1" colspan="1">0.025</td><td align="center" rowspan="1" colspan="1">0.057</td><td align="center" rowspan="1" colspan="1">0.037</td><td align="center" rowspan="1" colspan="1">3.30</td></tr><tr><td align="left" rowspan="1" colspan="1">98 Other and unspecified NCD</td><td align="center" rowspan="1" colspan="1">0.042</td><td align="center" rowspan="1" colspan="1">0.049</td><td align="center" rowspan="1" colspan="1">0.008</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.017</td><td align="center" rowspan="1" colspan="1">0.014</td><td align="center" rowspan="1" colspan="1">0.055</td><td align="center" rowspan="1" colspan="1">0.029</td><td align="center" rowspan="1" colspan="1">0.406</td><td align="center" rowspan="1" colspan="1">0.023</td><td align="center" rowspan="1" colspan="1">0.042</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.083</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">10.26</td></tr><tr><td align="left" rowspan="1" colspan="1">10.06 Congenital malformation</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.008</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.00</td></tr><tr><td align="left" rowspan="1" colspan="1">12.01 Road traffic accident</td><td align="center" rowspan="1" colspan="1">0.018</td><td align="center" rowspan="1" colspan="1">0.033</td><td align="center" rowspan="1" colspan="1">0.016</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.309</td><td align="center" rowspan="1" colspan="1">0.220</td><td align="center" rowspan="1" colspan="1">0.116</td><td align="center" rowspan="1" colspan="1">0.046</td><td align="center" rowspan="1" colspan="1">0.062</td><td align="center" rowspan="1" colspan="1">0.116</td><td align="center" rowspan="1" colspan="1">0.078</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.292</td><td align="center" rowspan="1" colspan="1">0.284</td><td align="center" rowspan="1" colspan="1">0.62</td></tr><tr><td align="left" rowspan="1" colspan="1">12.02 Other transport accident</td><td align="center" rowspan="1" colspan="1">0.012</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.00</td></tr><tr><td align="left" rowspan="1" colspan="1">12.03 Accid fall</td><td align="center" rowspan="1" colspan="1">0.003</td><td align="center" rowspan="1" colspan="1">0.004</td><td align="center" rowspan="1" colspan="1">0.013</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.106</td><td align="center" rowspan="1" colspan="1">0.048</td><td align="center" rowspan="1" colspan="1">0.212</td><td align="center" rowspan="1" colspan="1">0.008</td><td align="center" rowspan="1" colspan="1">0.015</td><td align="center" rowspan="1" colspan="1">0.013</td><td align="center" rowspan="1" colspan="1">0.024</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.00</td></tr><tr><td align="left" rowspan="1" colspan="1">12.04 Accid drowning and submersion</td><td align="center" rowspan="1" colspan="1">0.024</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.021</td><td align="center" rowspan="1" colspan="1">0.034</td><td align="center" rowspan="1" colspan="1">0.129</td><td align="center" rowspan="1" colspan="1">0.016</td><td align="center" rowspan="1" colspan="1">0.058</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.026</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.00</td></tr><tr><td align="left" rowspan="1" colspan="1">12.05 Accid expos to smoke, fire, and flame</td><td align="center" rowspan="1" colspan="1">0.012</td><td align="center" rowspan="1" colspan="1">0.016</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.008</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.008</td><td align="center" rowspan="1" colspan="1">0.015</td><td align="center" rowspan="1" colspan="1">0.166</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.040</td><td align="center" rowspan="1" colspan="1">0.024</td><td align="center" rowspan="1" colspan="1">0.00</td></tr><tr><td align="left" rowspan="1" colspan="1">12.06 Contact with venomous plant/animal</td><td align="center" rowspan="1" colspan="1">0.004</td><td align="center" rowspan="1" colspan="1">0.012</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.101</td><td align="center" rowspan="1" colspan="1">0.026</td><td align="center" rowspan="1" colspan="1">0.138</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.032</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.00</td></tr><tr><td align="left" rowspan="1" colspan="1">12.10 Exposure to force of nature</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.017</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.00</td></tr><tr><td align="left" rowspan="1" colspan="1">12.07 Accid poisoning and noxious subs</td><td align="center" rowspan="1" colspan="1">0.004</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.010</td><td align="center" rowspan="1" colspan="1">0.008</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.002</td><td align="center" rowspan="1" colspan="1">0.011</td><td align="center" rowspan="1" colspan="1">0.00</td></tr><tr><td align="left" rowspan="1" colspan="1">12.08 Intentional self-harm</td><td align="center" rowspan="1" colspan="1">0.254</td><td align="center" rowspan="1" colspan="1">0.469</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.090</td><td align="center" rowspan="1" colspan="1">0.025</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.025</td><td align="center" rowspan="1" colspan="1">0.079</td><td align="center" rowspan="1" colspan="1">0.046</td><td align="center" rowspan="1" colspan="1">0.074</td><td align="center" rowspan="1" colspan="1">0.018</td><td align="center" rowspan="1" colspan="1">0.379</td><td align="center" rowspan="1" colspan="1">0.121</td><td align="center" rowspan="1" colspan="1">1.06</td></tr><tr><td align="left" rowspan="1" colspan="1">12.09 Assault</td><td align="center" rowspan="1" colspan="1">0.069</td><td align="center" rowspan="1" colspan="1">0.032</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.080</td><td align="center" rowspan="1" colspan="1">0.061</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.030</td><td align="center" rowspan="1" colspan="1">0.070</td><td align="center" rowspan="1" colspan="1">0.100</td><td align="center" rowspan="1" colspan="1">0.053</td><td align="center" rowspan="1" colspan="1">0.020</td><td align="center" rowspan="1" colspan="1">0.372</td><td align="center" rowspan="1" colspan="1">0.461</td><td align="center" rowspan="1" colspan="1">2.83</td></tr><tr><td align="left" rowspan="1" colspan="1">12.99 Other and unspecified external CoD</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.003</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.00</td></tr><tr><td align="left" rowspan="1" colspan="1">09.01 Ectopic pregnancy</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.012</td><td align="center" rowspan="1" colspan="1">0.035</td><td align="center" rowspan="1" colspan="1">0.008</td><td align="center" rowspan="1" colspan="1">0.003</td><td align="center" rowspan="1" colspan="1">0.020</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.018</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.005</td><td align="center" rowspan="1" colspan="1">0.032</td><td align="center" rowspan="1" colspan="1">100.00</td></tr><tr><td align="left" rowspan="1" colspan="1">09.02 Abortion-related death</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.042</td><td align="center" rowspan="1" colspan="1">0.082</td><td align="center" rowspan="1" colspan="1">0.069</td><td align="center" rowspan="1" colspan="1">0.009</td><td align="center" rowspan="1" colspan="1">0.059</td><td align="center" rowspan="1" colspan="1">0.025</td><td align="center" rowspan="1" colspan="1">0.077</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.004</td><td align="center" rowspan="1" colspan="1">0.015</td><td align="center" rowspan="1" colspan="1">100.00</td></tr><tr><td align="left" rowspan="1" colspan="1">09.03 Pregnancy-induced hypertension</td><td align="center" rowspan="1" colspan="1">0.080</td><td align="center" rowspan="1" colspan="1">0.069</td><td align="center" rowspan="1" colspan="1">0.017</td><td align="center" rowspan="1" colspan="1">0.082</td><td align="center" rowspan="1" colspan="1">0.036</td><td align="center" rowspan="1" colspan="1">0.054</td><td align="center" rowspan="1" colspan="1">0.229</td><td align="center" rowspan="1" colspan="1">0.060</td><td align="center" rowspan="1" colspan="1">0.074</td><td align="center" rowspan="1" colspan="1">0.027</td><td align="center" rowspan="1" colspan="1">0.053</td><td align="center" rowspan="1" colspan="1">0.067</td><td align="center" rowspan="1" colspan="1">0.048</td><td align="center" rowspan="1" colspan="1">0.117</td><td align="center" rowspan="1" colspan="1">100.00</td></tr><tr><td align="left" rowspan="1" colspan="1">09.04 Obstetric haemorrhage</td><td align="center" rowspan="1" colspan="1">0.073</td><td align="center" rowspan="1" colspan="1">0.223</td><td align="center" rowspan="1" colspan="1">0.073</td><td align="center" rowspan="1" colspan="1">0.122</td><td align="center" rowspan="1" colspan="1">0.156</td><td align="center" rowspan="1" colspan="1">0.226</td><td align="left" rowspan="1" colspan="1">0.693</td><td align="left" rowspan="1" colspan="1">0.127</td><td align="left" rowspan="1" colspan="1">0.251</td><td align="left" rowspan="1" colspan="1">0.095</td><td align="left" rowspan="1" colspan="1">0.315</td><td align="left" rowspan="1" colspan="1">0.346</td><td align="left" rowspan="1" colspan="1">0.167</td><td align="left" rowspan="1" colspan="1">0.077</td><td align="left" rowspan="1" colspan="1">100.00</td></tr><tr><td align="left" rowspan="1" colspan="1">09.05 Obstructed labour</td><td align="center" rowspan="1" colspan="1">0.006</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.010</td><td align="center" rowspan="1" colspan="1">0.010</td><td align="center" rowspan="1" colspan="1">0.003</td><td align="center" rowspan="1" colspan="1">0.013</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.004</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.025</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.013</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">100.00</td></tr><tr><td align="left" rowspan="1" colspan="1">09.06 Pregnancy-related sepsis</td><td align="center" rowspan="1" colspan="1">0.024</td><td align="center" rowspan="1" colspan="1">0.041</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.039</td><td align="center" rowspan="1" colspan="1">0.028</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.516</td><td align="center" rowspan="1" colspan="1">0.024</td><td align="center" rowspan="1" colspan="1">0.090</td><td align="center" rowspan="1" colspan="1">0.141</td><td align="center" rowspan="1" colspan="1">0.036</td><td align="center" rowspan="1" colspan="1">0.211</td><td align="center" rowspan="1" colspan="1">0.039</td><td align="center" rowspan="1" colspan="1">0.051</td><td align="center" rowspan="1" colspan="1">100.00</td></tr><tr><td align="left" rowspan="1" colspan="1">09.07 Anaemia of pregnancy</td><td align="center" rowspan="1" colspan="1">0.002</td><td align="center" rowspan="1" colspan="1">0.007</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.038</td><td align="center" rowspan="1" colspan="1">0.009</td><td align="center" rowspan="1" colspan="1">0.007</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.009</td><td align="center" rowspan="1" colspan="1">0.071</td><td align="center" rowspan="1" colspan="1">0.015</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.195</td><td align="center" rowspan="1" colspan="1">0.040</td><td align="center" rowspan="1" colspan="1">0.036</td><td align="center" rowspan="1" colspan="1">100.00</td></tr><tr><td align="left" rowspan="1" colspan="1">09.08 Ruptured uterus</td><td align="center" rowspan="1" colspan="1">0.004</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.052</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">100.00</td></tr><tr><td align="left" rowspan="1" colspan="1">09.99 Other and unspecified maternal CoD</td><td align="center" rowspan="1" colspan="1">0.012</td><td align="center" rowspan="1" colspan="1">0.060</td><td align="center" rowspan="1" colspan="1">0.006</td><td align="center" rowspan="1" colspan="1">0.031</td><td align="center" rowspan="1" colspan="1">0.061</td><td align="center" rowspan="1" colspan="1">0.042</td><td align="center" rowspan="1" colspan="1">0.484</td><td align="center" rowspan="1" colspan="1">0.005</td><td align="center" rowspan="1" colspan="1">0.088</td><td align="center" rowspan="1" colspan="1">0.047</td><td align="center" rowspan="1" colspan="1">0.119</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.038</td><td align="center" rowspan="1" colspan="1">0.027</td><td align="center" rowspan="1" colspan="1">100.00</td></tr><tr><td align="left" rowspan="1" colspan="1">99 Indeterminate</td><td align="center" rowspan="1" colspan="1">0.263</td><td align="center" rowspan="1" colspan="1">0.181</td><td align="center" rowspan="1" colspan="1">0.154</td><td align="center" rowspan="1" colspan="1">0.304</td><td align="center" rowspan="1" colspan="1">1.567</td><td align="center" rowspan="1" colspan="1">1.329</td><td align="center" rowspan="1" colspan="1">1.559</td><td align="center" rowspan="1" colspan="1">0.177</td><td align="center" rowspan="1" colspan="1">2.016</td><td align="center" rowspan="1" colspan="1">0.749</td><td align="center" rowspan="1" colspan="1">0.824</td><td align="center" rowspan="1" colspan="1">1.538</td><td align="center" rowspan="1" colspan="1">2.891</td><td align="center" rowspan="1" colspan="1">0.967</td><td align="center" rowspan="1" colspan="1">10.67</td></tr></tbody></table></table-wrap><p>
<xref ref-type="fig" rid="F0002">Figure 2</xref> shows the proportions of maternal deaths (direct and indirect) for each site, along with proportions of non-maternal deaths in major cause groups. Major contributors to indirect maternal deaths were anaemia, pneumonia, malaria, and cardiovascular causes. The figure shows a substantial variation by site in the proportion of maternal deaths out of all WRA deaths (represented by the overall 100% bar for each site). Generally, sites with higher overall WRA mortality rates (<xref ref-type="table" rid="T0002">Table 2</xref>), and particularly those with a substantial HIV/AIDS and tuberculosis mortality component, consequently had lower proportions of maternal deaths.</p><fig id="F0002" position="float"><label>Fig. 2</label><caption><p>Proportions of maternal and non-maternal mortality among women aged 15–49 by cause category for 14 INDEPTH sites.</p></caption><graphic xlink:href="GHA-7-25368-g002"/></fig><p>
<xref ref-type="fig" rid="F0003">Figure 3</xref> shows a detailed breakdown of maternal mortality rates by various obstetric and indirect causes. Obstetric haemorrhage was the dominant direct obstetric cause at most sites.</p><fig id="F0003" position="float"><label>Fig. 3</label><caption><p>Maternal mortality rates per 1,000 person-years by WHO 2012 VA cause categories for 14 INDEPTH sites.</p></caption><graphic xlink:href="GHA-7-25368-g003"/></fig><p>Although not influencing the other results presented here, it was also possible to use the same methodology to see which causes of death might be related to a ‘healthy-pregnancy’ effect, by being under-represented among the PRD. The effects were not huge, but most sites reported proportionately less cancer deaths among pregnant women; in some sites, there were less HIV/AIDS and tuberculosis deaths. Sites in Bangladesh reported lower rates of suicides among pregnant women.</p></sec><sec sec-type="discussion" id="S0004"><title>Discussion</title><p>These analyses of pregnancy-related mortality, other than being numerically large and geographically wide, were strengthened by having cause-specific mortality and pregnancy status data for all deaths of women aged 15–49 in the site populations, rather than being based on surveys of maternal deaths. Thus, even though VA is not a method that facilitates distinguishing between indirect maternal deaths and incidental pregnancy-related mortality on an individual basis, it was possible using this dataset to account for maternal deaths in several different ways, including MMR; cause-specific mortality rates; and direct, indirect, and incidental maternal deaths.</p><p>Although the natural way to analyse and present maternal mortality from a longitudinal population-based dataset of this kind is in terms of mortality rates, in <xref ref-type="fig" rid="F0001">Fig. 1</xref> we also presented MMR estimates from these data in order to provide comparability with other sources of information. Although the 14 sites reporting here covered different surveillance periods, most of the data reported related to the period 2006–2012. Thus, we have compared MMR findings with those reported in UN estimates for 2010 (<xref rid="CIT0028" ref-type="bibr">28</xref>). For half of the sites, the MMR point estimates in <xref ref-type="fig" rid="F0001">Fig. 1</xref> lay within the range estimated for the country in 2010 by the United Nations. Other sites had MMR estimates slightly below the lower limit of the UN estimates. However, there is a lack of precision in comparing specific HDSS areas with national estimates.</p><p>The concepts of PRD that are ‘indirectly’ due to pregnancy or ‘incidental’ to pregnancy are somewhat fraught, and have become all the more difficult to interpret in populations with high HIV/AIDS mortality burdens. Because conceiving and successfully maintaining a pregnancy tend to require being reasonably healthy in the first place, the resulting selection effect means that pregnancy can appear to reduce women's mortality from many causes (<xref rid="CIT0029" ref-type="bibr">29</xref>). Certainly care has to be taken in interpreting population proportions of maternal mortality depending on other mortality pressures such as HIV/AIDS and malaria, as is evident from <xref ref-type="fig" rid="F0002">Fig. 2</xref>. Although the two South African sites (Agincourt and Africa Centre) in the figure had very low proportions of maternal deaths compared with overall WRA mortality, their MMRs were by no means low; but the massive burdens of HIV/AIDS mortality in the WRA populations in those locations minimised the maternal proportions. We believe that the approach we have taken for estimating indirect maternal deaths on the basis of specific cause proportions among pregnancy-related and non-pregnancy deaths is effective where VA data are available for all WRA deaths in a population. It offers a significant advantage over the standard DHS methods, which can only measure pregnancy-related mortality, rather than maternal mortality (<xref rid="CIT0003" ref-type="bibr">3</xref>).</p><p>There has also been considerable discussion, particularly in relation to arriving at realistic global estimates of maternal mortality, as to the role of HIV infection in PRD. Although it is well established that HIV-positive women are less likely to become pregnant, and that they are substantially more likely to die from numerous causes irrespective of pregnancy (<xref rid="CIT0030" ref-type="bibr">30</xref>), it has been a challenge to ascertain the mortality risks to HIV-positive women
who do become pregnant, in terms of possible interactions between HIV positivity and pregnancy (<xref rid="CIT0031" ref-type="bibr">31</xref>). Our results, shown in <xref ref-type="fig" rid="F0002">Fig. 2</xref>, suggest that HIV/AIDS does not constitute a major proportion of indirect maternal mortality, even in settings with high HIV/AIDS mortality burdens.</p><p>Apart from the obvious limitations of VA in any context, its use in relation to maternal mortality depends crucially on ascertaining pregnancy status reliably in the VA interview. Depending on how the VA interview is carried out, and who the available respondent is, there may be difficulties around capturing pregnancy status, particularly in relation to early or undisclosed pregnancies. Most sites, however, reported some cases of ectopic pregnancy and/or abortion-related deaths, which may indirectly be indicators of data reliability in relation to pregnancy. The proportions of direct maternal, indirect maternal, and incidental deaths making up total PRD here also suggests that many deaths not self-evidently connected with pregnancy were indeed identified as being pregnancy related in the VA interviews. Although it might be argued that prospective pregnancy registration could improve detection of pregnancies, and this is done in some INDEPTH sites, it is a hugely resource-intensive undertaking, probably only applicable in research settings. On the contrary, analysing total WRA cause of death data, which could in principle come from VA used in the context of civil registration of deaths, provides a more direct method for analysing and documenting maternal mortality (<xref rid="CIT0032" ref-type="bibr">32</xref>).</p></sec><sec sec-type="conclusions" id="S0005"><title>Conclusions</title><p>Although there are many potential difficulties in measuring maternal mortality at the population level, our findings here are generally plausible and in line with other estimates. They confirm the continuing unacceptably high levels of mortality in women in conjunction with giving birth across Africa and in parts of Asia. Measuring these high rates by recording the individual tragedies involved is not the solution to the problem, but understanding the details of what is happening at the population level is a pre-requisite to implementing and evaluating solutions.</p></sec> |
Causes of death among persons of all ages within the Kilifi Health and Demographic Surveillance System, Kenya, determined from verbal autopsies interpreted using the InterVA-4 model | <sec id="st1"><title>Background</title><p>The vast majority of deaths in the Kilifi study area are not recorded through official systems of vital registration. As a result, few data are available regarding causes of death in this population.</p></sec><sec id="st2"><title>Objective</title><p>To describe the causes of death (CODs) among residents of all ages within the Kilifi Health and Demographic Surveillance System (KHDSS) on the coast of Kenya.</p></sec><sec id="st3"><title>Design</title><p>Verbal autopsies (VAs) were conducted using the 2007 World Health Organization (WHO) standard VA questionnaires, and VA data further transformed to align with the 2012 WHO VA instrument. CODs were then determined using the InterVA-4 computer-based probabilistic model.</p></sec><sec id="st4"><title>Results</title><p>Five thousand one hundred and eighty seven deaths were recorded between January 2008 and December 2011. VA interviews were completed for 4,460 (86%) deaths. Neonatal pneumonia and birth asphyxia were the main CODs in neonates; pneumonia and malaria were the main CODs among infants and children aged 1–4, respectively, while HIV/AIDS was the main COD for adult women of reproductive age. Road traffic accidents were more commonly observed among men than women. Stroke and neoplasms were common CODs among the elderly over the age of 65.</p></sec><sec id="st5"><title>Conclusions</title><p>We have established the main CODs among people of all ages within the area served by the KHDSS on the coast of Kenya using the 2007 WHO VA questionnaire coded using InterVA-4. We hope that our data will allow local health planners to estimate the burden of various diseases and to allocate their limited resources more appropriately.</p></sec> | <contrib contrib-type="author"><name><surname>Ndila</surname><given-names>Carolyne</given-names></name><xref ref-type="aff" rid="AF0001">1</xref><xref ref-type="aff" rid="AF0002">2</xref><xref ref-type="corresp" rid="cor1">*</xref></contrib><contrib contrib-type="author"><name><surname>Bauni</surname><given-names>Evasius</given-names></name><xref ref-type="aff" rid="AF0001">1</xref><xref ref-type="aff" rid="AF0002">2</xref></contrib><contrib contrib-type="author"><name><surname>Mochamah</surname><given-names>George</given-names></name><xref ref-type="aff" rid="AF0001">1</xref><xref ref-type="aff" rid="AF0002">2</xref></contrib><contrib contrib-type="author"><name><surname>Nyirongo</surname><given-names>Vysaul</given-names></name><xref ref-type="aff" rid="AF0003">3</xref></contrib><contrib contrib-type="author"><name><surname>Makazi</surname><given-names>Alex</given-names></name><xref ref-type="aff" rid="AF0001">1</xref></contrib><contrib contrib-type="author"><name><surname>Kosgei</surname><given-names>Patrick</given-names></name><xref ref-type="aff" rid="AF0001">1</xref></contrib><contrib contrib-type="author"><name><surname>Tsofa</surname><given-names>Benjamin</given-names></name><xref ref-type="aff" rid="AF0001">1</xref></contrib><contrib contrib-type="author"><name><surname>Nyutu</surname><given-names>Gideon</given-names></name><xref ref-type="aff" rid="AF0001">1</xref><xref ref-type="aff" rid="AF0002">2</xref></contrib><contrib contrib-type="author"><name><surname>Etyang</surname><given-names>Anthony</given-names></name><xref ref-type="aff" rid="AF0001">1</xref></contrib><contrib contrib-type="author"><name><surname>Byass</surname><given-names>Peter</given-names></name><xref ref-type="aff" rid="AF0004">4</xref></contrib><contrib contrib-type="author"><name><surname>Williams</surname><given-names>Thomas N.</given-names></name><xref ref-type="aff" rid="AF0001">1</xref><xref ref-type="aff" rid="AF0002">2</xref><xref ref-type="aff" rid="AF0005">5</xref></contrib> | Global Health Action | <p>In developed countries, cause-specific mortality data are readily available from well-established vital registration systems. In developing countries, however, where a majority of the world’s deaths occur, vital registration systems are poor (<xref rid="CIT0001" ref-type="bibr">1</xref>), access to health care services is limited, and most deaths occur at home. Reliable medical records on causes of death (CODs) are inadequate, and therefore other sources of data must be used to investigate patterns of cause-specific mortality.</p><p>Despite the fact that some studies have questioned the reliability of the Verbal Autopsy (VA) approach, where close relatives of the deceased are asked about the signs and symptoms that precede deaths (<xref rid="CIT0002" ref-type="bibr">2</xref>, <xref rid="CIT0003" ref-type="bibr">3</xref>), it remains the only practical approach through which to estimate CODs in most resource-poor settings in which the majority of deaths occur outside the formal health system (<xref rid="CIT0002" ref-type="bibr">2</xref>, <xref rid="CIT0004" ref-type="bibr">4</xref>–<xref rid="CIT0008" ref-type="bibr">8</xref>)
. The approach has been used to determine CODs among people of all ages in a wide range of settings (<xref rid="CIT0002" ref-type="bibr">2</xref>, <xref rid="CIT0007" ref-type="bibr">7</xref>, <xref rid="CIT0009" ref-type="bibr">9</xref>–<xref rid="CIT0016" ref-type="bibr">16</xref>); however, in Kenya, although VAs have been widely used as a tool for documenting CODs (<xref rid="CIT0007" ref-type="bibr">7</xref>, <xref rid="CIT0012" ref-type="bibr">12</xref>–<xref rid="CIT0014" ref-type="bibr">14</xref>, <xref rid="CIT0017" ref-type="bibr">17</xref>–<xref rid="CIT0021" ref-type="bibr">21</xref>)
, no study has yet been published in which the WHO-compliant InterVA-4 has been applied across deaths of all ages. The InterVA-4 model uses posterior probabilities for CODs, given an a priori distribution of CODs in the population and conditional probabilities for circumstances leading to death. The current study was conducted with a view to using the InterVA-4 model to describe CODs among all residents of the area served by the Kilifi Health and Demographic Surveillance System (KHDSS), where close to 60% of deaths occur outside the formal health system. This study is also part of the multisite cause of death dataset (<xref rid="CIT0022" ref-type="bibr">22</xref>) available in the public domain at the INDEPTH Data Repository (<xref rid="CIT0023" ref-type="bibr">23</xref>).</p><sec sec-type="methods" id="S0001"><title>Methods</title><sec id="S0001-S20001"><title>Study population</title><p>The KHDSS study area has been described in detail previously (<xref rid="CIT0024" ref-type="bibr">24</xref>). Briefly, the KHDSS was established in 2000 and became a member of INDEPTH network in 2005. The KHDSS covers an area of 891 km<sup>2</sup> and has a current resident population of ~265,000. The population register is updated three times each year and records between 1,200 and 1,500 deaths annually.</p><p>VA, using the standard 2007 World Health Organization (WHO) VA tools (<xref rid="CIT0025" ref-type="bibr">25</xref>), were first introduced into the KHDSS activities in 2008 (<xref rid="CIT0012" ref-type="bibr">12</xref>). These tools include three separate VA questionnaires that are used to collect data on neonates (0–28 days old), children (29 days to 14 years old), and adolescents and adults (>15 years). Structurally, the questionnaires contain a short open narrative section that is followed by a cascade of closed questions. The narrative part provides the respondent’s chronological account of any illnesses and events that led to death, while the closed questions filter through the history and details of the illness. Age-appropriate VA questionnaires were generated for each death along with household maps and listings to guide interviewers to the appropriate respondents. With a view to minimizing transcription errors, we generated pre-filled VA questionnaires that included all basic demographic details such as names, personal identifiers, dates of birth and death for confirmation by the interviewers before their interviews. Completed VA forms were checked for errors by the field supervisor before entry into a database written in FileMaker Pro™ v11 (FileMaker, USA).</p></sec><sec id="S0001-S20002"><title>The InterVA-4 model</title><p>CODs were assigned using the InterVA-4 computer-based probabilistic model (<xref rid="CIT0025" ref-type="bibr">25</xref>). The original model used an expert panel to develop a coding system based on relatively broad COD categories. The current version (Version 4.02) (<xref rid="CIT0025" ref-type="bibr">25</xref>) has evolved, and has benefitted from testing and validation, largely using data from sub-Saharan Africa (<xref rid="CIT0004" ref-type="bibr">4</xref>, <xref rid="CIT0012" ref-type="bibr">12</xref>, <xref rid="CIT0013" ref-type="bibr">13</xref>). The InterVA-4 software is freely available in the public domain (<xref rid="CIT0025" ref-type="bibr">25</xref>). For our analysis, COD categories were obtained by running InterVA-4 in batch mode on the input indicators with both malaria and HIV prevalence set to ‘high’ for all the age categories.</p></sec><sec id="S0001-S20003"><title>Data management</title><p>All VA forms were scrutinized by a clinician for anomalies before double entry into a computer database written in FileMaker Pro™ v11 (FileMaker, USA). The VA data were further transformed to align with the WHO 2012 VA standard format and were then processed using the InterVA-4 model to assign COD. These VA standard formats and the specifications have been described in detail previously (<xref rid="CIT0026" ref-type="bibr">26</xref>). Where available, we supplemented the model with information from the open narrative section for input data variables that were not targeted systematically in the closed section of the VA questionnaires. Of particular note, we included data on a set of reported hospital diagnoses that were required as input data for InterVA-4 but that were not included on the list of previously known medical conditions in the 2007 WHO questionnaires. Examples included sickle cell disease (SCD) and congenital malformations. Such information was extracted by keyword searches of the free-text narrative section. For example, to create the congenital malformation (born_malf) indicator, we searched for the strings ‘heart’, ‘congenital’, ‘hydrocephalous’, ‘spina bifida’, and ‘hole in the heart’ within VAs collected from children who died at <5 years of age. The congenital malformation indicator was left unchanged if it had already been marked ‘YES’ but was converted from ‘NO’ to ‘YES’ in cases where heart disease had been selected in the list of previously known medical conditions. Similarly, injuries associated with sexual assault (rape) were not always captured in the structured questions but were sometimes described in the narrative section, facilitating mapping to the ‘assault’ indicator. Injury may either be intentional (e.g. assault or suicide) or accidental. The main warning messages flagged by the model when we input our data, involved cases where, for example, suicide was inconsistent with other injuries.</p><p>Finally, the variable ‘born_small’ requires either the birth weight or data to suggest that the child was small at birth as reported by the respondent. Some respondents indicated that the baby was born small while the birth weight recorded was more >2.5 kg or vice versa. Where responses were recorded for both indicators, therefore, we only considered the birth weight in mapping the ‘low birth weight’ (born_small) or ‘big baby’ (born_big) indicators.</p></sec><sec id="S0001-S20004"><title>Data analysis</title><p>Deaths were aggregated for all individuals in the study population for the period January 2008 to December 2011. An analytical dataset was constructed from the model’s output in which each VA case had one or more records. Each record having one cause and a weight corresponding to the likelihood of that cause for the particular VA case. The model assigned multiple CODs if they reached half of the likelihood of the leading cause. Any residual margin of likelihood not accounted for by the likelihood of the first, second or third causes was then considered as indeterminate. The possible CODs determined by the InterVA-4 model were derived for all ages. Cause-specific mortality fractions (CSMF) were determined as the proportion of all deaths that were attributable to any specific COD. We stratified mortality analyses by sex and seven age groups (neonates, infants, and ages 1–4, 5–14, 15–49, 50–64 and over 65 years). The age-group boundaries were chosen to reflect groups of public health importance. All statistical analyses were carried out using STATA V11 (Timberlake, USA) and the statistical software environment R (<ext-link ext-link-type="uri" xlink:href="http://www.r-project.org/">http://www.r-project.org/</ext-link>).</p></sec><sec id="S0001-S20005"><title>Ethical approval</title><p>Individual written informed consent was obtained by interviewers from all VA respondents. The study was approved by the KEMRI/Wellcome Trust Scientific Coordinating Committee (SCC) and by the KEMRI Scientific Steering Committee (SSC), both in Kilifi, and by the KEMRI/National Ethical Review Committee (ERC) in Nairobi.</p></sec></sec><sec sec-type="results" id="S0002"><title>Results</title><p>A total of 5,187 deaths were recorded among the resident population of the KHDSS between January 2008 and December 2011. One thousand one hundred and eighty one cases (23%) were among children <5 years old, 242 cases (5%) were aged 5–14 years, and 3,764 cases (73%) were >15 years of age, of which 1,665 cases (32%) were >65 years old (<xref ref-type="table" rid="T0001">Table 1</xref>). Of the deaths among children <5 years old, 810 (68%) occurred before the first year of life of which 469 (58%) were neonates. Male deaths out-numbered female deaths in most age groups. VA interviews were successfully completed for 4,460 (85%) of these deaths of which 2,304 (52%) were among males (<xref ref-type="table" rid="T0001">Table 1</xref>). Failure to identify an appropriate respondent was the main reason for missing data. Among those deaths in which a VA was performed, 57% occurred at home and 36% occurred in a health facility. Most neonatal deaths occurred in hospitals while more of the elderly died at home. The average recall period (time between VA interview and death) was 194 days. Among those aged >15 years (<italic>n=</italic>3,310), 10% were single, 55% were legally married, 28% were widowed, 4% were divorced, and 3% were separated. The InterVA-4 model assigned a single COD to 3,886 cases (87%), two CODs to 422 cases (9%), three CODs to 22 cases (1%), and 130 cases (3%) to ‘indeterminate’.</p><table-wrap id="T0001" position="float"><label>Table 1</label><caption><p>Total number of deaths and percentages with VA by age group and sex</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1"/><th align="center" colspan="14" rowspan="1">Age group</th><th align="center" rowspan="1" colspan="1"/><th align="center" rowspan="1" colspan="1"/></tr><tr><th align="left" rowspan="1" colspan="1"/><th colspan="14" rowspan="1">
<hr/>
</th></tr><tr><th align="left" rowspan="1" colspan="1">Sex</th><th align="center" colspan="2" rowspan="1">Neonate</th><th align="center" colspan="2" rowspan="1">Infant</th><th align="center" colspan="2" rowspan="1">1–4 years</th><th align="center" colspan="2" rowspan="1">5–14 years</th><th align="center" colspan="2" rowspan="1">15–49 years</th><th align="center" colspan="2" rowspan="1">50–64 years</th><th align="center" colspan="2" rowspan="1">65+ years</th><th align="center" colspan="2" rowspan="1">Total</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">Deaths</td><td align="center" rowspan="1" colspan="1">VA</td><td align="center" rowspan="1" colspan="1">Deaths</td><td align="center" rowspan="1" colspan="1">VA</td><td align="center" rowspan="1" colspan="1">Deaths</td><td align="center" rowspan="1" colspan="1">VA</td><td align="center" rowspan="1" colspan="1">Deaths</td><td align="center" rowspan="1" colspan="1">VA</td><td align="center" rowspan="1" colspan="1">Deaths</td><td align="center" rowspan="1" colspan="1">VA</td><td align="center" rowspan="1" colspan="1">Deaths</td><td align="center" rowspan="1" colspan="1">VA</td><td align="center" rowspan="1" colspan="1">Deaths</td><td align="center" rowspan="1" colspan="1">VA</td><td align="center" rowspan="1" colspan="1">Deaths</td><td align="center" rowspan="1" colspan="1">VA</td></tr><tr><td align="left" rowspan="1" colspan="1">Females</td><td align="center" rowspan="1" colspan="1">209</td><td align="center" rowspan="1" colspan="1">70</td><td align="center" rowspan="1" colspan="1">162</td><td align="center" rowspan="1" colspan="1">89</td><td align="center" rowspan="1" colspan="1">166</td><td align="center" rowspan="1" colspan="1">79</td><td align="center" rowspan="1" colspan="1">103</td><td align="center" rowspan="1" colspan="1">88</td><td align="center" rowspan="1" colspan="1">687</td><td align="center" rowspan="1" colspan="1">82</td><td align="center" rowspan="1" colspan="1">408</td><td align="center" rowspan="1" colspan="1">85</td><td align="center" rowspan="1" colspan="1">818</td><td align="center" rowspan="1" colspan="1">89</td><td align="center" rowspan="1" colspan="1">2,553</td><td align="center" rowspan="1" colspan="1">84</td></tr><tr><td align="left" rowspan="1" colspan="1">Males</td><td align="center" rowspan="1" colspan="1">260</td><td align="center" rowspan="1" colspan="1">76</td><td align="center" rowspan="1" colspan="1">179</td><td align="center" rowspan="1" colspan="1">79</td><td align="center" rowspan="1" colspan="1">205</td><td align="center" rowspan="1" colspan="1">83</td><td align="center" rowspan="1" colspan="1">139</td><td align="center" rowspan="1" colspan="1">91</td><td align="center" rowspan="1" colspan="1">569</td><td align="center" rowspan="1" colspan="1">88</td><td align="center" rowspan="1" colspan="1">435</td><td align="center" rowspan="1" colspan="1">88</td><td align="center" rowspan="1" colspan="1">847</td><td align="center" rowspan="1" colspan="1">92</td><td align="center" rowspan="1" colspan="1">2,634</td><td align="center" rowspan="1" colspan="1">87</td></tr><tr><td align="left" rowspan="1" colspan="1">Total</td><td align="center" rowspan="1" colspan="1">469</td><td align="center" rowspan="1" colspan="1">73</td><td align="center" rowspan="1" colspan="1">341</td><td align="center" rowspan="1" colspan="1">84</td><td align="center" rowspan="1" colspan="1">371</td><td align="center" rowspan="1" colspan="1">81</td><td align="center" rowspan="1" colspan="1">242</td><td align="center" rowspan="1" colspan="1">90</td><td align="center" rowspan="1" colspan="1">1256</td><td align="center" rowspan="1" colspan="1">85</td><td align="center" rowspan="1" colspan="1">843</td><td align="center" rowspan="1" colspan="1">86</td><td align="center" rowspan="1" colspan="1">1665</td><td align="center" rowspan="1" colspan="1">91</td><td align="center" rowspan="1" colspan="1">5,187</td><td align="center" rowspan="1" colspan="1">86</td></tr></tbody></table><table-wrap-foot><fn><p>Deaths: total deaths within the study area; VA: percentage of deaths for which a verbal autopsy was conducted.</p></fn></table-wrap-foot></table-wrap><sec id="S0002-S20001"><title>Cause-specific mortality fractions</title><p>The top five CODs for the whole population overall were: HIV-/AIDS-related causes, acute respiratory infections including pneumonia, malaria, and pulmonary tuberculosis. Pneumonia was the most common COD among the infants while malaria and HIV were the commonest CODs among children 1–4 years and adults aged 15–49 years, respectively. Pulmonary tuberculosis was the leading COD among the elderly age groups.</p><sec><title>Neonatal deaths (<italic>n</italic>=344)</title><p>The majority (78%) of neonatal deaths occurred in the first 7 days of life: 31% occurred within the first 24 hours, and 47% between 2 and 6 days of age. The leading CODs in neonates were pneumonia (26%), birth asphyxia (24%), prematurity (6%), and sepsis (6%) (<xref ref-type="table" rid="T0002">Table 2</xref>). Five percent of deaths were indeterminate. Death due to prematurity was more common among males than females (<xref ref-type="fig" rid="F0001">Fig. 1</xref>).</p><fig id="F0001" position="float"><label>Fig. 1</label><caption><p>Cause-specific mortality fractions by age group and sex.</p><p>The figure shows the top five cause-specific mortality fractions among each age group by sex as derived by the InterVA-4 model.</p></caption><graphic xlink:href="GHA-7-25593-g001"/></fig><table-wrap id="T0002" position="float"><label>Table 2</label><caption><p>Cause-specific mortality fractions by age group as assigned by the InterVA-4 model</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1">Cause of death<xref ref-type="table-fn" rid="TF0001">a</xref>
</th><th align="center" rowspan="1" colspan="1">Neonates</th><th align="center" rowspan="1" colspan="1">Infants</th><th align="center" rowspan="1" colspan="1">1–4 years</th><th align="center" rowspan="1" colspan="1">5–14 years</th><th align="center" rowspan="1" colspan="1">15–49 years</th><th align="center" rowspan="1" colspan="1">50–64 years</th><th align="center" rowspan="1" colspan="1">65+ years</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">01.01 Sepsis (non-obstetric)</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td></tr><tr><td align="left" rowspan="1" colspan="1">01.02 Acute resp infect incl. pneumonia</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">28</td><td align="center" rowspan="1" colspan="1">12</td><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">5</td></tr><tr><td align="left" rowspan="1" colspan="1">01.03 HIV-/AIDS-related death</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">21</td><td align="center" rowspan="1" colspan="1">18</td><td align="center" rowspan="1" colspan="1">23</td><td align="center" rowspan="1" colspan="1">21</td><td align="center" rowspan="1" colspan="1">12</td><td align="center" rowspan="1" colspan="1">3</td></tr><tr><td align="left" rowspan="1" colspan="1">01.04 Diarrheal diseases</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1</td></tr><tr><td align="left" rowspan="1" colspan="1">01.05 Malaria</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">12</td><td align="center" rowspan="1" colspan="1">23</td><td align="center" rowspan="1" colspan="1">13</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">1</td></tr><tr><td align="left" rowspan="1" colspan="1">01.06 Measles</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">01.07 Meningitis and encephalitis</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td></tr><tr><td align="left" rowspan="1" colspan="1">01.08 & 10.05 Tetanus</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">01.09 Pulmonary tuberculosis</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1">12</td><td align="center" rowspan="1" colspan="1">10</td></tr><tr><td align="left" rowspan="1" colspan="1">01.11 Hemorrhagic fever</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">01.99 Other and unspecified infect dis</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td></tr><tr><td align="left" rowspan="1" colspan="1">02.01 Oral neoplasms</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0</td></tr><tr><td align="left" rowspan="1" colspan="1">02.02 Digestive neoplasms</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1">3</td></tr><tr><td align="left" rowspan="1" colspan="1">02.03 Respiratory neoplasms</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1">6</td></tr><tr><td align="left" rowspan="1" colspan="1">02.04 Breast neoplasms</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">02.05 & 02.06 Reproductive neoplasms MF</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1</td></tr><tr><td align="left" rowspan="1" colspan="1">02.99 Other and unspecified neoplasms</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">4</td></tr><tr><td align="left" rowspan="1" colspan="1">03.01 Severe anemia</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0</td></tr><tr><td align="left" rowspan="1" colspan="1">03.02 Severe malnutrition</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">2</td></tr><tr><td align="left" rowspan="1" colspan="1">03.03 Diabetes mellitus</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">2</td></tr><tr><td align="left" rowspan="1" colspan="1">04.01 Acute cardiac disease</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1</td></tr><tr><td align="left" rowspan="1" colspan="1">04.02 Stroke</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">14</td></tr><tr><td align="left" rowspan="1" colspan="1">04.03 Sickle cell with crisis</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">04.99 Other and unspecified cardiac diseases</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1">9</td></tr><tr><td align="left" rowspan="1" colspan="1">05.01 Chronic obstructive pulmonary disease</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">3</td></tr><tr><td align="left" rowspan="1" colspan="1">05.02 Asthma</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1</td></tr><tr><td align="left" rowspan="1" colspan="1">06.01 Acute abdomen</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">2</td></tr><tr><td align="left" rowspan="1" colspan="1">06.02 Liver cirrhosis</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td></tr><tr><td align="left" rowspan="1" colspan="1">07.01 Renal failure</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1</td></tr><tr><td align="left" rowspan="1" colspan="1">08.01 Epilepsy</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td></tr><tr><td align="left" rowspan="1" colspan="1">09.01 Ectopic pregnancy</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">09.02 Abortion-related death</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">09.03 Pregnancy-induced hypertension</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">09.04 Obstetric hemorrhage</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">09.06 Pregnancy-related sepsis</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">09.07 Anemia of pregnancy</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">09.99 Other and unspecified maternal CoD</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">10.01 Prematurity</td><td align="center" rowspan="1" colspan="1">6</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">10.02 Birth asphyxia</td><td align="center" rowspan="1" colspan="1">24</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">10.03 Neonatal pneumonia</td><td align="center" rowspan="1" colspan="1">26</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">10.04 Neonatal sepsis</td><td align="center" rowspan="1" colspan="1">6</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">10.06 Congenital malformation</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">10.99 Other and unspecified neonatal CoD</td><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">12.01 Road traffic accident</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">7</td><td align="center" rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1</td></tr><tr><td align="left" rowspan="1" colspan="1">12.03 Accidental fall</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">1</td></tr><tr><td align="left" rowspan="1" colspan="1">12.04 Accidental drowning and submersion</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0</td></tr><tr><td align="left" rowspan="1" colspan="1">12.05 Accidental exposure to smoke fire & flame</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td></tr><tr><td align="left" rowspan="1" colspan="1">12.06 Contact with venomous plant/animal</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">12.07 Accidental poisoning & noxious substances</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">12.08 Intentional self-harm</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0</td></tr><tr><td align="left" rowspan="1" colspan="1">12.09 Assault</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">3</td></tr><tr><td align="left" rowspan="1" colspan="1">12.10 Exposure to force of nature</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">12.99 Other and unspecified external CoD</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td></tr><tr><td align="left" rowspan="1" colspan="1">98 Other and unspecified NCD</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">2</td></tr><tr><td align="left" rowspan="1" colspan="1">99 Indeterminate</td><td align="center" rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1">12</td><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1">7</td><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1">10</td></tr><tr><td align="left" rowspan="1" colspan="1">VA not completed</td><td align="center" rowspan="1" colspan="1">25</td><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1">23</td><td align="center" rowspan="1" colspan="1">13</td><td align="center" rowspan="1" colspan="1">17</td><td align="center" rowspan="1" colspan="1">14</td><td align="center" rowspan="1" colspan="1">10</td></tr><tr><td align="left" rowspan="1" colspan="1">Total</td><td align="center" rowspan="1" colspan="1">100</td><td align="center" rowspan="1" colspan="1">100</td><td align="center" rowspan="1" colspan="1">100</td><td align="center" rowspan="1" colspan="1">100</td><td align="center" rowspan="1" colspan="1">100</td><td align="center" rowspan="1" colspan="1">100</td><td align="center" rowspan="1" colspan="1">100</td></tr></tbody></table><table-wrap-foot><fn id="TF0001"><label>a</label><p>WHO 2012 VA standard categories (<xref rid="CIT0026" ref-type="bibr">26</xref>); CSMFs are rounded to nearest 1%, 0 representing a finite value <0.5%, and the blank cells are either impossibilities or cause/age combinations with no case assigned.</p></fn></table-wrap-foot></table-wrap></sec><sec><title>Infant deaths (<italic>n</italic>=286)</title><p>The top CODs in infants (excluding neonates) were pneumonia, HIV/AIDS, and malaria which accounted for 28, 21 and 12% of deaths, respectively. Five percent of deaths were attributed to sickle cell crisis while other COD categories totalled 14%. Twelve percent of deaths
were indeterminate (<xref ref-type="table" rid="T0002">Table 2</xref>). No sex-specific differences were noted with the exception of pneumonia, which was more common in female than male infants (<xref ref-type="fig" rid="F0001">Fig. 1</xref>).</p></sec><sec><title>Children 1–4 years old (<italic>n</italic>=302)</title><p>Malaria, HIV/AIDS, and pneumonia were the predominant CODs in this age group, causing 23, 18, and 12% of the childhood deaths, respectively, while diarrheal diseases, severe malnutrition, sickle cell with crisis, and road traffic accidents explained 3, 2, 2, and 1% of deaths, respectively. All other CODs accounted for 7% while a further 9% of deaths were indeterminate (<xref ref-type="table" rid="T0002">Table 2</xref>). A slightly higher proportion of girls than boys died of pneumonia while no differences were noted between the sexes for death due to malaria (<xref ref-type="fig" rid="F0001">Fig. 1</xref>).</p></sec><sec><title>5–14 years old (<italic>n</italic>=218)</title><p>Relatively fewer deaths were observed in this age group. The commonest CODs were HIV/AIDS related and malaria, which accounted for 23 and 13% of total deaths, respectively. Deaths due to pneumonia, road traffic accidents, meningitis/encephalitis, and sickle cell with crisis explained 8, 7, 5, and 4% of deaths, respectively. Eight percent of cases were indeterminate (<xref ref-type="table" rid="T0002">Table 2</xref>). Unlike the previous age group, in this age group a higher proportion of boys appeared to die of pneumonia and external CODs than girls (<xref ref-type="fig" rid="F0001">Fig. 1</xref>).</p></sec><sec><title>15–49 years old (<italic>n</italic>=1,068)</title><p>A third of deaths in this age group were attributed to HIV/AIDS and pulmonary tuberculosis. The majority of those who died of HIV/AIDS were either divorced or separated (60%). Road traffic accidents, assault, and stroke accounted for 5, 3, and 3%, respectively. Overall, 7% of deaths were indeterminate (<xref ref-type="table" rid="T0002">Table 2</xref>). A higher proportion of women (29.1%) than men (12.2%) in this age group died of HIV/AIDS while more men (23.8%) than women (2%) died of external CODs (<xref ref-type="fig" rid="F0001">Fig. 1</xref>). A total of 62/565 deaths among women of reproductive age were due to maternal causes of which the two most common were obstetric hemorrhage (51.6%) and pregnancy-induced hypertension (25.8%).</p></sec><sec><title>50–64 years old (<italic>n</italic>=729)</title><p>All neoplasms, pulmonary tuberculosis, HIV-/AIDS-related deaths and stroke were the predominant CODs among people of this age group, accounting for 16, 12, 12, and 10% of deaths, respectively. Other unspecified cardiac diseases explained 5% of deaths. Eight percent of the deaths were indeterminate (<xref ref-type="table" rid="T0002">Table 2</xref>). More males (14.7%) than females (8.6%) died of pulmonary tuberculosis whereas more females (18.1%) than males (3%) died of HIV/AIDS. Women were more likely to die of stroke than men in this age group (<xref ref-type="fig" rid="F0001">Fig. 1</xref>).</p></sec><sec><title>Above 65 years old (<italic>n</italic>=1,513)</title><p>All neoplasms, stroke, and pulmonary tuberculosis predominated, accounting respectively for 15, 14 and 10% of total deaths. Cardiac diseases accounted for 10% of deaths and 10% were indeterminate (<xref ref-type="table" rid="T0002">Table 2</xref>). A higher proportion of women (16.8%) than men (11.3%) died of stroke whereas in this age group men were more likely than women to die of pulmonary tuberculosis (<xref ref-type="fig" rid="F0001">Fig. 1</xref>).</p><p>The pattern of mortality as determined by the InterVA-4 model shows a high burden of infectious diseases, including HIV/AIDS, pneumonia, and pulmonary tuberculosis in the study population. These mortality patterns are consistent with existing knowledge on the burden of disease in many parts of sub-Saharan Africa.</p></sec></sec></sec><sec sec-type="discussion" id="S0003"><title>Discussion</title><p>The majority of previous studies that have reported CODs among rural populations in the developing world have used the physician certified verbal autopsy (PCVA) approach to COD assignment
(<xref rid="CIT0027" ref-type="bibr">27</xref>–<xref rid="CIT0033" ref-type="bibr">33</xref>). A VA programme based on PCVA requires the involvement of a minimum of three clinicians with skills in the method. As a result, the approach is expensive, a distraction to skilled personnel who represent a scarce resource in less developed countries, and is a frequent bottleneck in the provision of timely data (<xref rid="CIT0003" ref-type="bibr">3</xref>). Furthermore, the PCVA approach is vulnerable to bias from physician coders, who are prone to preconceptions about the common patterns of death in any given community that are not necessarily evidence based (<xref rid="CIT0034" ref-type="bibr">34</xref>). Such considerations have justified the more recent development of automated approaches to VA coding (<xref rid="CIT0025" ref-type="bibr">25</xref>, <xref rid="CIT0035" ref-type="bibr">35</xref>). One such method is the freely available, WHO-compliant, InterVA-4 model, a method that has recently been adopted by the INDEPTH Network of demographic surveillance sites as the only viable strategy currently available for the timely provision of comparable data from multiple populations (<xref rid="CIT0036" ref-type="bibr">36</xref>). In the current study, we have used this method as the basis for assigning CODs in 4,460 subjects of all ages who were residents of the KHDSS area on the coast of Kenya, where we used the 2012 version of the InterVA-4 model to interpret VA data collected using the 2007 version of the WHO VA questionnaires.</p><p>In general, our COD estimates appear credible and conform to expected patterns. As anticipated, we found a slight excess of deaths among males (2,304; 52%) in comparison to females (2,156; 48%). This observation was consistent across all age groups individually with the exception of the 15–49 years age group where, predictably, we found an excess of deaths among women, predominantly from pregnancy-related causes and HIV/AIDS. Of particular interest, external CODs such as road traffic accidents were more commonly observed among men than women. Since Kenya has recently experienced a surge in the use of motorcycles as a mean of transport, and a majority of riders lack proper training, this is, perhaps, not surprising, but is nevertheless a matter of considerable concern. Similarly, palm wine tapping, a common socioeconomic activity for men within the KHDSS, can sometimes result in fatal accidental falls, especially when men climb the trees having sampled the product. In the elderly, 65+ age group, malignancies and stroke were key concerns, the latter being consistent with data from our previous study in which we reported hypertension and diabetes as major risk factors (<xref rid="CIT0012" ref-type="bibr">12</xref>).</p><p>While in general the process of VA coding was straight forward, we did face some challenges with regard to the input and interpretation of our data. First, in comparison to that reported from previous studies (<xref rid="CIT0004" ref-type="bibr">4</xref>, <xref rid="CIT0037" ref-type="bibr">37</xref>), a higher proportion of cases in our analysis were reported as ‘indeterminate’. The majority of these cases were associated with scanty signs and symptoms obtained from the respondents, a phenomenon that was particularly common where deaths were sudden or where subjects were found dead. Further research will be needed to establish more reliable methods for COD assignment in such cases. Second, HIV varies considerably from place to place and according to the model, it is necessary to specify whether HIV in the area is high, low or very low, corresponding to ratios of 1:100, 1:1,000 and 1:10,000 of all deaths, respectively (<xref rid="CIT0025" ref-type="bibr">25</xref>). Since the prevalence of HIV infection in well-nourished and severely malnourished children within the KHDSS has been reported at 2 and 14%, respectively (<xref rid="CIT0038" ref-type="bibr">38</xref>), we set the HIV variable within the model to ‘high’, as described in the InterVA-4 user guide (<xref rid="CIT0025" ref-type="bibr">25</xref>). We suspect, however, that HIV may be overestimated as a cause of death among children within the current analysis, reflecting how difficult it can be to discriminate between HIV and malnutrition on the basis of clinical features alone. On the contrary, the high frequency of HIV/AIDS among adult women of reproductive age seems more plausible and is consistent with previous reports (<xref rid="CIT0012" ref-type="bibr">12</xref>, <xref rid="CIT0037" ref-type="bibr">37</xref>). Third, because the InterVA-4 model is based on a more recent version of the WHO VA tools, we needed to map our input indicators from the format in which they were collected (on the 2007 version of the WHO VA questionnaire) onto the relevant indicators within the InterVA-4 model, a process that proved to be straight forward with regard to the majority of indicators. Finally, although in general the pattern of deaths reported in our study seems generally plausible, and is supported by our previous validation study conducted in adults within the same area (<xref rid="CIT0012" ref-type="bibr">12</xref>), in the current study, COD interpretation has not been validated by any other method. In the future, we plan to undertake further work with this aim in mind.</p><p>A major advantage of computer-based coding methods such as InterVA-4 is their potential for providing standardized data across multiple sites and over long periods of time. For this to be viable, it will be important to agree on standardized methods for data input. For example, in our own data set some questions were missing from the WHO 2007 VA instrument but were present in the WHO 2012 instrument. However, these data were partially captured in the free-text sections of the VA form, for which we developed an automated search for the ‘keyword’ and mapped it onto the corresponding indicator. This was particularly important in capturing specific diagnoses such as SCD and specific forms of injury. While reviewing and modifying the questionnaires from time to time has certain advantages, it also leads to problems in the analysis and interpretation of longitudinal data. If the method is to be used successfully for the purpose of comparisons between multiple sites, collaborative discussion will be needed to agree on protocols for data input and interpretation.</p></sec><sec sec-type="conclusions" id="S0004"><title>Conclusions</title><p>Our study suggests that both the WHO 2012 instrument and the InterVA-4 model are feasible tools to measure cause-specific mortality, which may potentially inform both health policy and program interventions in resource-limited settings. The model requires minimal time and labor resources, especially in comparison with the PCVA method. The current analysis, using InterVA-4, returned COD patterns that were generally credible at the population level and in subjects of all ages. While further work will be required to fine-tune and validate COD analysis using InterVA-4, particularly in children, we hope that our results will prove useful for informing health intervention policies both locally and internationally.</p></sec> |
Distribution of cause of death in rural Bangladesh during 2003–2010: evidence from two rural areas within Matlab Health and Demographic Surveillance site | <sec id="st1"><title>Objective</title><p>This study used the InterVA-4 computerised model to assign probable cause of death (CoD) to verbal autopsies (VAs) generated from two rural areas, with a difference in health service provision, within the Matlab Health and Demographic Surveillance site (HDSS). This study aimed to compare CoD by gender, as well as discussing possible factors which could influence differences in the distribution of CoD between the two areas.</p></sec><sec id="st2"><title>Design</title><p>Data for this study came from the Matlab the HDSS maintained by the International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b) since 1966. In late 1977, icddr,b divided HDSS and implemented a high-quality maternal, newborn and child health and family planning (MNCH-FP) services project in one half, called the icddr,b service area (SA), in addition to the usual public and private MNCH-FP services that serve the other half, called the government SA. HDSS field workers registered 12,144 deaths during 2003–2010, and trained interviewers obtained VA for 98.9% of them. The probabilistic model InterVA-4 probabilistic model (version 4.02) was used to derive probable CoD from VA symptoms. Cause-specific mortality rates and fractions were compared across gender and areas. Appropriate statistical tests were applied for significance testing.</p></sec><sec id="st3"><title>Results</title><p>Mortality rates due to neonatal causes and communicable diseases (CDs) were lower in the icddr,b SA than in the government SA, where mortality rates due to non-communicable diseases (NCDs) were lower. Cause-specific mortality fractions (CSMFs) due to CDs (23.2% versus 18.8%) and neonatal causes (7.4% versus 6%) were higher in the government SA, whereas CSMFs due to NCDs were higher (58.2% versus 50.7%) in the icddr,b SA. The rank-order of CSMFs by age group showed marked variations, the largest category being acute respiratory infection/pneumonia in infancy, injury in 1–4 and 5–14 years, neoplasms in 15–49 and 50–64 years, and stroke in 65+ years.</p></sec><sec id="st4"><title>Conclusions</title><p>Automated InterVA-4 coding of VA to determine probable CoD revealed the difference in the structure of CoD between areas with prominence of NCDs in both areas. Such information can help local planning of health services for prevention and management of disease burden.</p></sec> | <contrib contrib-type="author"><name><surname>Alam</surname><given-names>Nurul</given-names></name><xref ref-type="aff" rid="AF0001">1</xref><xref ref-type="corresp" rid="cor1">*</xref></contrib><contrib contrib-type="author"><name><surname>Chowdhury</surname><given-names>Hafizur R.</given-names></name><xref ref-type="aff" rid="AF0002">2</xref></contrib><contrib contrib-type="author"><name><surname>Ahmed</surname><given-names>Ali</given-names></name><xref ref-type="aff" rid="AF0001">1</xref></contrib><contrib contrib-type="author"><name><surname>Rahman</surname><given-names>Mahfuzur</given-names></name><xref ref-type="aff" rid="AF0001">1</xref></contrib><contrib contrib-type="author"><name><surname>Streatfield</surname><given-names>P. Kim</given-names></name><xref ref-type="aff" rid="AF0001">1</xref></contrib> | Global Health Action | <p>Bangladesh is the most densely populated and the eighth most populous country in the world (<xref rid="CIT0001" ref-type="bibr">1</xref>). Despite its widespread poverty, turbulent past, and occasionally extreme climate events, the country has made remarkable progress in the last four decades in providing vaccines to children and mothers; reducing vitamin A deficiency; lowering fertility, as well as mortality of infants, children and mothers; increasing life expectancy; and reducing gender and economic inequalities in health outcomes (<xref rid="CIT0002" ref-type="bibr">2</xref>, <xref rid="CIT0003" ref-type="bibr">3</xref>). Since independence in 1971 the government has been pursuing a policy of health development that ensures provision of basic services to the entire population, particularly to the underserved population in rural areas. Within the overall development policy framework community-based approaches in health service delivery; partnership of the public sector with non-government organizations (NGOs) to reach deprived populations and address service gaps; and rapid adoption of context specific innovative technologies and policies have been instrumental in improving the health service coverage and health outcomes (<xref rid="CIT0004" ref-type="bibr">4</xref>). All of these are accelerating the demographic and epidemiologic transitions, and the country now faces new population and health challenges as the burden of infectious diseases has shifted towards non-communicable diseases (NCDs) in the last decades (<xref rid="CIT0005" ref-type="bibr">5</xref>, <xref rid="CIT0006" ref-type="bibr">6</xref>).</p><p>The demographic and epidemiologic transitions are likely to lower not only the overall risk of dying, but also the risk of dying from certain diseases more than others. The distribution of the risk factors and lifestyle can, however, change the structure of cause of death (CoD), but there is little empirical evidence due to a lack of reliable data. Introduction of a community-based integrated maternal, newborn and child health, and family planning (MNCH-FP) services project by icddr,b in late 1977 in half of the Matlab Health and Demographic Surveillance site (HDSS) that has been functioning since 1966, resulted in differences in the fertility and mortality (infant, child, and maternal) rates between the icddr,b service area (SA) and the government SA during 1978–2009 (<xref rid="CIT0007" ref-type="bibr">7</xref>–<xref rid="CIT0009" ref-type="bibr">9</xref>). The majority of the differences in the health outcomes between the two areas of comparable socioeconomic and demographic conditions were due to the differences in the quality of MNCH-FP services (<xref rid="CIT0010" ref-type="bibr">10</xref>, <xref rid="CIT0011" ref-type="bibr">11</xref>). The differences in the fertility and mortality rates in these two areas for a longer period may have changed the structure of CoD, but this has not been examined systematically. Introduction of the WHO verbal autopsy (VA) into the HDSS in 2003 and development of the computerized automated algorithms for processing VA symptoms to reliably determine probable CoDs, provide a unique opportunity to compare CoD between the two rural areas with a different MNCH-FP services delivery provision within the Matlab HDSS during 2003–2010. This study aimed to exploit this opportunity using the HDSS mortality and VA data and compare CoD across gender in these two rural areas. The study results may help planning of health services for areas with reference to the local health service delivery system in order to prioritise disease burden considering local sociodemographic conditions.</p><sec sec-type="methods" id="S0002"><title>Methods</title><sec id="S0002-S20001"><title>The study site and local health service delivery system</title><p>The study site is Matlab, a sub-district of Chandpur district, where icddr,b maintains a HDSS since 1966. Matlab is 55 km southwest of the capital city Dhaka, Bangladesh. The government of Bangladesh provides primary health care services for a nominal fee through a three-tiered health service delivery system at the sub-district level: community clinics, each serving about 6,000 people, the health and family welfare centres, each serving 25,000 people, and the Upazila (sub-district) health complexes, each serving 250,000 people. The Upazila Health Complex situated at Upazila headquarters coordinates services between different tiers.</p><p>In late 1977, the icddr,b divided the HDSS into an icddr,b SA and a government SA (<xref ref-type="fig" rid="F0001">Fig. 1</xref>) and implemented a community-based MNCH-FP services project in the icddr,b SA in addition to usual public and private MNCH-FP services that serve the government SA. The icddr,b has set up at Matlab Upazila headquarters a primary care hospital (70 beds for managing patients with diarrhoeal diseases coming from anywhere and 60 beds for managing patients with maternal, newborn, and child health problems coming from the icddr,b SA only) and four community-based treatment centres in support of the hospital, which provide health services to women of reproductive age and children under 5 years old. These primary care hospital and four community-based treatment centres are located within the icddr,b SA. Over the years new service components complimentary to the MNCH-FP services were added in phases. The 50-bed public Upazila Health Complex with provision for outpatient and inpatient care facilities, and an operation theatre for minor surgery and caesarean section is also located at Matlab headquarters and falls within the icddr,b SA. Also more private fee-for-service clinics and unqualified health care providers (i.e. village doctors, drug sellers, homeopaths, and herbalists) are available in the icddr,b SA. The icddr,b primary care hospital and the four treatment centres are better equipped compared to public health facilities in terms of staff attendance, supervision, supply of medicines and logistics, quality of care, and effective referral service (<xref rid="CIT0010" ref-type="bibr">10</xref>, <xref rid="CIT0011" ref-type="bibr">11</xref>). The icddr,b SA has greatly enhanced quality of health care services compared to the Government SA.</p></sec><sec id="S0002-S20002"><title>Identification of deaths and training of VA interviewers for VA collection</title><p>Female community health research workers (CHRWs) having at least 10th grade education, visited households monthly up to 2006 and bi-monthly since then to record health and demographic events: births, deaths, migrations, marriages, and marital disruptions. Households with deaths registered by CHRWs during routine household visits have been visited by Field Research Supervisors (FRS) with WHO standard VA questionnaires since 2003. VA refers to interviewing close family members of the deceased about the events surrounding the fatal illness episodes or conditions. It contains both open narratives related to death and close-ended leading questions to elicit symptoms and signs of illness or conditions leading to death. For neonatal deaths, it contains a description of the mother's pregnancy and delivery care. The VA questionnaires in English were customized to suit local conditions, by reducing number of questions on HIV/AIDs and malaria, as prevalence of these diseases in the HDSS is very low, and adding few additional questions on arsenic related disease symptoms because ground water in the HDSS is arsenic contaminated. The customized VA questionnaires were translated into Bangla for training in the field data collection.</p><p>A public health physician and a medical demographer provided 4 days training to FRSs of non-medical background and a field research officer (FRO with 3 years training in medicine) on VA questionnaires, followed by 2 days of field practice. FRSs interviewed, with the VA questionnaires, the closest caretakers/relatives who had lived with the deceased in the same household around the time of terminal illness or death, within 6–12 weeks after the date of death. The FRO regularly supervised the fieldwork and the public health physician provided technical support in terms of clarification of the questions or disease symptoms when required. Informed standard consent forms were used to inform the respondents about the purpose of the study and guarantee confidentiality of information provided. Willingness to take part was expressed by signature or thumb impression. The Matlab HDSS data collection is approved by the Ethical Review Committee of icddr,b.</p></sec><sec id="S0002-S20003"><title>Quality control</title><p>Scheduled revisits to 5% of randomly selected VA questionnaires were part of the quality control measures. The FRO visited the FRSs during data collection and reviewed the surveillance data collected at the household level, including VA filled-in questionnaires. Immediate feedback was provided. The FRO then completed mandatory checks and edited all filled-in VA questionnaires before sending them for coding and computer entry.</p></sec><sec id="S0002-S20004"><title>Assessment of CoD from VA</title><p>The computer automated models are increasingly used for analysing VA symptom data to derive probable CoD, for being fast, low-cost and reliable compared to the physician-coded CoD (<xref rid="CIT0012" ref-type="bibr">12</xref>–<xref rid="CIT0014" ref-type="bibr">14</xref>). These models processed a range of items of VA information about the background characteristics and circumstances of a death, details of any illness (signs and symptoms) or conditions leading to death, previous medical history, etc. in a mathematical model based on Bayes’ theorem, and produced likely cause(s) of death (<xref rid="CIT0015" ref-type="bibr">15</xref>, <xref rid="CIT0016" ref-type="bibr">16</xref>). The signs and symptoms or conditions, alone or in combinations, are highly indicative of specific diseases and infer the ‘most likely’ biomedical cause(s). VA data on symptoms and signs of illness or conditions that led to death collected under earlier standards of VA were converted to the WHO 2012 standard (<xref rid="CIT0017" ref-type="bibr">17</xref>). The probabilistic model InterVA-4 (version 4.02) with options of low HIV/AIDS and malaria in the HDSS was applied to the individual VA information to assign probable CoD categories as defined by the WHO 2012 VA standards. The InterVA-4 yields, for each case, up to three possible CoD or an indeterminate result. It thus gives, if not indeterminate, the most likely CoD with an estimated probability for this cause. If the sum of the estimated probabilities for the first, second, and third most likely causes of death was less than 100%, the residual component was then assigned as being indeterminate.</p></sec><sec id="S0002-S20005"><title>VA data analysis</title><p>Matlab HDSS provided VA data generated during 2003–2010 to the INDEPTH multisite dataset (<xref rid="CIT0018" ref-type="bibr">18</xref>). Of 12,144 deaths that occurred in the HDSS area, VA symptom data could not be obtained for 129 deaths: 31 cases for non-response and 98 cases for no symptoms or signs of illness prior to death. The InterVA-4 coded CoD were then broadly grouped into communicable diseases (CDs), NCDs, perinatal and neonatal causes, pregnancy-related deaths, injury and other external causes, or indeterminate. Estimated broad cause-specific mortality rates for children (<15 years), adults (aged 15–64 years) and elderly were compared between the two areas. Also estimated cause-specific mortality fractions (CSMFs) per 100 deaths by sex within the areas were used to examine differentials in burden of disease. The difference between the two death rates was compared using Chi-square test and the difference between the two fractions was compared by Z-test for statistical significance at 80% power and p<0.05.</p></sec></sec><sec sec-type="results" id="S0003"><title>Results</title><sec id="S0003-S20001"><title>Demographic, social, and health profiles in two service areas</title><p>Age distributions (in%), mean ages, and sex ratios of the populations in the two adjacent areas in 2010 were comparable (<xref ref-type="table" rid="T0001">Table 1</xref>). Social indicators, such as level of education of youth aged 15–24 years, are comparable between the two areas. Although total fertility rates since 2005 were comparable between the two areas, the infant and child mortality rates were lower in the icddr,b SA. Coverage of immunizations of children and mothers with tetanus toxoid were very high in both areas, but the antenatal care coverage, facility-based delivery, caesarean sections, pneumonia treatment from a well-trained provider, and management of diarrhoea with oral rehydration solution and with zinc tablets were very low in the government SA. Both the areas were similar in demographic and social indicators, but different in health indicators.</p><fig id="F0001" position="float"><label>Fig. 1</label><caption><p>Map of Matlab demographic surveillance area showing icddr,b (yellow) and the government (green) service areas.</p></caption><graphic xlink:href="GHA-7-25510-g001"/></fig><table-wrap id="T0001" position="float"><label>Table 1</label><caption><p>Demographic, social and health indicators in the icddr,b and government service areas, Matlab HDSS 2010</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1">Demographic, social and health indicators</th><th align="center" rowspan="1" colspan="1">icddr,b service area</th><th align="center" rowspan="1" colspan="1">Government service area</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">Mean age±standard deviation</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1"> Male</td><td align="center" rowspan="1" colspan="1">28.2±21.4</td><td align="center" rowspan="1" colspan="1">27.7±21.3</td></tr><tr><td align="left" rowspan="1" colspan="1"> Female</td><td align="center" rowspan="1" colspan="1">28.8±20.3</td><td align="center" rowspan="1" colspan="1">29.0±20.6</td></tr><tr><td align="left" rowspan="1" colspan="1">Sex ratio of males per 100 females</td><td align="center" rowspan="1" colspan="1">87.2</td><td align="center" rowspan="1" colspan="1">86.5</td></tr><tr><td align="left" rowspan="1" colspan="1">Age distribution (in %) of the population:</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1"> Children aged <15 years (%)</td><td align="center" rowspan="1" colspan="1">32.8</td><td align="center" rowspan="1" colspan="1">33.4</td></tr><tr><td align="left" rowspan="1" colspan="1"> Adults aged 15–64 years (%)</td><td align="center" rowspan="1" colspan="1">60.6</td><td align="center" rowspan="1" colspan="1">59.7</td></tr><tr><td align="left" rowspan="1" colspan="1"> Elderly aged 65+years (%)</td><td align="center" rowspan="1" colspan="1">6.5</td><td align="center" rowspan="1" colspan="1">6.8</td></tr><tr><td align="left" rowspan="1" colspan="1">Education of youth aged 15–24 years in 2013:</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1"> None (%)</td><td align="center" rowspan="1" colspan="1">3.2</td><td align="center" rowspan="1" colspan="1">3.6</td></tr><tr><td align="left" rowspan="1" colspan="1"> Primary (up to class V) (%)</td><td align="center" rowspan="1" colspan="1">18.0</td><td align="center" rowspan="1" colspan="1">20.3</td></tr><tr><td align="left" rowspan="1" colspan="1"> Secondary & above (class VI+) (%)</td><td align="center" rowspan="1" colspan="1">78.8</td><td align="center" rowspan="1" colspan="1">76.1</td></tr><tr><td align="left" rowspan="1" colspan="1">Total fertility rate per woman</td><td align="center" rowspan="1" colspan="1">2.6</td><td align="center" rowspan="1" colspan="1">2.5</td></tr><tr><td align="left" rowspan="1" colspan="1">Infant mortality rate per 1000 live births</td><td align="center" rowspan="1" colspan="1">25.1</td><td align="center" rowspan="1" colspan="1">35.4</td></tr><tr><td align="left" rowspan="1" colspan="1">Child (1–4yrs) mortality rate per 1000 children</td><td align="center" rowspan="1" colspan="1">2.1</td><td align="center" rowspan="1" colspan="1">2.5</td></tr><tr><td align="left" rowspan="1" colspan="1">Crude death rate per 1000 populationa<xref ref-type="table-fn" rid="TF0001">a</xref>
</td><td align="center" rowspan="1" colspan="1">6.7</td><td align="center" rowspan="1" colspan="1">6.7</td></tr><tr><td align="left" rowspan="1" colspan="1">Coverage of full immunization:</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1"> Children aged 12–23 months (%)</td><td align="center" rowspan="1" colspan="1">88.6</td><td align="center" rowspan="1" colspan="1">87.4</td></tr><tr><td align="left" rowspan="1" colspan="1"> Mother with tetanus toxoid (%)</td><td align="center" rowspan="1" colspan="1">97.0</td><td align="center" rowspan="1" colspan="1">86.4</td></tr><tr><td align="left" rowspan="1" colspan="1">Antenatal care received from skilled providers:</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1"> 1<sup>st</sup> trimester (%)</td><td align="center" rowspan="1" colspan="1">47.9</td><td align="center" rowspan="1" colspan="1">12.0</td></tr><tr><td align="left" rowspan="1" colspan="1"> 2<sup>nd</sup> trimester (%)</td><td align="center" rowspan="1" colspan="1">96.2</td><td align="center" rowspan="1" colspan="1">60.4</td></tr><tr><td align="left" rowspan="1" colspan="1"> 3<sup>rd</sup> trimester (%)</td><td align="center" rowspan="1" colspan="1">97.5</td><td align="center" rowspan="1" colspan="1">78.7</td></tr><tr><td align="left" rowspan="1" colspan="1">Delivery in a health facility (%)</td><td align="center" rowspan="1" colspan="1">80.7</td><td align="center" rowspan="1" colspan="1">28.1</td></tr><tr><td align="left" rowspan="1" colspan="1"> C- section (%)</td><td align="center" rowspan="1" colspan="1">22.8</td><td align="center" rowspan="1" colspan="1">15.2</td></tr><tr><td align="left" rowspan="1" colspan="1">Seeking treatment from trained health providers:</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1"> for ARI/pneumonia (%)</td><td align="center" rowspan="1" colspan="1">39.8</td><td align="center" rowspan="1" colspan="1">31.8</td></tr><tr><td align="left" rowspan="1" colspan="1">Management of diarrhoeal episode:</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1"> with oral rehydration solution (ORS) (%)</td><td align="center" rowspan="1" colspan="1">36.6</td><td align="center" rowspan="1" colspan="1">24.0</td></tr><tr><td align="left" rowspan="1" colspan="1"> ORS+zinc tablet (%)</td><td align="center" rowspan="1" colspan="1">17.2</td><td align="center" rowspan="1" colspan="1">10.6</td></tr></tbody></table><table-wrap-foot><fn><p>Source: Matlab HDSS annual reports published in 2012 and 2014, icddr,b, Dhaka, Bangladesh.</p></fn><fn id="TF0001"><label>a</label><p>The rate was lower in the icddr,b SA than the government SA for in last 10 years except in 2010.</p></fn></table-wrap-foot></table-wrap></sec><sec id="S0003-S20002"><title>Mortality rates in 2003–2010 by area</title><p>The HDSS recorded 5,965 deaths in the icddr,b SA and 6,179 in the government SA during 2003–2010. Yearly death rates by age and area showed declining trends in infant and child (age 1–14 years) mortality rates in both areas and the declines were faster in the icddr,b SA (<xref ref-type="fig" rid="F0002">Fig. 2</xref>). Mortality rates in the age groups 15–64 years were stable in the icddr,b area, but in 2003–2004 in the government SA were little higher and showed a slow declining trend over the years. In the age group 65 years and above, mortality rates were comparable between the two areas and did not show declining trends.</p><fig id="F0002" position="float"><label>Fig. 2</label><caption><p>Trends in mortality (per 1,000 person-years) in difference age groups by area.</p></caption><graphic xlink:href="GHA-7-25510-g002"/></fig><p>Percentage distribution of deaths and the death rates (per 1,000 person-years) during 2003–2010 by age and area revealed lower neonatal and post-neonatal mortality rates in the icddr,b SA than in the government SA (<xref ref-type="table" rid="T0002">Table 2</xref>). Crude death rate was a little higher in the government SA, mainly due to the higher death rates in the neonatal and post-neonatal periods. Mortality was higher for males than females in the neonatal period and in the age groups 15–49 and 50–64 years in each area.</p><table-wrap id="T0002" position="float"><label>Table 2</label><caption><p>Distribution of deaths, death rates<xref ref-type="table-fn" rid="TF0002">a</xref> and sex ratios<xref ref-type="table-fn" rid="TF0003">b</xref> by age and area, Matlab HDSS 2003–2010</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1"/><th align="center" colspan="3" rowspan="1">icddr,b service area</th><th align="center" colspan="3" rowspan="1">Government service area</th></tr><tr><th align="left" rowspan="1" colspan="1"/><th align="center" colspan="3" rowspan="1">
<hr/>
</th><th align="center" colspan="3" rowspan="1">
<hr/>
</th></tr><tr><th align="left" rowspan="1" colspan="1">Age group</th><th align="center" rowspan="1" colspan="1"># deaths (%)</th><th align="center" rowspan="1" colspan="1">Rate<xref ref-type="table-fn" rid="TF0002">a</xref>
</th><th align="center" rowspan="1" colspan="1">Sex ratio<xref ref-type="table-fn" rid="TF0003">b</xref>
</th><th align="center" rowspan="1" colspan="1">% (# deaths)</th><th align="center" rowspan="1" colspan="1">Rate<xref ref-type="table-fn" rid="TF0002">a</xref>
</th><th align="center" rowspan="1" colspan="1">Sex ratio<xref ref-type="table-fn" rid="TF0003">b</xref>
</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">0–28 days</td><td align="center" rowspan="1" colspan="1">485 (8.1)</td><td align="center" rowspan="1" colspan="1">299.9<xref ref-type="table-fn" rid="TF0005">*</xref>
</td><td align="center" rowspan="1" colspan="1">139.8</td><td align="center" rowspan="1" colspan="1">637 (10.3)</td><td align="center" rowspan="1" colspan="1">418.9</td><td align="center" rowspan="1" colspan="1">117.6</td></tr><tr><td align="left" rowspan="1" colspan="1">29 days to 11 months<xref ref-type="table-fn" rid="TF0004">c</xref>
</td><td align="center" rowspan="1" colspan="1">161 (2.7)</td><td align="center" rowspan="1" colspan="1">8.2<xref ref-type="table-fn" rid="TF0005">*</xref>
</td><td align="center" rowspan="1" colspan="1">94.4</td><td align="center" rowspan="1" colspan="1">210 (3.4)</td><td align="center" rowspan="1" colspan="1">11.4</td><td align="center" rowspan="1" colspan="1">76.7</td></tr><tr><td align="left" rowspan="1" colspan="1">1–4 yr</td><td align="center" rowspan="1" colspan="1">240 (4.0)</td><td align="center" rowspan="1" colspan="1">2.8</td><td align="center" rowspan="1" colspan="1">95.7</td><td align="center" rowspan="1" colspan="1">249 (4.0)</td><td align="center" rowspan="1" colspan="1">3.0</td><td align="center" rowspan="1" colspan="1">106.4</td></tr><tr><td align="left" rowspan="1" colspan="1">5–14 yr</td><td align="center" rowspan="1" colspan="1">121 (2.0)</td><td align="center" rowspan="1" colspan="1">0.6</td><td align="center" rowspan="1" colspan="1">104.9</td><td align="center" rowspan="1" colspan="1">145 (2.3)</td><td align="center" rowspan="1" colspan="1">0.7</td><td align="center" rowspan="1" colspan="1">109.4</td></tr><tr><td align="left" rowspan="1" colspan="1">15–49 yr</td><td align="center" rowspan="1" colspan="1">711 (11.9)</td><td align="center" rowspan="1" colspan="1">1.6</td><td align="center" rowspan="1" colspan="1">147.4</td><td align="center" rowspan="1" colspan="1">738 (11.9)</td><td align="center" rowspan="1" colspan="1">1.7</td><td align="center" rowspan="1" colspan="1">150.0</td></tr><tr><td align="left" rowspan="1" colspan="1">50–64yr</td><td align="center" rowspan="1" colspan="1">989 (16.6)</td><td align="center" rowspan="1" colspan="1">10.3</td><td align="center" rowspan="1" colspan="1">183.3</td><td align="center" rowspan="1" colspan="1">1005 (16.3)</td><td align="center" rowspan="1" colspan="1">10.8</td><td align="center" rowspan="1" colspan="1">193.0</td></tr><tr><td align="left" rowspan="1" colspan="1">65+ yr</td><td align="center" rowspan="1" colspan="1">3258 (54.6)</td><td align="center" rowspan="1" colspan="1">59.9</td><td align="center" rowspan="1" colspan="1">108.3</td><td align="center" rowspan="1" colspan="1">3196 (51.7)</td><td align="center" rowspan="1" colspan="1">59.7</td><td align="center" rowspan="1" colspan="1">121.5</td></tr><tr><td align="left" rowspan="1" colspan="1">All ages</td><td align="center" rowspan="1" colspan="1">5965 (100.0)</td><td align="center" rowspan="1" colspan="1">6.6</td><td align="center" rowspan="1" colspan="1">132.0</td><td align="center" rowspan="1" colspan="1">6179 (100.0)</td><td align="center" rowspan="1" colspan="1">7.0</td><td align="center" rowspan="1" colspan="1">132.4</td></tr><tr><td align="left" rowspan="1" colspan="1"># person years</td><td colspan="3" align="center" rowspan="1">909319</td><td colspan="3" align="center" rowspan="1">884583</td></tr></tbody></table><table-wrap-foot><fn id="TF0002"><label>a</label><p>per 1000 person years of observation.</p></fn><fn id="TF0003"><label>b</label><p>ratio of male to female death rates (100*M/F).</p></fn><fn id="TF0004"><label>c</label><p>per 1000 person years in infancy.</p></fn><fn id="TF0005"><label>*</label><p>p<0.01(compared between the two areas).</p></fn></table-wrap-foot></table-wrap></sec><sec id="S0003-S20003"><title>Difference in cause-specific mortality by area</title><p>Mortality rates by broad age- and cause-category showed lower mortality rates due to CDs among children (aged 0–14 years), adults (aged 15–64 years), and elderly in the icddr,b SA than in the government SA, where the mortality rate due to non-NCDs was lower among elderly (<xref ref-type="table" rid="T0003">Table 3</xref>). No death was assigned to the vaccine preventable diseases. Mortality rates due to neonatal causes and pregnancy-related causes in adult females were lower in the icddr,b SA, whereas mortality rates due to external causes were comparable in each age group between these two areas. The rate of indeterminate, however, was higher in the government SA.</p><table-wrap id="T0003" position="float"><label>Table 3</label><caption><p>Death rates<xref ref-type="table-fn" rid="TF0006">a</xref> by broad age and cause categories in the icddr,b and government service</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1"/><th align="center" colspan="2" rowspan="1">Age 0–14 years</th><th align="center" colspan="2" rowspan="1">Age 15–64 years</th><th align="center" colspan="2" rowspan="1">Age 65+ years</th></tr><tr><th align="left" rowspan="1" colspan="1"/><th align="center" colspan="2" rowspan="1">
<hr/>
</th><th align="center" colspan="2" rowspan="1">
<hr/>
</th><th align="center" colspan="2" rowspan="1">
<hr/>
</th></tr><tr><th align="left" rowspan="1" colspan="1">Disease category</th><th align="center" rowspan="1" colspan="1">icddr,b</th><th align="center" rowspan="1" colspan="1">Government</th><th align="center" rowspan="1" colspan="1">icddr,b</th><th align="center" rowspan="1" colspan="1">Government</th><th align="center" rowspan="1" colspan="1">icddr,b</th><th align="center" rowspan="1" colspan="1">Government</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">Communicable diseases</td><td align="center" rowspan="1" colspan="1">0.72</td><td align="center" rowspan="1" colspan="1">0.99<xref ref-type="table-fn" rid="TF0009">**</xref>
</td><td align="center" rowspan="1" colspan="1">0.59</td><td align="center" rowspan="1" colspan="1">0.74<xref ref-type="table-fn" rid="TF0008">*</xref>
</td><td align="center" rowspan="1" colspan="1">10.55</td><td align="center" rowspan="1" colspan="1">13.55<xref ref-type="table-fn" rid="TF0009">**</xref>
</td></tr><tr><td align="left" rowspan="1" colspan="1">Non-communicable diseases</td><td align="center" rowspan="1" colspan="1">0.34</td><td align="center" rowspan="1" colspan="1">0.35</td><td align="center" rowspan="1" colspan="1">1.95</td><td align="center" rowspan="1" colspan="1">1.87</td><td align="center" rowspan="1" colspan="1">41.87</td><td align="center" rowspan="1" colspan="1">38.14<xref ref-type="table-fn" rid="TF0009">**</xref>
</td></tr><tr><td align="left" rowspan="1" colspan="1">Neonatal/maternal causes</td><td align="center" rowspan="1" colspan="1">13.18</td><td align="center" rowspan="1" colspan="1">1.49<xref ref-type="table-fn" rid="TF0009">**</xref>
</td><td align="center" rowspan="1" colspan="1">0.08</td><td align="center" rowspan="1" colspan="1">0.12<xref ref-type="table-fn" rid="TF0007">$</xref>
</td><td align="center" rowspan="1" colspan="1">NA</td><td align="center" rowspan="1" colspan="1">NA</td></tr><tr><td align="left" rowspan="1" colspan="1">Injury and external causes</td><td align="center" rowspan="1" colspan="1">0.58</td><td align="center" rowspan="1" colspan="1">0.55</td><td align="center" rowspan="1" colspan="1">0.20</td><td align="center" rowspan="1" colspan="1">0.28</td><td align="center" rowspan="1" colspan="1">0.66</td><td align="center" rowspan="1" colspan="1">0.80</td></tr><tr><td align="left" rowspan="1" colspan="1">Indeterminate</td><td align="center" rowspan="1" colspan="1">0.49</td><td align="center" rowspan="1" colspan="1">0.66<xref ref-type="table-fn" rid="TF0009">**</xref>
</td><td align="center" rowspan="1" colspan="1">0.29</td><td align="center" rowspan="1" colspan="1">0.37</td><td align="center" rowspan="1" colspan="1">6.59</td><td align="center" rowspan="1" colspan="1">7.11</td></tr><tr><td align="left" rowspan="1" colspan="1">All causes</td><td align="center" rowspan="1" colspan="1">3.31</td><td align="center" rowspan="1" colspan="1">4.03<xref ref-type="table-fn" rid="TF0009">**</xref>
</td><td align="center" rowspan="1" colspan="1">3.29</td><td align="center" rowspan="1" colspan="1">3.60</td><td align="center" rowspan="1" colspan="1">59.66</td><td align="center" rowspan="1" colspan="1">59.60</td></tr><tr><td align="left" rowspan="1" colspan="1">Person years</td><td align="center" rowspan="1" colspan="1">303110</td><td align="center" rowspan="1" colspan="1">306829</td><td align="center" rowspan="1" colspan="1">551789</td><td align="center" rowspan="1" colspan="1">524230</td><td align="center" rowspan="1" colspan="1">54421</td><td align="center" rowspan="1" colspan="1">53523</td></tr><tr><td align="left" rowspan="1" colspan="1">Number of deaths</td><td align="center" rowspan="1" colspan="1">1004</td><td align="center" rowspan="1" colspan="1">1235</td><td align="center" rowspan="1" colspan="1">1816</td><td align="center" rowspan="1" colspan="1">1886</td><td align="center" rowspan="1" colspan="1">3247</td><td align="center" rowspan="1" colspan="1">3190</td></tr></tbody></table><table-wrap-foot><fn id="TF0006"><label>a</label><p>per 1000 person years of observation;</p></fn><fn id="TF0007"><label>$</label><p>p<0.1</p></fn><fn id="TF0008"><label>*</label><p>p<0.05</p></fn><fn id="TF0009"><label>**</label><p>p<0.01, (compared between the two areas).</p></fn></table-wrap-foot></table-wrap><p>The all-age CSMFs due to CDs were less frequent (18.8% versus 22.9%) in the icddr,b SA, and those due to NCDs were less frequent (50.7% versus 58.2%) in the government SA (<xref ref-type="table" rid="T0004">Table 4</xref>). The most common CDs were acute respiratory infection (ARI)/pneumonia and pulmonary tuberculosis, and these were less frequent in the icddr,b SA. Some NCDs exhibited area differences; stroke (19.9% versus 15.6%), cardiac diseases (8.1% versus 6.1%) and chronic obstetric pulmonary diseases including asthma (6.1% versus 5.1%) were more frequent in the icddr,b SA than the government SA. Malignant neoplasms accounted one in seven deaths (14.2–15.3%), and malignancies were more frequent in the digestive system (5.5–6.0%) followed by the respiratory system (4.1–5.2%) in both areas. Neonatal causes were less frequent (6.0% versus 7.4%) in the icddr,b SA due to less frequent deaths from infections (ARI, pneumonia, or sepsis) and birth asphyxia. Fraction of deaths due to injury and other external causes was comparable (5.4–5.8%) between these two areas. Common external causes were accidental drowning (2.6–2.8%), followed by intentional self-harm (0.7–1.0%).</p><table-wrap id="T0004" position="float"><label>Table 4</label><caption><p>Cause-specific mortality fractions (in %) by sex and area, Matlab HDSS 2003–2010</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1"/><th align="center" colspan="3" rowspan="1">icddr,b service area</th><th align="center" colspan="3" rowspan="1">Government service area</th></tr><tr><th align="left" rowspan="1" colspan="1"/><th align="center" colspan="3" rowspan="1">
<hr/>
</th><th align="center" colspan="3" rowspan="1">
<hr/>
</th></tr><tr><th align="left" rowspan="1" colspan="1">Cause of death</th><th align="center" rowspan="1" colspan="1">Male</th><th align="center" rowspan="1" colspan="1">Female</th><th align="center" rowspan="1" colspan="1">Total</th><th align="center" rowspan="1" colspan="1">Male</th><th align="center" rowspan="1" colspan="1">Female</th><th align="center" rowspan="1" colspan="1">Total</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">Communicable diseases (CDs)</td><td align="center" rowspan="1" colspan="1">20.5</td><td align="center" rowspan="1" colspan="1">16.8<xref ref-type="table-fn" rid="TF0012">*</xref>
</td><td align="center" rowspan="1" colspan="1">18.8</td><td align="center" rowspan="1" colspan="1">24.3</td><td align="center" rowspan="1" colspan="1">21.3<xref ref-type="table-fn" rid="TF0012">*</xref>
</td><td align="center" rowspan="1" colspan="1">22.9<xref ref-type="table-fn" rid="TF0012">*</xref>
</td></tr><tr><td align="left" rowspan="1" colspan="1"> ARI/pneumonia</td><td align="center" rowspan="1" colspan="1">8.3</td><td align="center" rowspan="1" colspan="1">8.3</td><td align="center" rowspan="1" colspan="1">8.3</td><td align="center" rowspan="1" colspan="1">10.6</td><td align="center" rowspan="1" colspan="1">10.8</td><td align="center" rowspan="1" colspan="1">10.7<xref ref-type="table-fn" rid="TF0012">*</xref>
</td></tr><tr><td align="left" rowspan="1" colspan="1"> Pulmonary tuberculosis</td><td align="center" rowspan="1" colspan="1">10.0</td><td align="center" rowspan="1" colspan="1">5.8<xref ref-type="table-fn" rid="TF0012">*</xref>
</td><td align="center" rowspan="1" colspan="1">8.1</td><td align="center" rowspan="1" colspan="1">11.0</td><td align="center" rowspan="1" colspan="1">6.8<xref ref-type="table-fn" rid="TF0012">*</xref>
</td><td align="center" rowspan="1" colspan="1">9.1<xref ref-type="table-fn" rid="TF0011">$</xref>
</td></tr><tr><td align="left" rowspan="1" colspan="1"> Other CDs</td><td align="center" rowspan="1" colspan="1">2.3</td><td align="center" rowspan="1" colspan="1">2.7</td><td align="center" rowspan="1" colspan="1">2.5</td><td align="center" rowspan="1" colspan="1">2.7</td><td align="center" rowspan="1" colspan="1">3.6<xref ref-type="table-fn" rid="TF0011">$</xref>
</td><td align="center" rowspan="1" colspan="1">3.1<xref ref-type="table-fn" rid="TF0011">$</xref>
</td></tr><tr><td align="left" rowspan="1" colspan="1">Non-communicable diseases (NCDs)</td><td align="center" rowspan="1" colspan="1">55.9</td><td align="center" rowspan="1" colspan="1">60.9<xref ref-type="table-fn" rid="TF0012">*</xref>
</td><td align="center" rowspan="1" colspan="1">58.2</td><td align="center" rowspan="1" colspan="1">49.6</td><td align="center" rowspan="1" colspan="1">52.1<xref ref-type="table-fn" rid="TF0011">$</xref>
</td><td align="center" rowspan="1" colspan="1">50.7<xref ref-type="table-fn" rid="TF0012">*</xref>
</td></tr><tr><td align="left" rowspan="1" colspan="1"> Stroke</td><td align="center" rowspan="1" colspan="1">15.9</td><td align="center" rowspan="1" colspan="1">24.6<xref ref-type="table-fn" rid="TF0012">*</xref>
</td><td align="center" rowspan="1" colspan="1">19.9</td><td align="center" rowspan="1" colspan="1">13.9</td><td align="center" rowspan="1" colspan="1">17.6<xref ref-type="table-fn" rid="TF0012">*</xref>
</td><td align="center" rowspan="1" colspan="1">15.6<xref ref-type="table-fn" rid="TF0012">*</xref>
</td></tr><tr><td align="left" rowspan="1" colspan="1"> Cardiac disease</td><td align="center" rowspan="1" colspan="1">9.8</td><td align="center" rowspan="1" colspan="1">6.2<xref ref-type="table-fn" rid="TF0012">*</xref>
</td><td align="center" rowspan="1" colspan="1">8.1</td><td align="center" rowspan="1" colspan="1">7.6</td><td align="center" rowspan="1" colspan="1">4.4<xref ref-type="table-fn" rid="TF0012">*</xref>
</td><td align="center" rowspan="1" colspan="1">6.1<xref ref-type="table-fn" rid="TF0012">*</xref>
</td></tr><tr><td align="left" rowspan="1" colspan="1"> COPD and asthma</td><td align="center" rowspan="1" colspan="1">6.1</td><td align="center" rowspan="1" colspan="1">6.1</td><td align="center" rowspan="1" colspan="1">6.1</td><td align="center" rowspan="1" colspan="1">5.3</td><td align="center" rowspan="1" colspan="1">5.0</td><td align="center" rowspan="1" colspan="1">5.1<xref ref-type="table-fn" rid="TF0012">*</xref>
</td></tr><tr><td align="left" rowspan="1" colspan="1">Malignant neoplasm</td><td align="center" rowspan="1" colspan="1">16.2</td><td align="center" rowspan="1" colspan="1">11.8<xref ref-type="table-fn" rid="TF0012">*</xref>
</td><td align="center" rowspan="1" colspan="1">14.2</td><td align="center" rowspan="1" colspan="1">15.9</td><td align="center" rowspan="1" colspan="1">14.6</td><td align="center" rowspan="1" colspan="1">15.3</td></tr><tr><td align="left" rowspan="1" colspan="1"> Digestive system</td><td align="center" rowspan="1" colspan="1">6.1</td><td align="center" rowspan="1" colspan="1">4.7<xref ref-type="table-fn" rid="TF0012">*</xref>
</td><td align="center" rowspan="1" colspan="1">5.5</td><td align="center" rowspan="1" colspan="1">6.3</td><td align="center" rowspan="1" colspan="1">5.7</td><td align="center" rowspan="1" colspan="1">6.0</td></tr><tr><td align="left" rowspan="1" colspan="1"> Respiratory system</td><td align="center" rowspan="1" colspan="1">5.8</td><td align="center" rowspan="1" colspan="1">2.2<xref ref-type="table-fn" rid="TF0012">*</xref>
</td><td align="center" rowspan="1" colspan="1">4.1</td><td align="center" rowspan="1" colspan="1">6.1</td><td align="center" rowspan="1" colspan="1">4.2<xref ref-type="table-fn" rid="TF0012">*</xref>
</td><td align="center" rowspan="1" colspan="1">5.2<xref ref-type="table-fn" rid="TF0012">*</xref>
</td></tr><tr><td align="left" rowspan="1" colspan="1"> Other neoplasm</td><td align="center" rowspan="1" colspan="1">4.3</td><td align="center" rowspan="1" colspan="1">4.9</td><td align="center" rowspan="1" colspan="1">4.6</td><td align="center" rowspan="1" colspan="1">3.5</td><td align="center" rowspan="1" colspan="1">4.7<xref ref-type="table-fn" rid="TF0011">$</xref>
</td><td align="center" rowspan="1" colspan="1">4.1</td></tr><tr><td align="left" rowspan="1" colspan="1"> Acute abdomen</td><td align="center" rowspan="1" colspan="1">2.8</td><td align="center" rowspan="1" colspan="1">4.0<xref ref-type="table-fn" rid="TF0012">*</xref>
</td><td align="center" rowspan="1" colspan="1">3.4</td><td align="center" rowspan="1" colspan="1">2.7</td><td align="center" rowspan="1" colspan="1">3.3</td><td align="center" rowspan="1" colspan="1">3.0</td></tr><tr><td align="left" rowspan="1" colspan="1"> Liver cirrhosis</td><td align="center" rowspan="1" colspan="1">0.5</td><td align="center" rowspan="1" colspan="1">0.4</td><td align="center" rowspan="1" colspan="1">0.5</td><td align="center" rowspan="1" colspan="1">0.6</td><td align="center" rowspan="1" colspan="1">0.8</td><td align="center" rowspan="1" colspan="1">0.7</td></tr><tr><td align="left" rowspan="1" colspan="1"> Diabetes mellitus</td><td align="center" rowspan="1" colspan="1">1.6</td><td align="center" rowspan="1" colspan="1">2.6<xref ref-type="table-fn" rid="TF0012">*</xref>
</td><td align="center" rowspan="1" colspan="1">2.0</td><td align="center" rowspan="1" colspan="1">1.2</td><td align="center" rowspan="1" colspan="1">2.0<xref ref-type="table-fn" rid="TF0011">$</xref>
</td><td align="center" rowspan="1" colspan="1">1.5<xref ref-type="table-fn" rid="TF0011">$</xref>
</td></tr><tr><td align="left" rowspan="1" colspan="1"> Anaemia/malnutrition</td><td align="center" rowspan="1" colspan="1">1.1</td><td align="center" rowspan="1" colspan="1">2.4<xref ref-type="table-fn" rid="TF0012">*</xref>
</td><td align="center" rowspan="1" colspan="1">1.7</td><td align="center" rowspan="1" colspan="1">1.2</td><td align="center" rowspan="1" colspan="1">2.4<xref ref-type="table-fn" rid="TF0012">*</xref>
</td><td align="center" rowspan="1" colspan="1">1.8</td></tr><tr><td align="left" rowspan="1" colspan="1"> Other NCD</td><td align="center" rowspan="1" colspan="1">1.2</td><td align="center" rowspan="1" colspan="1">1.7</td><td align="center" rowspan="1" colspan="1">1.4</td><td align="center" rowspan="1" colspan="1">0.7</td><td align="center" rowspan="1" colspan="1">0.9</td><td align="center" rowspan="1" colspan="1">0.8<xref ref-type="table-fn" rid="TF0012">*</xref>
</td></tr><tr><td align="left" rowspan="1" colspan="1">Perinatal and neonatal causes</td><td align="center" rowspan="1" colspan="1">6.8</td><td align="center" rowspan="1" colspan="1">5.0<xref ref-type="table-fn" rid="TF0012">*</xref>
</td><td align="center" rowspan="1" colspan="1">6.0</td><td align="center" rowspan="1" colspan="1">7.5</td><td align="center" rowspan="1" colspan="1">7.3</td><td align="center" rowspan="1" colspan="1">7.4<xref ref-type="table-fn" rid="TF0012">*</xref>
</td></tr><tr><td align="left" rowspan="1" colspan="1"> ARI, pneumonia, sepsis</td><td align="center" rowspan="1" colspan="1">3.2</td><td align="center" rowspan="1" colspan="1">2.1<xref ref-type="table-fn" rid="TF0012">*</xref>
</td><td align="center" rowspan="1" colspan="1">2.7</td><td align="center" rowspan="1" colspan="1">3.7</td><td align="center" rowspan="1" colspan="1">2.8<xref ref-type="table-fn" rid="TF0011">$</xref>
</td><td align="center" rowspan="1" colspan="1">3.3<xref ref-type="table-fn" rid="TF0011">$</xref>
</td></tr><tr><td align="left" rowspan="1" colspan="1"> Birth asphyxia</td><td align="center" rowspan="1" colspan="1">0.9</td><td align="center" rowspan="1" colspan="1">0.7</td><td align="center" rowspan="1" colspan="1">0.8</td><td align="center" rowspan="1" colspan="1">1.4</td><td align="center" rowspan="1" colspan="1">1.4</td><td align="center" rowspan="1" colspan="1">1.4<xref ref-type="table-fn" rid="TF0012">*</xref>
</td></tr><tr><td align="left" rowspan="1" colspan="1"> Prematurity</td><td align="center" rowspan="1" colspan="1">1.9</td><td align="center" rowspan="1" colspan="1">1.6</td><td align="center" rowspan="1" colspan="1">1.8</td><td align="center" rowspan="1" colspan="1">1.3</td><td align="center" rowspan="1" colspan="1">2.1</td><td align="center" rowspan="1" colspan="1">1.7</td></tr><tr><td align="left" rowspan="1" colspan="1"> Other neonatal cause</td><td align="center" rowspan="1" colspan="1">0.9</td><td align="center" rowspan="1" colspan="1">0.6</td><td align="center" rowspan="1" colspan="1">0.8</td><td align="center" rowspan="1" colspan="1">1.1</td><td align="center" rowspan="1" colspan="1">0.9</td><td align="center" rowspan="1" colspan="1">1.0</td></tr><tr><td align="left" rowspan="1" colspan="1">Cause related to pregnancy</td><td align="center" rowspan="1" colspan="1">NA</td><td align="center" rowspan="1" colspan="1">0.7</td><td align="center" rowspan="1" colspan="1">0.3</td><td align="center" rowspan="1" colspan="1">Na</td><td align="center" rowspan="1" colspan="1">1.0</td><td align="center" rowspan="1" colspan="1">0.5</td></tr><tr><td align="left" rowspan="1" colspan="1">Injury and other external cause</td><td align="center" rowspan="1" colspan="1">5.4</td><td align="center" rowspan="1" colspan="1">5.5</td><td align="center" rowspan="1" colspan="1">5.4</td><td align="center" rowspan="1" colspan="1">6.1</td><td align="center" rowspan="1" colspan="1">5.4</td><td align="center" rowspan="1" colspan="1">5.8</td></tr><tr><td align="left" rowspan="1" colspan="1"> Accidental drowning</td><td align="center" rowspan="1" colspan="1">2.8</td><td align="center" rowspan="1" colspan="1">2.7</td><td align="center" rowspan="1" colspan="1">2.8</td><td align="center" rowspan="1" colspan="1">2.8</td><td align="center" rowspan="1" colspan="1">2.3</td><td align="center" rowspan="1" colspan="1">2.6</td></tr><tr><td align="left" rowspan="1" colspan="1"> Road traffic accident</td><td align="center" rowspan="1" colspan="1">0.7</td><td align="center" rowspan="1" colspan="1">0.3<xref ref-type="table-fn" rid="TF0012">*</xref>
</td><td align="center" rowspan="1" colspan="1">0.5</td><td align="center" rowspan="1" colspan="1">0.9</td><td align="center" rowspan="1" colspan="1">0.2<xref ref-type="table-fn" rid="TF0012">*</xref>
</td><td align="center" rowspan="1" colspan="1">0.6</td></tr><tr><td align="left" rowspan="1" colspan="1"> International self-harm</td><td align="center" rowspan="1" colspan="1">0.4</td><td align="center" rowspan="1" colspan="1">1.2<xref ref-type="table-fn" rid="TF0012">*</xref>
</td><td align="center" rowspan="1" colspan="1">0.7</td><td align="center" rowspan="1" colspan="1">0.5</td><td align="center" rowspan="1" colspan="1">1.5<xref ref-type="table-fn" rid="TF0012">*</xref>
</td><td align="center" rowspan="1" colspan="1">1.0</td></tr><tr><td align="left" rowspan="1" colspan="1"> Assault</td><td align="center" rowspan="1" colspan="1">0.3</td><td align="center" rowspan="1" colspan="1">0.5</td><td align="center" rowspan="1" colspan="1">0.4</td><td align="center" rowspan="1" colspan="1">0.4</td><td align="center" rowspan="1" colspan="1">0.5</td><td align="center" rowspan="1" colspan="1">0.5</td></tr><tr><td align="left" rowspan="1" colspan="1"> Other cause</td><td align="center" rowspan="1" colspan="1">1.1</td><td align="center" rowspan="1" colspan="1">0.8</td><td align="center" rowspan="1" colspan="1">1.0</td><td align="center" rowspan="1" colspan="1">1.4</td><td align="center" rowspan="1" colspan="1">1.0</td><td align="center" rowspan="1" colspan="1">1.2</td></tr><tr><td align="left" rowspan="1" colspan="1">Indeterminate</td><td align="center" rowspan="1" colspan="1">11.4</td><td align="center" rowspan="1" colspan="1">11.0</td><td align="center" rowspan="1" colspan="1">11.2</td><td align="center" rowspan="1" colspan="1">12.4</td><td align="center" rowspan="1" colspan="1">12.8</td><td align="center" rowspan="1" colspan="1">12.6<xref ref-type="table-fn" rid="TF0011">$</xref>
</td></tr><tr><td align="left" rowspan="1" colspan="1"> # VA with disease symptom<xref ref-type="table-fn" rid="TF0010">a</xref>
</td><td align="center" rowspan="1" colspan="1">3217</td><td align="center" rowspan="1" colspan="1">2729</td><td align="center" rowspan="1" colspan="1">5,946</td><td align="center" rowspan="1" colspan="1">3,348</td><td align="center" rowspan="1" colspan="1">2,819</td><td align="center" rowspan="1" colspan="1">6,167</td></tr></tbody></table><table-wrap-foot><fn><p>Note: ARI=acute respiratory infection; COPD=chronic obstetric pulmonary diseases.</p></fn><fn id="TF0010"><label>a</label><p>Excluded from analysis was 129 deaths: 31 cases for no response to VA and 98 cases for no disease symptom or sign prior to death.</p></fn><fn id="TF0011"><label>$</label><p>p<0.05</p></fn><fn id="TF0012"><label>*</label><p>p<0.01 (compared between males and females within the areas or between area totals).</p></fn></table-wrap-foot></table-wrap></sec><sec id="S0003-S20004"><title>Gender differences in CSMF by area</title><p>The distribution of CSMFs by gender shows biosocial differences in mortality risks (<xref ref-type="table" rid="T0004">Table 4</xref>). The frequency of deaths due to CDs was higher among males than females (20.5% versus 16.8% in the icddr,b SA and 24.3% versus 21.3% in the government SA), due mostly to a higher frequency of pulmonary tuberculosis deaths in both areas. Frequencies of ARI/pneumonia and other infections were comparable between males and females. Share of deaths due to NCDs was lower (55.9% versus 60.9% in the icddr,b SA and 49.6% versus 52.1% in the government SA) for males than females. Stroke was more frequent among females than males among whom cardiac diseases were higher in both areas. Frequency of overall malignant neoplasms, particularly neoplasms in respiratory system, was higher among males. CSMFs for acute abdomen, diabetes mellitus, and severe anaemia or malnutrition were relatively low, but significantly higher among females than males in each area.</p><p>As expected, the share of neonatal deaths was a little higher (6.8% versus 5.0%) among male deaths than female deaths in the icddr,b SA. The most common neonatal cause was ARI/pneumonia/sepsis. Injury and other external causes were comparable between males and females with half of them being due to accidental drowning, mostly in the age group 1–4 years. Road traffic accident deaths were less common, but higher among males than females, among whom self-harm and assaults were higher.</p></sec><sec id="S0003-S20005"><title>Rank-order of CoD by age</title><p>
<xref ref-type="table" rid="T0005">Table 5</xref> shows marked variations in the rank-order of top causes of death by age group, as well as the percentage of indeterminate cause. Indeterminate was the highest for neonates (23.2%) and the lowest for children aged 1–4 years (6.8%). Neonatal deaths were due mostly to ARI/pneumonia (26.8%), followed by prematurity (18.6%), birth asphyxia (11.9%), and sepsis (5.5%), summing up to 62.8% of the deaths. In the post-neonatal period, the leading cause was ARI/pneumonia (60.9%), followed by malnutrition (8.5%), diarrhoea (6.3%), meningitis or encephalitis (3.8%), and injury (3.1%), totalling 82.6% of the deaths. Injury and other external cause (49.4%, accidental drowning accounted for 46.4%) were the leading cause of child (age 1–4 years) deaths followed by ARI/pneumonia (16.2%), malnutrition (15.5%), tuberculosis (2.9%), meningitis or encephalitis (2.3%), and diarrhoea (1.7%), totalling 88% of the child deaths. Among older children (age 5–14 years), the leading causes were injury and other external causes (33.5%, accidental drowning accounted for 17.6%), malnutrition (7.4%), acute abdomen (7.4%), ARI/pneumonia (7.1%), neoplasm (5.9%), tuberculosis (5.0%), and meningitis or encephalitis (2.5%); totalling 70.8% of the deaths .</p><table-wrap id="T0005" position="float"><label>Table 5</label><caption><p>Cause-specific mortality fractions<xref ref-type="table-fn" rid="TF0013">a</xref> [in %] by age group of the deceased in Matlab HDSS, 2003–2010</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1">0–28 days<break/>(# deaths=1116)</th><th align="center" rowspan="1" colspan="1">29 days-<1 year<break/>(# deaths=368)</th><th align="center" rowspan="1" colspan="1">1–4 years<break/>(# deaths=489)</th><th align="center" rowspan="1" colspan="1">5–14 years<break/>(# deaths=265)</th><th align="center" rowspan="1" colspan="1">15–49 years<break/>(# deaths=1429)</th><th align="center" rowspan="1" colspan="1">50–64 years<break/>(# deaths=1974)</th><th align="center" rowspan="1" colspan="1">65+ years (# deaths=6374)</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">ARI or pneumonia [26.8%]</td><td align="left" rowspan="1" colspan="1">ARI/pneumonia [60.9%]</td><td align="left" rowspan="1" colspan="1">Injury [49.4%]<break/>Drowning [46.4%]</td><td align="left" rowspan="1" colspan="1">Injury [33.5%]<break/>Drowning [17.6%]</td><td align="left" rowspan="1" colspan="1">Neoplasm [20.5%]</td><td align="left" rowspan="1" colspan="1">Neoplasm [22.8%]</td><td align="left" rowspan="1" colspan="1">Stroke [25.8%]</td></tr><tr><td align="left" rowspan="1" colspan="1">Prematurity [18.6%]</td><td align="left" rowspan="1" colspan="1">Malnutrition [8.5%]</td><td align="left" rowspan="1" colspan="1">ARI/pneumonia [16.2%]</td><td align="left" rowspan="1" colspan="1">Malnutrition [7.4%]</td><td align="left" rowspan="1" colspan="1">Injury [14.0%]<break/>Self-harm [5.8%]</td><td align="left" rowspan="1" colspan="1">Stroke [17.3%]</td><td align="left" rowspan="1" colspan="1">Neoplasm [15.7%]</td></tr><tr><td align="left" rowspan="1" colspan="1">Birth asphyxia [11.9%]</td><td align="left" rowspan="1" colspan="1">Diarrhoea [6.3%]</td><td align="left" rowspan="1" colspan="1">Malnutrition (15.5%)</td><td align="left" rowspan="1" colspan="1">Acute abdomen [7.4%]</td><td align="left" rowspan="1" colspan="1">Tuberculosis [11.6%]</td><td align="left" rowspan="1" colspan="1">Tuberculosis [12.5%]</td><td align="left" rowspan="1" colspan="1">ARI/pneumonia [9.3%]</td></tr><tr><td align="left" rowspan="1" colspan="1">Sepsis [5.5%]</td><td align="left" rowspan="1" colspan="1">Meningitis/encephalitis [3.8%]</td><td align="left" rowspan="1" colspan="1">Tuberculosis [2.9%]</td><td align="left" rowspan="1" colspan="1">ARI/pneumonia [7.1%]</td><td align="left" rowspan="1" colspan="1">Stroke [9.6%]</td><td align="left" rowspan="1" colspan="1">Cardiac diseases [11.0%]</td><td align="left" rowspan="1" colspan="1">Tuberculosis [9.2%]</td></tr><tr><td align="left" rowspan="1" colspan="1">Meningitis/encephalitis [3.3%]</td><td align="left" rowspan="1" colspan="1">Injury [3.1%]</td><td align="left" rowspan="1" colspan="1">Meningitis/encephalitis [2.3%]</td><td align="left" rowspan="1" colspan="1">Neoplasm [5.9%]</td><td align="left" rowspan="1" colspan="1">Cardiac diseases [7.3%]</td><td align="left" rowspan="1" colspan="1">COPD/asthma [7.7%]</td><td align="left" rowspan="1" colspan="1">Cardiac diseases [8.2%]</td></tr><tr><td align="left" rowspan="1" colspan="1">Malformation [1.2%]</td><td align="left" rowspan="1" colspan="1">Sepsis [2.2%]</td><td align="left" rowspan="1" colspan="1">Diarrhoea [1.7%]</td><td align="left" rowspan="1" colspan="1">Tuberculosis [5.0%]</td><td align="left" rowspan="1" colspan="1">Acute abdomen [5.5%]</td><td align="left" rowspan="1" colspan="1">ARI/pneumonia [7.4%]</td><td align="left" rowspan="1" colspan="1">COPD/asthma [7.6%]</td></tr><tr><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1">Meningitis/encephalitis [2.5%]</td><td align="left" rowspan="1" colspan="1">ARI/pneumonia [5.3%]</td><td align="left" rowspan="1" colspan="1">Acute abdomen [3.6%]</td><td align="left" rowspan="1" colspan="1">Acute abdomen [3.1%]</td></tr><tr><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1">Epilepsy [2.0]</td><td align="left" rowspan="1" colspan="1">Maternal cause [3.4%]</td><td align="left" rowspan="1" colspan="1">Injury [2.8%]</td><td align="left" rowspan="1" colspan="1">Diabetes [2.7%]</td></tr><tr><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1">COPD/asthma [2.3%]</td><td align="left" rowspan="1" colspan="1">Diabetes [1.6%]</td><td align="left" rowspan="1" colspan="1">Renal failure [1.2%]</td></tr><tr><td align="left" rowspan="1" colspan="1">Others [9.4%]</td><td align="left" rowspan="1" colspan="1">Other [7.3%]</td><td align="left" rowspan="1" colspan="1">Other [6.4%]</td><td align="left" rowspan="1" colspan="1">Other [15.2%]</td><td align="left" rowspan="1" colspan="1">Other [7.7%]</td><td align="left" rowspan="1" colspan="1">Other [4.2%]</td><td align="left" rowspan="1" colspan="1">Other [5.6%]</td></tr><tr><td align="left" rowspan="1" colspan="1">Indeterminate [23.1%]</td><td align="left" rowspan="1" colspan="1">Indeterminate [7.9%]</td><td align="left" rowspan="1" colspan="1">Indeterminate [6.8%]</td><td align="left" rowspan="1" colspan="1">Indeterminate [14.0%]</td><td align="left" rowspan="1" colspan="1">Indeterminate [12.8%]</td><td align="left" rowspan="1" colspan="1">Indeterminate [9.1%]</td><td align="left" rowspan="1" colspan="1">Indeterminate [11.6%]</td></tr></tbody></table><table-wrap-foot><fn id="TF0013"><label>a</label><p>Included VA with reported disease symptoms.</p></fn></table-wrap-foot></table-wrap><p>The most common CoD among younger adults (age 15–49 years) was malignant neoplasm (20.5%), followed by injury and other external causes (14.0%), tuberculosis (11.6%), stroke (9.6%) and cardiac diseases (7.3%), acute abdomen (5.5%), and ARI/pneumonia (5.3%). These causes accounted for 73.8% of the deaths. Deaths from pregnancy-related causes accounted for 7.5% of the adult female deaths. Malignant neoplasms (22.8%) continued to be the leading cause among older adults (50–64 years), followed by stroke (17.3%), tuberculosis (12.5%), cardiac disease (11.0%), COPD/asthma (7.7%), ARI/ pneumonia (7.4%), acute abdomen (3.6%), and injury (2.8%), accounting for 85.1% of the deaths. The leading causes of elderly (65 years and older) deaths were stroke (25.8%), followed by malignant neoplasm (15.7%), ARI/pneumonia (9.3%), tuberculosis (9.2%), cardiac diseases (8.2%), and COPD or asthma (7.6%), totalling 75.8% of all deaths.</p></sec></sec><sec sec-type="discussion" id="S0004"><title>Discussion</title><p>The results showed the differences in health burdens, particularly drop in mortality fractions due to CDs and perinatal and neonatal causes, and a rise in mortality fractions due to NCDs in the icddr,b SA, as compared to the government SA. Earlier studies reported lower infant and child mortality rates in 1982–2002, and lower perinatal and neonatal mortality in 2005–2009 in the icddr,b SA than in the government SA, and the majority of the differences were due to the high quality of MNCH services in the icddr,b area (<xref rid="CIT0010" ref-type="bibr">10</xref>, <xref rid="CIT0011" ref-type="bibr">11</xref>). A few studies reported the lower risk of dying from ARI/pneumonia in children under 5 years old in the icddr,b area with enhanced health care system (<xref rid="CIT0019" ref-type="bibr">19</xref>, <xref rid="CIT0020" ref-type="bibr">20</xref>). Our results, in support of the previous studies, revealed the simultaneous decrease in mortality from CDs in all ages and increase in mortality rates from NCDs in older ages in the icddr,b SA as compared to the government SA during 2003–2010. This clearly suggests an age shift in mortality towards old age with some compensatory effect on NCDs and on overall death rate in the icddr,b SA, possibly due to the enhanced MNCH-FP services project for a long period. The distribution of the InterVA-4-coded CoDs in broad cause categories was more similar than dissimilar with the physician-coded CoD (<xref rid="CIT0006" ref-type="bibr">6</xref>, <xref rid="CIT0021" ref-type="bibr">21</xref>, <xref rid="CIT0022" ref-type="bibr">22</xref>).</p><p>The study findings showed a gender difference in mortality fractions due to specific CDs and NCDs, and injuries/external causes in rural areas, indicating a need for differential priorities and public health responses. The finding is consistent with the gender difference found in the distribution of physician-coded CoD of adults and elderly (<xref rid="CIT0006" ref-type="bibr">6</xref>). The risk factors of NCDs showed sex difference in the prevalence of hypertension (32% in women and 19% in men aged 35 years and older) in 2011 (<xref rid="CIT0021" ref-type="bibr">21</xref>). More than half of the diseased were not aware that they had the disease (<xref rid="CIT0021" ref-type="bibr">21</xref>). More men aged 25–64 years used tobacco products (68.2% versus 32.7%) than women of similar ages, who were more often overweight (15.2% versus 10.8%) and more often had self-reported heart problems (10.5% versus 4.9%) (<xref rid="CIT0023" ref-type="bibr">23</xref>).</p><p>Management of NCDs is available at tertiary-level hospitals, which are very expensive. Many NCDs are, however, amenable to prevention through behaviour changes. Lifestyle and behaviours are linked to 20–25% of the global burden of disease, which is likely to increase rapidly in poorer countries (<xref rid="CIT0024" ref-type="bibr">24</xref>). In Bangladesh, use of tobacco products, excessive intake of salt, and abuse of substances are substantially high; and consumption of vegetables and fruits and regular exercise are very low (<xref rid="CIT0023" ref-type="bibr">23</xref>). A number of ‘conventional’ NCDs share these same risk factors, and interventions directed towards these will address the diseases simultaneously (<xref rid="CIT0025" ref-type="bibr">25</xref>).</p><p>Injury and other external causes were the leading cause of mortality in the age groups 1–4 and 5–14 years in this rural community. This finding reconfirmed drowning as the leading CoD for children and recommended undertaking an appropriate intervention for preventing such deaths in rural Bangladesh. An intervention project called ‘Saving lives from drowning’ is currently underway, but the results are yet to be seen. One-fourth (24%) of the deaths in the 15–49 years age group were due to injury and other external causes, half of which were due to self-harm and another 14% were due to assault. Assaults (physical and mental) often provoke self-harm, thus underestimating the share of assault. Violence against women, particularly young women, is quite high in South Asia including Bangladesh and is often perpetrated by the woman's husband or his family members (<xref rid="CIT0026" ref-type="bibr">26</xref>–<xref rid="CIT0028" ref-type="bibr">28</xref>). Assessment of ‘self-harm and assault’ obtained from family members through VA may underestimate the true burden, therefore indicating a limitation of VA in divulging the true cause in some cases.</p><p>The government Health, Population and Nutrition Sector Development Programs (HPNSDPs) for 2011–2016 have an operational plan to control NCDs by expanding access to health services (<xref rid="CIT0029" ref-type="bibr">29</xref>). The operation plan of HPNSDP includes conducting training on NCD screening and management for health care providers at district and sub-district levels, organizing awareness-building workshops on injuries, and pilot screening as well as management of selected NCDs at the sub-district level facilities, and gradually expanding to the Union Health and Family Welfare Centre and to the Community Clinic (CC). The private sector, particularly pharmacies in urban and rural areas and ‘workplace based prevention and screening intervention’ can play an important role in screening and referral. Prevention, early detection, and compliance with effective medication can save national health expenditure, improve population health, and reduce household's unnecessary medical costs and loss of productivity. Strengthening behaviour change communications activities at the community level for controlling unhealthy diet and lifestyle and promoting healthy lifestyle and injury prevention may further lower disease and economic burdens. As NCDs bring catastrophic economic consequences and greatly exacerbate poverty, their control must receive due importance to alleviate poverty and improve population health.</p><p>In conclusion, the InterVA-4 coded CoD revealed double burdens of CDs and NCDs, with difference in burdens between the two areas and a shift towards NCDs and external causes. The present public health system is focused mainly on infectious diseases that remain of utmost concern, but it needs to be equipped to address the massive burdens of NCDs in rural areas.</p></sec> |
Immunomodulatory Potential of Human Adipose Mesenchymal Stem Cells Derived Exosomes on <italic>in vitro</italic> Stimulated T Cells | <p>In the recent years, it has been demonstrated that the biological activity of mesenchymal stem cells (MSCs) is mediated through the release of paracrine factors. Many of these factors are released into exosomes, which are small membranous vesicles that participate in cell–cell communication. Exosomes from MSCs are thought to have similar functions to MSCs such as repairing and regeneration of damaged tissue, but little is known about the immunomodulatory effect of these vesicles. Based on an extensive bibliography where the immunomodulatory capacity of MSCs has been demonstrated, here we hypothesized that released exosomes from MSCs may have an immunomodulatory role on the differentiation, activation and function of different lymphocyte subsets. According to this hypothesis, <italic>in vitro</italic> experiments were performed to characterize the immunomodulatory effect of human adipose MSCs derived exosomes (exo-hASCs) on <italic>in vitro</italic> stimulated T cells. The phenotypic characterization of cytotoxic and helper T cells (activation and differentiation markers) together with functional assays (proliferation and IFN-γ production) demonstrated that exo-hASCs exerted an inhibitory effect in the differentiation and activation of T cells as well as a reduced T cell proliferation and IFN-γ release on <italic>in vitro</italic> stimulated cells. In summary, here we demonstrate that MSCs-derived exosomes are a cell-derived product that could be considered as a therapeutic agent for the treatment of inflammation-related diseases.</p> | <contrib contrib-type="author"><name><surname>Blazquez</surname><given-names>Rebeca</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><uri xlink:type="simple" xlink:href="http://frontiersin.org/people/u/191128"/></contrib><contrib contrib-type="author"><name><surname>Sanchez-Margallo</surname><given-names>Francisco Miguel</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><uri xlink:type="simple" xlink:href="http://frontiersin.org/people/u/56922"/></contrib><contrib contrib-type="author"><name><surname>de la Rosa</surname><given-names>Olga</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref><uri xlink:type="simple" xlink:href="http://frontiersin.org/people/u/50424"/></contrib><contrib contrib-type="author"><name><surname>Dalemans</surname><given-names>Wilfried</given-names></name><xref ref-type="aff" rid="aff3"><sup>3</sup></xref><uri xlink:type="simple" xlink:href="http://frontiersin.org/people/u/191120"/></contrib><contrib contrib-type="author"><name><surname>Álvarez</surname><given-names>Verónica</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><uri xlink:type="simple" xlink:href="http://frontiersin.org/people/u/191125"/></contrib><contrib contrib-type="author"><name><surname>Tarazona</surname><given-names>Raquel</given-names></name><xref ref-type="aff" rid="aff4"><sup>4</sup></xref><uri xlink:type="simple" xlink:href="http://frontiersin.org/people/u/169130"/></contrib><contrib contrib-type="author"><name><surname>Casado</surname><given-names>Javier G.</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="corresp" rid="cor1">*</xref><uri xlink:type="simple" xlink:href="http://frontiersin.org/people/u/84942"/></contrib> | Frontiers in Immunology | <sec sec-type="introduction" id="S1"><title>Introduction</title><p>Exosomes are small membranous vesicles secreted by most cell types. These vesicles participate in cell–cell communication and their content consists of RNA, lipids, and proteins. Some of these proteins (i.e., CD9, CD63, or CD81) are ubiquitously expressed, but depending on the cell source, cell type-specific proteins can be found being responsible of their functionality. The proteins, lipids, and RNA expression of exosomes from different cells and organisms are extensively described in ExoCarta database (<xref rid="B1" ref-type="bibr">1</xref>).</p><p>Exosomes can be easily isolated by ultracentrifugation from <italic>in vitro</italic> cultured cells but different isolation protocols have been described in the literature (<xref rid="B2" ref-type="bibr">2</xref>). All these protocols differ from each other on the basis of particular types of research being divided as procedures for discovery, diagnostic, or preparative research (<xref rid="B3" ref-type="bibr">3</xref>). For a clinical-grade production of exosomes, safe technologies for large scale production are an absolute prerequisite (<xref rid="B4" ref-type="bibr">4</xref>).</p><p>In preclinical settings, especially in murine models, exosomes have been applied for the treatment of many different diseases such as infections (<xref rid="B5" ref-type="bibr">5</xref>, <xref rid="B6" ref-type="bibr">6</xref>), allergies (<xref rid="B7" ref-type="bibr">7</xref>) as well as autoimmune diseases (<xref rid="B8" ref-type="bibr">8</xref>, <xref rid="B9" ref-type="bibr">9</xref>). Regarding the immunomodulatory potential of these vesicles, the first <italic>in vivo</italic> studies were conducted by Pêche et al. using bone marrow dendritic cell-derived exosomes (<xref rid="B10" ref-type="bibr">10</xref>, <xref rid="B11" ref-type="bibr">11</xref>). Compared to preclinical studies, only a few clinical trials have been conducted using exosomes. Some of the first clinical trials were conducted in cancer patients using dendritic cell-derived exosomes (<xref rid="B12" ref-type="bibr">12</xref>) and ascites-derived exosomes (<xref rid="B13" ref-type="bibr">13</xref>) where the safety, tolerability, and efficacy of the treatments were demonstrated.</p><p>At the present, the therapeutic potential of exosomes derived from MSCs (Exo-MSCs) has been successfully applied in murine models for the treatment of cardiovascular diseases (<xref rid="B14" ref-type="bibr">14</xref>). In this sense, the proangiogenic effect described in different stem cell subsets may be the responsible of this therapeutic effect (<xref rid="B15" ref-type="bibr">15</xref>).</p><p>There are no differences in terms of morphological features, isolation, and storage conditions between exosomes derived from MSCs and other sources. As to the identification, exo-MSCs express not only the common surface markers of exosomes, such as CD9 and CD81, but also some adhesion molecules, including CD29, CD44, and CD73, which are expressed on the membrane of MSCs (<xref rid="B16" ref-type="bibr">16</xref>).</p><p>Accumulative evidences have established that, the effect of MSC transplantation is thought to be mediated in part, by a paracrine effect. Indeed, in the context of myocardial infarct it was experimentally quantified that the overall beneficial effect of paracrine mechanisms accounted between 50 and 80% (<xref rid="B17" ref-type="bibr">17</xref>). Several advantages of using released factors from MSCs have been described. For example, transferred cells may die or not fully home into the site of damaged tissue whereas biological factors can be locally administered with a controlled dosage (<xref rid="B18" ref-type="bibr">18</xref>).</p><p>Current preclinical trials with exo-MSCs have been driven for repairing damaged tissues, but few reports have been focused on the immunomodulatory effect of these vesicles. Here, we hypothesize that exo-MSCs may have similar regulatory functions than the original MSCs source on the differentiation, activation and function of different T cell subsets (<xref rid="B16" ref-type="bibr">16</xref>).</p><p>Supporting this idea, previous reports have demonstrated that the immunomodulatory capacity of MSCs against NK cells (<xref rid="B19" ref-type="bibr">19</xref>, <xref rid="B20" ref-type="bibr">20</xref>), cytotoxic T lymphocytes (<xref rid="B21" ref-type="bibr">21</xref>), γδ T cells (<xref rid="B22" ref-type="bibr">22</xref>), dendritic cells (<xref rid="B23" ref-type="bibr">23</xref>, <xref rid="B24" ref-type="bibr">24</xref>), or invariant NKT cells (<xref rid="B25" ref-type="bibr">25</xref>) is mediated by a paracrine mechanism.</p><p>In order to address this hypothesis, <italic>in vitro</italic> experiments were performed to characterize the immunomodulatory effect of exo-MSCs on <italic>in vitro</italic> stimulated T cells. The phenotypic characterization of cytotoxic and helper T cells (activation and differentiation markers) together with functional assays (proliferation and IFN-γ production) demonstrated that exo-MSCs exerted an inhibitory effect in the differentiation and activation of T cells as well as a reduced proliferation and IFN-γ release on <italic>in vitro</italic> expanded T cells. In summary, our results suggest that, exo-MSCs are a cell-derived product that could be considered as an immunomodulatory therapeutic agent for the treatment of immunological diseases.</p></sec><sec sec-type="materials|methods" id="S2"><title>Materials and Methods</title><sec id="S2-1"><title>Human adipose mesenchymal stem cells isolation and expansion</title><p>The human adipose mesenchymal stem cells (hASCs) were isolated from lipoaspirates obtained from human adipose tissue from healthy adult donors. Lipoaspirates were washed with PBS, and digested with collagenase type I in PBS. The digested sample was washed with 10% of fetal bovine serum (FBS), treated with ammonium chloride 160 mM, suspended in culture medium (DMEM containing 10% FBS), and filtered through a 40 μm nylon mesh. Cells were seeded onto tissue culture flasks and expanded at 37°C and 5% CO<sub>2</sub>, changing the culture medium every 7 days. Cells were passed to a new culture flask when cultures reached 90% of confluence. In addition, hASCs were tested by flow cytometry using specific surface markers being negative for CD14, CD31, CD34, CD45 and positive for CD29, CD59, CD90, and CD105 (data not shown). Cell lines from two healthy donors were used in the study. The biological samples were obtained after informed consent under the auspices of the appropriate Research and Ethics Committees.</p></sec><sec id="S2-2"><title>Isolation and purification of exosomes from hASCs</title><p>An enriched fraction of exosomes from hASCs (exo-hASCs) was obtained from hASCs cultured in 175 cm<sup>2</sup> flasks. When cells reached a confluence of 80%, culture medium (DMEM containing 10% FBS) was replaced by exosome isolation medium (DMEM containing 1% insulin–transferrin–selenium). The hASCs supernatants were collected every 3–4 days. Exosomes were isolated from supernatants by two successive centrifugations at 1000 × <italic>g</italic> (10 min) and 5000 × <italic>g</italic> (20 min) at 4°C to eliminate cells and debris, followed by an ultracentrifugation at 100,000 × g for 6 h to precipitate exosomes. The pellets were resuspended in 250 μL of PBS and stored at −20°C. Prior to <italic>in vitro</italic> experiments, exosomes were quantified by Bradford assays and characterized by nanoparticle tracking analysis.</p></sec><sec id="S2-3"><title>Characterization of exo-hASCs</title><p>The concentration and size of purified exosomes were measured by nanoparticle tracking analysis (NanoSight Ltd, Amesbury, UK) that relates the rate of Brownian motion to particle size. Results were analyzed using the nanoparticle tracking analysis software package version 2.2. Triplicate samples were diluted 1:10 in sterile-filtered PBS and analyzed.</p></sec><sec id="S2-4"><title>Bradford assay</title><p>Exosome concentrations were indirectly measured by protein quantification in a Bradford assay. To quantify protein concentration, 20 μL of exosomes sample were incubated with 180 μL of Bradford reagent (Bio Rad Laboratories, Hercules, CA) at RT. Absorbance was read 5 min after at 595 nm, and protein concentration was extrapolated from a standard concentration curve of Bovine Serum Albumin.</p></sec><sec id="S2-5"><title>Lymphocytes isolation and preservation</title><p>Peripheral blood lymphocytes (PBLs) from healthy donors were obtained by centrifugation over Histopaque-1077 (Sigma, St. Louis, MO, USA) and washed twice with PBS. The PBLs were frozen and stored in liquid nitrogen. For <italic>in vitro</italic> experiments, cell aliquots were thawed at 37°C, added to 10 mL of RPMI 1640 and centrifuged at 1500 rpm for 5 min to eliminate DMSO. Pellet was resuspended in RPMI 1640 supplemented with 10% of FBS.</p></sec><sec id="S2-6"><title><italic>In vitro</italic> stimulation of T cells and co-culture with exosomes</title><p>To determine the immunomodulatory effect of exo-hASCs on <italic>in vitro</italic> stimulated PBLs, 2 × 10<sup>5</sup> purified PBLs were seeded in a 96 wells plate (200 μl per well). To stimulate PBLs, a T cell activation/expansion kit (Miltenyi Biotec Inc, San Diego, CA, USA) was used, adding 5 μL of microbeads coated with anti-CD2/anti-CD3/anti-CD28 to each well. Finally, exosomes at different concentrations (4, 8, and 16 μg/10<sup>6</sup> PBLs) were added to wells. The PBLs were cultured for 6 days. Negative controls (non-stimulated PBLs) and positive controls (stimulated PBLs without exosomes) were used in all the experiments.</p></sec><sec id="S2-7"><title>CFSE proliferation assay</title><p>The proliferative behavior of T cells was quantified by carboxyfluorescein succinimidyl ester (CFSE) dilution. The CFSE staining was performed before seeding, using the CFSE cell proliferation kit (Invitrogen, Eugene, OR) at a final concentration of 10 μM for 10 min at 37°C, followed by immediate quenching with culture medium. After 6 days, <italic>in vitro</italic> stimulated PBLs in the presence or absence of exo-hASCs were tested for CFSE dilution by flow cytometry.</p></sec><sec id="S2-8"><title>Differentiation/activation markers expression analysis on <italic>in vitro</italic> stimulated PBLs</title><p>For flow cytometric analysis of <italic>in vitro</italic> stimulated PBLs, the cells were collected from wells after 6 days by pipetting up and down. The cells were stained with fluorescence-labeled human monoclonal antibodies against CD3 (SK7), CD4 (SK3), CD8 (SK1), CCR7 (3D12), CD45RA (L48) (BD Biosciences, San Jose, CA, USA). The markers expression analysis was performed as follows: 2 × 10<sup>5</sup> cells were incubated for 30 min at 4°C with appropriate concentrations of monoclonal antibodies in the presence of PBS containing 2% FBS. The cells were washed and resuspended in PBS. The flow cytometric analysis was performed on a FACScalibur cytometer (BD Biosciences, San Jose, CA, USA) after acquisition of 10<sup>5</sup> events. Cells were primarily selected using forward and side scatter characteristics and fluorescence was analyzed using CellQuest software (BD Biosciences, San Jose, CA, USA). Isotype-matched negative control antibodies were used in all the experiments. The mean relative fluorescence intensity was calculated by dividing the mean fluorescent intensity (MFI) by the MFI of its negative control.</p></sec><sec id="S2-9"><title>Intracellular gamma-interferon assay</title><p>For IFN-γ assays, the PBLs were <italic>in vitro</italic> stimulated with the T cell activation/expansion kit (Miltenyi Biotec Inc, San Diego, CA, USA) for 6 days in the presence of exo-hASCs at 16 μg/10<sup>6</sup> PBLs. The PBLs were then incubated for 6 h with BD GolgiStop. PBLs were stained with PerCP-labeled anti-CD4 (SK3) and APC-labeled anti-CD8 (SK1), fixed and permeabilized using BD Cytofix/Cytoperm fixation/permeabilization kit. Finally, cells were stained with PE-labeled anti-IFN-γ antibody (all reagents from BD Biosciences, San Jose, CA, USA). Analysis by flow cytometry was performed by measuring the frequency of IFN-γ expression on gated CD3<sup>+</sup>CD4<sup>+</sup> and CD3<sup>+</sup>CD8<sup>+</sup> cells.</p></sec><sec id="S2-10"><title>Statistical analysis</title><p>Data were statistically analyzed using the Student’s <italic>t</italic>-test for variables with parametric distribution. For the proliferation assay, an ANOVA with <italic>post hoc</italic> Bonferroni test was performed. The <italic>p</italic>-values ≤0.10 or ≤0.05 were considered statistically significant. All the statistical determinations were made using SPSS-21 software (SPSS, Chicago, IL, USA).</p></sec></sec><sec id="S3"><title>Results</title><sec id="S3-11"><title>Size distribution and concentration of exo-hASCs</title><p>An enriched fraction of exosomes was collected from hASCs by ultracentrifugation. The protein concentration of exosomes was determined by Bradford assay. Three independently performed nanoparticle tracking analysis were performed for each exosome sample to quantify size distribution and particle concentration. Firstly, the total protein concentration allowed us to quantify exosomes for <italic>in vitro</italic> assays. Secondly, the nanoparticle tracking analysis allowed us to characterize the released vesicles. The size of isolated vesicles ranged from 223 to 282 nm and the mean size and standard deviation was 246.8 ± 25.05 nm. Representative results of exo-hASCs are displayed as a frequency size distribution graph (Figure <xref ref-type="fig" rid="F1">1</xref>). The corresponding nanoparticle tracking analysis video frame is included as Video <xref ref-type="supplementary-material" rid="SM1">S1</xref> in Supplementary Material. Finally, the concentration of exosomes (<italic>n</italic> = 6) was determined by nanoparticle tracking analysis and ranged between 8.4 and 9.7 (×10<sup>9</sup>) particles per milliliter and the mean concentration was 9.1 ± 0.5 (×10<sup>9</sup>) particles per milliliter.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p><bold>Frequency size distribution graph of exo-hASCs</bold>. The nanoparticle tracking analysis was performed on exosome samples to quantify size distribution and particle concentration (<italic>n</italic> = 6). A representative graph of nanoparticle tracking analysis is shown.</p></caption><graphic xlink:href="fimmu-05-00556-g001"/></fig></sec><sec id="S3-12"><title>Proliferative ability of <italic>in vitro</italic> stimulated T cells co-cultured in the presence of exo-hASCs</title><p>In order to assess the biological activity of exo-hASCs, we aimed to determine their effect over the proliferation rate of lymphocyte subsets. For that, a total of 2 × 10<sup>6</sup> PBLs were stimulated with anti-CD2/anti-CD3/anti-CD28 as described in Section “<xref ref-type="sec" rid="S2">Materials and Methods</xref>” and co-cultured with different concentrations of exo-hASCs (4, 8, and 16 μg/10<sup>6</sup> PBLs) during 6 days. The proliferation ability was determined by CFSE dilution. Non-stimulated PBLs were used as negative control, and stimulated PBLs without exosomes constituted the positive control. As expected, the proliferation rate of non-stimulated PBLs was very low (data not shown) and the maximum proliferation rate was reached by stimulated PBLs without exosomes. A total of eight cell divisions were detected by CFSE fluorescence. As shown in the Figure <xref ref-type="fig" rid="F2">2</xref>A, when <italic>in vitro</italic> stimulated lymphocytes were cultured in the presence of different concentrations of exo-hASCs, the proliferation rate was proportionally decreased both in CD4<sup>+</sup> and CD8<sup>+</sup> T cells. A large percentage of cells presented a low number of cell divisions, while the highest number of cell divisions was reached by a lower percentage of cells. A detailed representation showing the percentage of cells in each division cycle is provided in the Figure <xref ref-type="fig" rid="F2">2</xref>A. A representative histogram (Figure <xref ref-type="fig" rid="F2">2</xref>B) and a detailed representation showing the percentage of cells in each division cycle is also provided (Figure <xref ref-type="fig" rid="F2">2</xref>C).</p><fig id="F2" position="float"><label>Figure 2</label><caption><p><bold>The proliferative ability of <italic>in vitro</italic> stimulated PBLs is reduced by exo-hASCs</bold>. The PBLs were cultured either alone or co-cultured with different batches of exo-hASCs (<italic>n</italic> = 8) at different concentrations (4, 8, and 16 μg of exosomes per million of PBLs). At day six, PBLs were collected and T-lymphocytes subsets were stained with anti-CD3, anti-CD4, and anti-CD8. Fluorescence profiles of CFSE-labeled cells allowed us to identify eight divisions. A detailed representation of CD4<sup>+</sup> T cells and CD8<sup>+</sup> T cells showing the percentage of the total population in each cell division cycle (indicated as #) is provided <bold>(A)</bold>, as well as a representative histogram <bold>(B)</bold>. The statistical comparison of lymphocyte subsets at different cell division cycles is also provided <bold>(C)</bold>. Horizontal bars represent statistically significant differences between the groups (significant at <italic>p</italic> ≤ 0.05).</p></caption><graphic xlink:href="fimmu-05-00556-g002"/></fig><p>Here, it can be seen how increasing concentrations of exosomes are arresting both CD4 and CD8 proliferation from eight generations to seven. Moreover exosomes are retaining the cells in the earlier division cycles 4, 5, and 6, in where the percentage of cells are significantly higher in the presence of exosomes, however, division cycles 7 and 8 have a significantly reduced percentage of cells when higher doses of exosomes were used. The first two division cycles contain a very low percentage of T cells both in the presence or absence of exosomes indicating that the effect of the polyclonal stimulation starts after these two division cycles, nevertheless the presence of exosomes are still significantly retaining cells in these firs two division cycles (although this is happening in a group of T cells below 10%). The statistical analysis showed that, significant differences were found in different division cycles either in CD4<sup>+</sup> and CD8<sup>+</sup> T cells. Finally, the stimulation index was calculated on CD4<sup>+</sup> and CD8<sup>+</sup> T cells as frequencies of CFSE-low T cells among unstimulated T cells. The stimulation index of CD4<sup>+</sup> and CD8<sup>+</sup> T cells stimulated with anti-CD2/anti-CD3/anti-CD28 was 692.3 and 655.6, respectively. However, when PBLs were stimulated in the presence of exosomes, the stain index significantly decreased on CD4<sup>+</sup> T cells (589.93 ± 39.31, 585 ± 80.27, 529.14 ± 58.88 at 4, 8, and 16 μg) as well as in CD8<sup>+</sup> T cells (519.75 ± 60.97, 488.03 ± 107.32, 437.4 ± 79.25 at 4, 8, and 16 μg).</p></sec><sec id="S3-13"><title>T cells subsets distribution of <italic>in vitro</italic> stimulated T cells co-cultured in the presence of exo-hASCs</title><p>The CD45RA isoform and chemokine receptor CCR7 are surface marker commonly used to identify the differentiation stages of CD4<sup>+</sup> and CD8<sup>+</sup> T cells. In order to study the effect of exo-hASCs over lymphocyte subsets, a total of 2 × 10<sup>6</sup> stimulated PBLs were cultured in the presence of exo-hASCs (from two different donors) at 16 μg/10<sup>6</sup> PBLs. At day 6, flow cytometry was performed using a commercial antibody against CD45RA and CCR7. The quantitative expression of CD45RA and CCR7 was normalized referred to control (<italic>in vitro</italic> stimulated T cells in the absence of exo-hASCs). Our results showed a significant decrease CD45RA<sup>+</sup> and CCR7<sup>+</sup> cells both in the CD4<sup>+</sup> and CD8<sup>+</sup> T cells in the positive control (stimulated PBLs). However, the loss of CD45RA and CCR7 on <italic>in vitro</italic> stimulated PBLs was partially compensated by the presence of exo-hASCs (Figure <xref ref-type="fig" rid="F3">3</xref>). Representative histograms are provided in Figure <xref ref-type="supplementary-material" rid="SM2">S1</xref> in Supplementary Material.</p><fig id="F3" position="float"><label>Figure 3</label><caption><p><bold>Percentage of CD45RA and CCR7 expression on <italic>in vitro</italic> stimulated T cells co-cultured in the presence of exo-hASCs</bold>. At day 6, <italic>in vitro</italic> stimulated PBLs were analyzed for CD45RA and CCR7 on CD8<sup>+</sup> and CD4<sup>+</sup> T cell subsets. Two different exo-hASCs at 16 μg/10<sup>6</sup> cells from different donors were used in these experiments (Exos#1 and Exos#2). The graphs show the normalized quantitative expression referred to control (<italic>in vitro</italic> stimulated T cells in the absence of exo-hASCs). Values shown in the bars represent mean ± SD of three independently performed experiments. Horizontal bars represent statistically significant differences between the stimulated PBLs groups (significant at <italic>p</italic> ≤ 0.1).</p></caption><graphic xlink:href="fimmu-05-00556-g003"/></fig><p>In the model proposed by Lanzavecchia and Sallusto, four different stages have been defined within CD8<sup>+</sup> T cells according to the combined analysis of CD45RA and CCR7 expression, namely: naïve (CD45RA<sup>+</sup>CCR7<sup>+</sup>), central memory (CD45RA<sup>−</sup>CCR7<sup>+</sup>) and at least two subset of effector-memory cells: effector-memory cells (CD45RA<sup>−</sup>CCR7<sup>−</sup>) and terminally differentiated effector-memory cells (CD45RA<sup>+</sup>CCR7<sup>−</sup>) (<xref rid="B26" ref-type="bibr">26</xref>, <xref rid="B27" ref-type="bibr">27</xref>). To study the effect of exo-hASCs over this distribution, the co-expression of CD45RA and CCR7 was analyzed by flow cytometry on CD4<sup>+</sup> and CD8<sup>+</sup> T cell subsets. As shown in Figure <xref ref-type="fig" rid="F4">4</xref>, although the percentage of naïve cells was not significantly modified by the presence of exo-hASCs, a significant decrease of terminally differentiated effector-memory cells (CD45RA<sup>+</sup>CCR7<sup>−</sup>) was observed on <italic>in vitro</italic> stimulated CD8<sup>+</sup> T cells cultured in the presence of exo-hASCs. In the case of CD4<sup>+</sup> T cells, exo-hASCs reduced the percentage of effector-memory cells (CD45RA<sup>−</sup>CCR7<sup>−</sup>) and significantly increased the percentage of central memory cells (CD45RA<sup>−</sup>CCR7<sup>+</sup>).</p><fig id="F4" position="float"><label>Figure 4</label><caption><p><bold>CD45RA and CCR7 co-expression on <italic>in vitro</italic> stimulated T cells co-cultured in the presence of exo-hASCs</bold>. At day 6, <italic>in vitro</italic> stimulated PBLs were analyzed for the co-expression of CD45RA and CCR7. The CD45RA isoform and CCR7 distinguishes four subsets of T cells: terminally differentiated RA<sup>+</sup> T cells (TEMRA, CD45RA<sup>+</sup> CCR7<sup>−</sup>), naïve T cells (NAIVE, CCR7<sup>+</sup> CD45RA<sup>+</sup>), and two memory subsets: effector memory (EM, CD45RA<sup>−</sup> CCR7<sup>−</sup>) and central memory (CM, CD45RA<sup>−</sup> CCR7<sup>+</sup>). Two different exosomes from different donors were used in these experiments (Exos#1 and Exos#2). Values shown represent mean ± SD of 3 independently performed experiments.</p></caption><graphic xlink:href="fimmu-05-00556-g004"/></fig></sec><sec id="S3-14"><title>IFN-γ production on <italic>in vitro</italic> stimulated T cells co-cultured in the presence of human adipose mesenchymal stem cells derived exosomes</title><p>The IFN-γ is a pro-inflammatory cytokine secreted by immune cells under certain conditions of activation. There is a direct correlation between IFN-γ secretion and the level of T cell activation. In order to determine the effect of exosomes on the secretory IFN-γ response of T cells, PBLs were cultured in the presence and absence of exo-hASCs during 6 days and intracellular levels of IFN-γ were determined on CD4<sup>+</sup> and CD8<sup>+</sup> T cell subsets. Our results showed that, at day 6, the percentage of intracellular IFN-γ was reduced when PBLs were cultured with exosomes, in comparison to positive control, in both T cell subsets. However, this reduction was only statistically significant on gated CD4<sup>+</sup> T cells (Figure <xref ref-type="fig" rid="F5">5</xref>). These results demonstrated that exo-hASCs impaired not only the differentiation phenotype of lymphocytes but also their IFN-γ secretion.</p><fig id="F5" position="float"><label>Figure 5</label><caption><p><bold>The exo-hASCs inhibit the IFN-γ production of <italic>in vitro</italic> stimulated T cells</bold>. Two different exosomes from different donors were used in these experiments (Exos#1 and Exos#2). Graphs represent the mean ± SD of 3 independently performed experiments. A representative dot plot of each condition is represented below each graph, and numbers in the quadrants indicate the percentage of IFN-γ in gated CD4<sup>+</sup>
<bold>(A)</bold> and CD8<sup>+</sup> T cells <bold>(B)</bold> (significant at <italic>p</italic> ≤ 0.05).</p></caption><graphic xlink:href="fimmu-05-00556-g005"/></fig></sec></sec><sec sec-type="discussion" id="S4"><title>Discussion</title><p>The hASCs have been successfully used for the treatment of numerous diseases (<xref rid="B28" ref-type="bibr">28</xref>). These cells can be isolated from the adult liposuctioned tissue and efficiently expanded <italic>in vitro</italic> to be used as an “off-the-shelf” cellular product. At the present, there are quite a few stem cell products in the market (<xref rid="B29" ref-type="bibr">29</xref>) but hASCs are currently considered one of the most promising cell types for therapeutic applications that fulfill regulatory requirements.</p><p>The hASCs have immunomodulatory properties, which are directly mediated by cell–cell contact or indirectly mediated through the release of immunosuppressive factors. The release of these factors is enhanced under inflammatory conditions and results in suppression of T cell function (proliferation, differentiation, and cytotoxicity) (<xref rid="B30" ref-type="bibr">30</xref>), B cell functions (<xref rid="B31" ref-type="bibr">31</xref>), NK cell cytotoxicity (<xref rid="B20" ref-type="bibr">20</xref>), decrease in maturation, and activation of dendritic cells (<xref rid="B32" ref-type="bibr">32</xref>) as well as an increase of regulatory T cells (<xref rid="B33" ref-type="bibr">33</xref>, <xref rid="B34" ref-type="bibr">34</xref>).</p><p>Among the released factors, the exosomes from MSCs have been considered as a promising candidate for a novel cell-free therapy. At the present, these microvesicles have been tested in preclinical settings for the treatment of cardiovascular diseases (<xref rid="B35" ref-type="bibr">35</xref>), kidney injury (<xref rid="B36" ref-type="bibr">36</xref>), graft-versus-host disease (<xref rid="B37" ref-type="bibr">37</xref>), and neurological diseases (<xref rid="B38" ref-type="bibr">38</xref>). In clinical settings, the exo-MSCs have been tested in graft-versus-host disease patients, which experienced improvement in symptoms for 5 months (<xref rid="B39" ref-type="bibr">39</xref>).</p><p>In these studies, exosomes from MSCs have demonstrated a biological effect for repairing tissue damage. From an immunological point of view, these exosomes have demonstrated immunomodulatory properties inducing peripheral tolerance (<xref rid="B40" ref-type="bibr">40</xref>) and modulating the immune response (<xref rid="B41" ref-type="bibr">41</xref>).</p><p>In this work, we aimed to investigate the immunomodulatory role of an enriched fraction of exo-hASCs on T cell subsets under <italic>in vitro</italic> conditions. For this purpose, the T cells were <italic>in vitro</italic> expanded and activated with anti-CD2, anti-CD3, and anti-CD28 that partially mimic stimulation by antigen-presenting cells (<xref rid="B42" ref-type="bibr">42</xref>). The immunomodulation was assessed by measuring the proliferative behavior of T cells, their differentiation toward the memory lineage and IFN-γ secretory response.</p><p>Although this is a preliminary study, our results demonstrated that exo-hASCs significantly inhibited the proliferation of CD4 and CD8 T cells. These results are very similar to our previously published results using hASCs co-cultured with <italic>in vitro</italic> activated PBLs (<xref rid="B30" ref-type="bibr">30</xref>). Recent reports have demonstrated that TNF-α/NF-κB signaling in MSCs are required for the inhibition of T-cell proliferation (<xref rid="B43" ref-type="bibr">43</xref>). In this sense, future studies will be conducted to evaluate if the anti-proliferative activity of exo-hASCs could also be related with the activation of NF-κB.</p><p>Together with the inhibition of T cell proliferation, here we hypothesized that exo-hASCs may arrest the T cell differentiation toward effector or memory cell phenotypes. In order to confirm this hypothesis, <italic>in vitro</italic> activated lymphocytes were co-cultured in the presence of exo-hASCs and the differentiation profile of CD4<sup>+</sup> and CD8<sup>+</sup> T cells was monitored according to co-expression of CD45RA and CCR7 molecules. The combined usage of CD45RA and CCR7 allowed us the identification of different CD8<sup>+</sup> T cell subsets (<xref rid="B26" ref-type="bibr">26</xref>) and CD4<sup>+</sup> T cell subsets (<xref rid="B27" ref-type="bibr">27</xref>): naive (CCR7<sup>+</sup>CD45RA<sup>+</sup>), central memory (CCR7<sup>+</sup>CD45RA<sup>−</sup>), effector memory (CCR7<sup>−</sup>CD45RA<sup>−</sup>), and terminally differentiated effector-memory cells (CD45RA<sup>+</sup>CCR7<sup>−</sup>). Our results evidenced that exo-hASCs hamper the <italic>in vitro</italic> differentiation mediated by anti-CD3/CD2/CD28 stimuli. Actually, in the case of CD8<sup>+</sup> and CD4<sup>+</sup> T cells, exo-hASCs have an inhibitory effect in the differentiation of toward a terminally differentiated phenotype and effector-memory phenotype, respectively.</p><p>These <italic>in vitro</italic> results with exo-hASCs showed similarities with previous reports using MSCs and <italic>in vitro</italic> stimulated PBLs where the T lymphocytes showed reduced-memory responses after a tetanus toxoid boost (<xref rid="B44" ref-type="bibr">44</xref>). Current experiments are being conducted in a relevant animal model of T cell-mediated disease (collagen-induced arthritis experimental model for rheumatoid arthritis). Once completed, these results will give us a more complete understanding of exo-hASCs as a therapeutic agent for the control of local T cell responses.</p><p>Finally, the immunomodulatory activity of exo-hASCs was determined by measuring IFN-γ production on CD4<sup>+</sup> and CD8<sup>+</sup> T cells. These experiments confirmed that, similarly to <italic>in vitro</italic> experiments using hASCs, both in contact or separated by transwells (<xref rid="B30" ref-type="bibr">30</xref>), the IFN-γ production was significantly reduced by exo-hASCs. Considering that IFN-γ is crucial for protection against immune-mediated inflammatory disorders, we could assume that exo-hASCs could be used as ideal vehicles for a local immunosuppression. Moreover, in contrast to cell therapy, where the viability, homing, or implantation of individual cells is compromised, the usage of well-characterized exo-hASCs in a dosing regimen that can be controlled and defined in space and time could be considered an advantage (<xref rid="B45" ref-type="bibr">45</xref>). Additionally, several authors have reported the susceptibility of allogeneic cells to CD8<sup>+</sup> T cells and NK cells, which is an important issue for the clinical efficacy of MSCs (<xref rid="B46" ref-type="bibr">46</xref>). In the case of exo-hASCs, these microvesicles will not be affected by cell-mediated lysis, which is an advantage for their therapeutic effectiveness.</p><p>An important aspect to be discussed here is the role of MHC molecules on exosomes. It has been previously described by Pêche et al. that, exosomes from bone marrow dendritic cells (exo-DCs) induce regulatory responses and allograft tolerance through the presentation of donor-MHC antigens (<xref rid="B10" ref-type="bibr">10</xref>). These exo-DCs are positives for MHC class I, MHC class II, and co-stimulatory molecules and the <italic>in vivo</italic> administration of allogeneic exosomes induced tolerance (<xref rid="B11" ref-type="bibr">11</xref>).</p><p>Contrary to exo-DCs, the exo-hASCs are characterized by the absence of MHC class II and co-stimulatory molecules. Regarding to MHC class I, the hASCs cell lines contains very low levels of MHC class I and the expression of this molecule in exo-hASCs is still under debate. Indeed, in Exocarta (a database of exosomal proteins, <uri xlink:type="simple" xlink:href="http://www.exocarta.org/">http://www.exocarta.org/</uri>), the presence of HLA-A, HLA-B, or HLA-C has not been detected in the 939 proteins analyzed from exo-MSCs. However, the proteomic analysis of exosomes from human embryonic stem cell-derived MSCs have demonstrated the presence of HLA-A molecules in these vesicles (<xref rid="B47" ref-type="bibr">47</xref>).</p><p>On the other hand, considering that human clinical trials using allogeneic hASC is not causing early, aggressive immunological rejection, we could assume that allogeneic exo-hASCs from non-activated hASCs might have similar consequences. The absence of MHC class II and co-stimulatory molecules on exo-hASCs may also indicate that these vesicles have a direct inhibitory effect on T cells being independent from antigen presentation. Nevertheless, although the inhibitory effect was only tested in allogeneic setting (using exo-hASCs from clinically established hASCs), future experiments are being conducted to determine if inhibitory effect is stronger in autologous T cells.</p><p>The limitations of the study are evident as this paper has been purely focused on the <italic>in vitro</italic> role of exo-hASCs against <italic>in vitro</italic> activated T cell subsets, and future experiments should confirm these observations on <italic>in vivo</italic> animal models. These <italic>in vivo</italic> animal models will help us to define the optimal the route, dosing and frequencies of exo-hASCs administration.</p><p>In summary, this paper provides a better understanding of exo-hASCs for their future applicability in clinical practice. Moreover, in terms of immunomodulation, our <italic>in vitro</italic> results demonstrated a parallelism between hASCs and exo-hASCs.</p></sec><sec id="S5"><title>Conflict of Interest Statement</title><p>Olga de la Rosa and Wilfried Dalemans are full time employees of TiGenix. The other co-authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec><sec sec-type="supplementary-material" id="S6"><title>Supplementary Material</title><p>The Supplementary Material for this article can be found online at <uri xlink:type="simple" xlink:href="http://www.frontiersin.org/Journal/10.3389/fimmu.2014.00556/abstract">http://www.frontiersin.org/Journal/10.3389/fimmu.2014.00556/abstract</uri></p><supplementary-material content-type="local-data" id="SM1"><label>Supplementary Video S1</label><caption><p><bold>Nanoparticle tracking analysis video frame</bold>. A representative video of nanoparticle tracking analysis of exo-hASCs is shown.</p></caption><media xlink:href="Video_1.WMV"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="SM2"><label>Supplementary Figure S1</label><caption><p><bold>Percentage of CD45RA and CCR7 expression on in vitro stimulated T cells co-cultured in the presence of exo-hASCs</bold>. At day 6, <italic>in vitro</italic> stimulated PBLs were analyzed for CD45RA and CCR7 on CD8<sup>+</sup> and CD4<sup>+</sup> T cell subsets. Two different exo-hASCs at 16 μg/10<sup>6</sup> cells from different donors were used in these experiments (Exos#1 and Exos#2). The figure shows representative histograms from the three different experiments. Percentage of positive cells is shown in each histogram.</p></caption><media xlink:href="Image_1.TIF"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec> |
Moving towards better cause of death registration in Africa and Asia | Could not extract abstract | <contrib contrib-type="author"><name><surname>Boerma</surname><given-names>Ties</given-names></name><role>Director</role></contrib> | Global Health Action | <p>Mortality statistics by age, sex, and cause of death are the foundations of public health programmes. They are essential for planning, programming, and monitoring progress. Ideally, mortality statistics should be available for small areas, to detect differences in the distribution of mortality risks and plan health programmes accordingly. Yet, the majority of countries in sub-Saharan Africa and Asia do not have reliable mortality statistics by age, sex, and cause.</p><p>The main reason is that national civil registration and vital statistics (CRVS) systems function poorly in most low- and middle-income countries. Only a small proportion of deaths are registered and reported. An even smaller proportion of deaths is medically certified with a cause of death using the World Health Organization's International Classification of Diseases (ICD).</p><p>Special efforts are needed to fill this critical information gap in countries. Some countries have succeeded in improving cause of death data that are generated in hospitals. This is an important starting point but will only partially address the information deficit as in most countries less than one-fifth of deaths occur in hospitals. The cause patterns of hospital deaths are also likely to differ from those in the community. Household surveys can provide an important vehicle for collecting retrospective information on mortality levels, trends, and differentials. Verbal autopsies (carrying out interviews with witnesses of deaths to estimate likely causes) provide a strategy for documenting deaths that are not otherwise certified. Some countries have included verbal autopsy modules in household surveys to obtain a general picture of the causes of death.</p><p>Local health and demographic surveillance system (HDSS) sites are another important source of mortality statistics. Their strength lies in the quality of the information generated and the measurement of trends over time. This volume of <italic>Global Health Action</italic> presents a unique collection of verbal autopsies conducted in 22 different HDSS sites that are part of the INDEPTH Network in 13 countries in sub-Saharan Africa and south and south-east Asia. To my knowledge, the combined total of almost 100,000 verbal autopsies, mostly conducted since 2006, presents the largest data set of this nature ever and provides a nice overview of leading causes of death in mostly rural settings in low- and middle-income countries.</p><p>In 2006, a predecessor of this set of papers was published, based on 38,000 verbal autopsies from 12 INDEPTH sites (<xref rid="CIT0001" ref-type="bibr">1</xref>). At the time, considerable resources had to be spent to maximise the comparability of the data between study sites, and physicians were used to ascertain the probable cause of death. In this volume of <italic>Global Health Action</italic>, the application of the WHO 2012 verbal autopsy standard (<xref rid="CIT0002" ref-type="bibr">2</xref>) and the use of automated assignment of cause of death using the InterVA-4 model (<xref rid="CIT0003" ref-type="bibr">3</xref>) ensure more comparable and consistent results. In addition, the replicability and opportunities for further analyses are enhanced by the recent commendable actions of the INDEPTH Network to put cause of death and other data sets into the public domain.</p><p>The work of INDEPTH on mortality and causes of death needs to be considered in the context of the need to strengthen country CRVS systems. Increasingly, it is realised that strong CRVS systems are critical for good governance, establishing people's rights and those of their children. It should be a central element in the post-2015 development agenda. The health sector would be a beneficiary of complete and reliable CRVS systems, but it also needs to make a substantial contribution to the recording and reporting of events in a CRVS system (<xref rid="CIT0004" ref-type="bibr">4</xref>). The HDSS sites should play important roles in strengthening of CRVS systems. This includes the sharing of their experiences with often innovative methods of data collection on vital events, such as the use of verbal autopsy or technologies; the national capacity strengthening related to generating vital statistics; and the assessment of completeness of an expanding national CRVS system within HDSS populations. This special collection of papers on mortality and causes of death using verbal autopsy presents a significant example of how INDEPTH HDSS sites can contribute to stronger CRVS systems in countries.</p><sig-block><sig><italic>Ties Boerma</italic><break/>Director<break/>Health Statistics and Information Systems<break/>World Health Organization<break/>Geneva, Switzerland<break/></sig></sig-block> |
Two decades of mortality change in rural northeast South Africa | <sec id="st1"><title>Background</title><p>The MRC/Wits University Agincourt research centre, part of the INDEPTH Network, has documented mortality in a defined population in the rural northeast of South Africa for 20 years (1992–2011) using long-term health and socio-demographic surveillance. Detail on the unfolding, at times unpredicted, mortality pattern has been published. This experience is reviewed here and updated using more recent data.</p></sec><sec id="st2"><title>Objective</title><p>To present a review and summary of mortality patterns across all age-sex groups in the Agincourt sub-district population for the period 1992–2011 as a comprehensive basis for public health action.</p></sec><sec id="st3"><title>Design</title><p>Vital events in the Agincourt population have been updated in annual surveys undertaken since 1992. All deaths have been rigorously recorded and followed by verbal autopsy interviews. Responses to questions from these interviews have been processed retrospectively using the WHO 2012 verbal autopsy standard and the InterVA-4 model for assigning causes of death in a standardised manner.</p></sec><sec id="st4"><title>Results</title><p>Between 1992 and 2011, a total of 12,209 deaths were registered over 1,436,195 person-years of follow-up, giving a crude mortality rate of 8.5 per 1,000 person-years. During the 20-year period, the population experienced a major HIV epidemic, which resulted in more than doubling of overall mortality for an extended period. Recent years show signs of declining mortality, but levels remain above the 1992 baseline recorded using the surveillance system.</p></sec><sec id="st5"><title>Conclusions</title><p>The Agincourt population has experienced a major mortality shock over the past two decades from which it will take time to recover. The basic epidemic patterns are consistent with generalised mortality patterns observed in South Africa as a whole, but the detailed individual surveillance behind these analyses allows finer-grained analyses of specific causes, age-related risks, and trends over time. These demonstrate the complex, somewhat unpredicted course of mortality transition over the years since the dawn of South Africa's democratic era in 1994.</p></sec> | <contrib contrib-type="author"><name><surname>Kabudula</surname><given-names>Chodziwadziwa W.</given-names></name><xref ref-type="aff" rid="AF0001">1</xref><xref ref-type="aff" rid="AF0002">2</xref><xref ref-type="corresp" rid="cor1">*</xref></contrib><contrib contrib-type="author"><name><surname>Tollman</surname><given-names>Stephen</given-names></name><xref ref-type="aff" rid="AF0001">1</xref><xref ref-type="aff" rid="AF0002">2</xref><xref ref-type="aff" rid="AF0003">3</xref></contrib><contrib contrib-type="author"><name><surname>Mee</surname><given-names>Paul</given-names></name><xref ref-type="aff" rid="AF0001">1</xref><xref ref-type="aff" rid="AF0003">3</xref></contrib><contrib contrib-type="author"><name><surname>Ngobeni</surname><given-names>Sizzy</given-names></name><xref ref-type="aff" rid="AF0001">1</xref></contrib><contrib contrib-type="author"><name><surname>Silaule</surname><given-names>Bernard</given-names></name><xref ref-type="aff" rid="AF0001">1</xref></contrib><contrib contrib-type="author"><name><surname>Gómez-Olivé</surname><given-names>F. Xavier</given-names></name><xref ref-type="aff" rid="AF0001">1</xref><xref ref-type="aff" rid="AF0002">2</xref></contrib><contrib contrib-type="author"><name><surname>Collinson</surname><given-names>Mark</given-names></name><xref ref-type="aff" rid="AF0001">1</xref><xref ref-type="aff" rid="AF0002">2</xref><xref ref-type="aff" rid="AF0003">3</xref></contrib><contrib contrib-type="author"><name><surname>Kahn</surname><given-names>Kathleen</given-names></name><xref ref-type="aff" rid="AF0001">1</xref><xref ref-type="aff" rid="AF0002">2</xref><xref ref-type="aff" rid="AF0003">3</xref></contrib><contrib contrib-type="author"><name><surname>Byass</surname><given-names>Peter</given-names></name><xref ref-type="aff" rid="AF0001">1</xref><xref ref-type="aff" rid="AF0002">2</xref><xref ref-type="aff" rid="AF0003">3</xref></contrib> | Global Health Action | <p>The Agincourt Health and Socio-Demographic Surveillance System (HDSS) of the MRC/Wits Rural Public Health and Health Transitions Research Unit is an INDEPTH member site situated in the northeast of South Africa. The HDSS has been documenting mortality in a rural population since 1992. As of 2011, the population comprised some 90,000 individuals residing in 16,000 households in 27 villages (<xref rid="CIT0001" ref-type="bibr">1</xref>). This paper both reviews the published outputs from Agincourt HDSS relating to mortality and presents detailed overall results based on the 20 year period 1992–2011, as contributed to the INDEPTH Network pooled cause of death analyses covering the Agincourt HDSS and 21 other INDEPTH HDSS sites (<xref rid="CIT0002" ref-type="bibr">2</xref>).</p><p>Over the two decades the Agincourt site has operated, more than 40 papers have reported on particular aspects of mortality patterns and transitions, and related issues. Several papers have taken an overview of mortality at different points in time. The first signs of a reversal of mortality declines were documented at an early stage of the local HIV/AIDS epidemic (<xref rid="CIT0003" ref-type="bibr">3</xref>). Subsequently, the complexities of HIV and TB co-infection were also documented (<xref rid="CIT0004" ref-type="bibr">4</xref>), alongside South Africa's new struggle with increasing mortality (<xref rid="CIT0005" ref-type="bibr">5</xref>). Throughout, mortality patterns have been seen to be shaped by competing forces of: 1) HIV/AIDS, 2) other communicable and nutritional diseases, 3) non-communicable diseases, and 4) violence and injuries (<xref rid="CIT0006" ref-type="bibr">6</xref>, <xref rid="CIT0007" ref-type="bibr">7</xref>). Given the high proportions of HIV and TB-related mortality in this population, this has been perceived as a major issue (<xref rid="CIT0008" ref-type="bibr">8</xref>–<xref rid="CIT0011" ref-type="bibr">11</xref>). Maternal health and fertility, including the effects of the HIV epidemic, have also been important issues (<xref rid="CIT0012" ref-type="bibr">12</xref>–<xref rid="CIT0014" ref-type="bibr">14</xref>). Child mortality has been explored in relation to various risk factors (<xref rid="CIT0015" ref-type="bibr">15</xref>) and adult mortality similarly (<xref rid="CIT0016" ref-type="bibr">16</xref>). The relationship between cause of death and beliefs in witchcraft has also been explored (<xref rid="CIT0017" ref-type="bibr">17</xref>), as well as mortality related to sleep disorders (<xref rid="CIT0018" ref-type="bibr">18</xref>). Connections between mortality and migration, as geographic patterns of residence and work have gradually evolved in the post-apartheid era, have been shown to continue to be important (<xref rid="CIT0019" ref-type="bibr">19</xref>–<xref rid="CIT0021" ref-type="bibr">21</xref>). Finally, there has been a substantial volume of work that has capitalised on the rich detail of the Agincourt HDSS data in terms of undertaking spatio-temporal analyses of mortality patterns (<xref rid="CIT0022" ref-type="bibr">22</xref>–<xref rid="CIT0031" ref-type="bibr">31</xref>).</p><p>At the same time as measuring mortality patterns, the MRC/Wits Agincourt Research Unit has also served as something of a methodological incubator for mortality surveillance, starting well before the INDEPTH Network was founded in 1998. From the initial descriptions of the Agincourt HDSS and its methods (<xref rid="CIT0032" ref-type="bibr">32</xref>, <xref rid="CIT0033" ref-type="bibr">33</xref>), a focus on determining cause of death became an important theme (<xref rid="CIT0034" ref-type="bibr">34</xref>–<xref rid="CIT0037" ref-type="bibr">37</xref>). Agincourt verbal autopsy (VA) data contributed to early work on automated cause of death assignment (<xref rid="CIT0038" ref-type="bibr">38</xref>, <xref rid="CIT0039" ref-type="bibr">39</xref>). It became clear that computer-coded VA methods were indeed able to track a mortality transition as dramatic as the evolving HIV epidemic in Agincourt (<xref rid="CIT0040" ref-type="bibr">40</xref>). Agincourt data again contributed substantially to the development and evaluation of the InterVA-4 VA interpretation model (<xref rid="CIT0041" ref-type="bibr">41</xref>) following the release of new WHO standards for VA in 2012 (<xref rid="CIT0042" ref-type="bibr">42</xref>). Pilot studies of handheld technology for undertaking VA interviews followed (<xref rid="CIT0043" ref-type="bibr">43</xref>).</p><p>Against this detailed and complex background of mortality levels and trends in the Agincourt HDSS, and methodological developments in automated cause of death assignment, we present here an overview of cause-specific mortality findings over the 20-year period 1992–2011 for all age groups.</p><sec sec-type="methods" id="S0002"><title>Methods</title><p>The MRC/Wits Agincourt Research Unit has maintained individual health and socio-demographic surveillance among a rural population in the Agincourt sub-district, in northeast South Africa, starting in 1992. A detailed description of the site has been presented elsewhere (<xref rid="CIT0001" ref-type="bibr">1</xref>). Annual household surveillance update rounds have identified all deaths occurring in the population, and these have been followed up with VA interviews, undertaken by specifically trained interviewers 1–11 months after death. Local physicians have assigned causes of death to the VA material, which has also more recently been processed retrospectively using the InterVA-4 probabilistic model (version 4.02) (<xref rid="CIT0041" ref-type="bibr">41</xref>). As far as is possible, the VA instrument and InterVA-4 model aim to arrive at underlying causes of death, but this is not always possible with certainty using VA methods. Nevertheless, it has been established that causes of death based on results generated by the InterVA-4 model do not differ substantially from physician findings (<xref rid="CIT0040" ref-type="bibr">40</xref>). In addition, the InterVA-4 model assigns causes of death in a standardised automated manner that is much quicker and more consistent than physician coding. Rates of cause-specific HIV associated mortality using the InterVA-4 model have been explored in a multisite study (<xref rid="CIT0044" ref-type="bibr">44</xref>).</p><p>The outputs from the InterVA-4 model, up to three causes for each case plus a possible indeterminate residual fraction, for Agincourt contained in the INDEPTH Network Cause-Specific Mortality multisite dataset (<xref rid="CIT0045" ref-type="bibr">45</xref>) have been analysed with the same age-sex-time standardisation used for the INDEPTH cross-site comparisons (<xref rid="CIT0046" ref-type="bibr">46</xref>). The standardisation was necessary because the age-sex population structure in the Agincourt site changed appreciably over the 20-year period (<xref rid="CIT0001" ref-type="bibr">1</xref>). Consequently, in considering changing patterns of causes of death, it is important to standardise the age-sex structure to ensure that changes observed in particular causes are not due to age-sex changes in the population over time. Because the site covers an entire defined population, it is not meaningful to ascribe confidence intervals, although individual-level uncertainty in cause of death is captured through the indeterminate component of the InterVA-4 output.</p><p>The Agincourt HDSS was reviewed and approved by the Committee for Research on Human Subjects (Medical) of the University of the Witwatersrand (protocol M960720 and M081145). Community consent from civic and traditional leadership was secured at the start of surveillance and is reaffirmed from time to time, and informed verbal consent is obtained at individual and household level at each annual follow-up visit. A record is kept of the household respondent who consented to be interviewed as well as the responsible fieldworker.</p></sec><sec sec-type="results" id="S0003"><title>Results</title><p>Over the period 1992–2011 a total of 12,209 deaths were registered over 1,436,195 person-years of follow-up, corresponding to a crude mortality rate of 8.5 per 1,000 person-years. After standardisation, these amounted to 13,153 deaths, of which 12,010 (91.3%) had VA interviews successfully completed. For 607 (5.1%) of these, the InterVA-4 model was unable to reach any conclusive cause of death.</p><p>
<xref ref-type="table" rid="T0001">Table 1</xref> presents detailed standardised mortality rates by age group and causes of death according to the WHO 2012 VA standard categories (<xref rid="CIT0047" ref-type="bibr">47</xref>). <xref ref-type="fig" rid="F0001">Figure 1</xref> shows the development of standardised mortality rates by cause categories over time, clearly showing the overall mortality epidemic peak associated with HIV in this population around 2007 and reduction in the most recent time period. <xref ref-type="fig" rid="F0002">Figure 2</xref> shows the same data for the separate WHO 2012 age groups (with neonatal and 1–11 month age groups combined into a single infancy category). In this figure, the vertical scales for mortality rates are appreciably different between the different age groups, with highest rates for infants and over-65 year olds, and lowest rates for the 5–14 year age group. All age groups except the over-65 year olds experienced reduction in mortality rate from around 2007. Mortality increased steadily in the over-65 year olds over the 20-year period. <xref ref-type="fig" rid="F0003">Figure 3</xref> shows trends in non-communicable disease (NCD) mortality in adult age groups. NCD mortality steadily increased in all adult age groups from the late 1990s and declined in the most recent time period only in the 15–49 year age group. The increase in NCD mortality in the 50–64 and 65+ year age groups are largely driven by increases in cardiovascular conditions (including stroke and acute cardiac disease) followed by respiratory conditions (including chronic obstructive pulmonary disease). In the 15–49 year age group, trends in NCD mortality mirror trends in HIV/AIDS mortality.</p><fig id="F0001" position="float"><label>Fig. 1</label><caption><p>Age-sex-time standardised mortality rates by broad cause categories, Agincourt HDSS.</p></caption><graphic xlink:href="GHA-7-25596-g001"/></fig><fig id="F0002" position="float"><label>Fig. 2</label><caption><p>Age-sex-time standardised mortality rates by broad cause categories and age group, Agincourt HDSS.</p></caption><graphic xlink:href="GHA-7-25596-g002"/></fig><fig id="F0003" position="float"><label>Fig. 3</label><caption><p>Age-sex-time standardised non-communicable disease (NCD) mortality in adult age groups, Agincourt HDSS.</p></caption><graphic xlink:href="GHA-7-25596-g003"/></fig><table-wrap id="T0001" position="float"><label>Table 1</label><caption><p>Age-sex-time standardised cause-specific mortality rates per 1,000 person-years by WHO 2012 causes and age group, for a total of 12,209 deaths registered over 1,436,195 person-years at the Agincourt HDSS, 1992–2011</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1">Cause of death</th><th align="center" rowspan="1" colspan="1">Neonate</th><th align="center" rowspan="1" colspan="1">1–11 mo</th><th align="center" rowspan="1" colspan="1">1–4 yr</th><th align="center" rowspan="1" colspan="1">5–14 yr</th><th align="center" rowspan="1" colspan="1">15–49 yr M</th><th align="center" rowspan="1" colspan="1">15–49 yr F</th><th align="center" rowspan="1" colspan="1">50–64 yr</th><th align="center" rowspan="1" colspan="1">65+ yr</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">01.01 Sepsis (non-obstetric)</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.601</td><td align="center" rowspan="1" colspan="1">0.006</td><td align="center" rowspan="1" colspan="1">0.001</td><td align="center" rowspan="1" colspan="1">0.004</td><td align="center" rowspan="1" colspan="1">0.003</td><td align="center" rowspan="1" colspan="1">0.028</td><td align="center" rowspan="1" colspan="1">0.109</td></tr><tr><td align="left" rowspan="1" colspan="1">01.02 Acute resp infect, incl pneumonia</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">10.563</td><td align="center" rowspan="1" colspan="1">0.788</td><td align="center" rowspan="1" colspan="1">0.124</td><td align="center" rowspan="1" colspan="1">0.268</td><td align="center" rowspan="1" colspan="1">0.275</td><td align="center" rowspan="1" colspan="1">1.102</td><td align="center" rowspan="1" colspan="1">3.177</td></tr><tr><td align="left" rowspan="1" colspan="1">01.03 HIV/AIDS related death</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">5.172</td><td align="center" rowspan="1" colspan="1">2.361</td><td align="center" rowspan="1" colspan="1">0.193</td><td align="center" rowspan="1" colspan="1">1.130</td><td align="center" rowspan="1" colspan="1">2.474</td><td align="center" rowspan="1" colspan="1">4.393</td><td align="center" rowspan="1" colspan="1">2.883</td></tr><tr><td align="left" rowspan="1" colspan="1">01.04 Diarrhoeal diseases</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">4.087</td><td align="center" rowspan="1" colspan="1">0.500</td><td align="center" rowspan="1" colspan="1">0.012</td><td align="center" rowspan="1" colspan="1">0.035</td><td align="center" rowspan="1" colspan="1">0.035</td><td align="center" rowspan="1" colspan="1">0.158</td><td align="center" rowspan="1" colspan="1">0.548</td></tr><tr><td align="left" rowspan="1" colspan="1">01.05 Malaria</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.461</td><td align="center" rowspan="1" colspan="1">0.233</td><td align="center" rowspan="1" colspan="1">0.052</td><td align="center" rowspan="1" colspan="1">0.057</td><td align="center" rowspan="1" colspan="1">0.078</td><td align="center" rowspan="1" colspan="1">0.094</td><td align="center" rowspan="1" colspan="1">0.109</td></tr><tr><td align="left" rowspan="1" colspan="1">01.06 Measles</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.196</td><td align="center" rowspan="1" colspan="1">0.015</td><td align="center" rowspan="1" colspan="1">0.003</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">01.07 Meningitis and encephalitis</td><td align="center" rowspan="1" colspan="1">1.849</td><td align="center" rowspan="1" colspan="1">0.196</td><td align="center" rowspan="1" colspan="1">0.068</td><td align="center" rowspan="1" colspan="1">0.009</td><td align="center" rowspan="1" colspan="1">0.020</td><td align="center" rowspan="1" colspan="1">0.023</td><td align="center" rowspan="1" colspan="1">0.024</td><td align="center" rowspan="1" colspan="1">0.011</td></tr><tr><td align="left" rowspan="1" colspan="1">01.09 Pulmonary tuberculosis</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.187</td><td align="center" rowspan="1" colspan="1">0.237</td><td align="center" rowspan="1" colspan="1">0.103</td><td align="center" rowspan="1" colspan="1">1.656</td><td align="center" rowspan="1" colspan="1">1.279</td><td align="center" rowspan="1" colspan="1">4.796</td><td align="center" rowspan="1" colspan="1">6.977</td></tr><tr><td align="left" rowspan="1" colspan="1">01.10 Pertussis</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.653</td><td align="center" rowspan="1" colspan="1">0.033</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">01.11 Haemorrhagic fever</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.006</td><td align="center" rowspan="1" colspan="1">0.005</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">01.99 Other and unspecified infect dis</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.121</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.018</td><td align="center" rowspan="1" colspan="1">0.021</td><td align="center" rowspan="1" colspan="1">0.013</td><td align="center" rowspan="1" colspan="1">0.097</td><td align="center" rowspan="1" colspan="1">0.212</td></tr><tr><td align="left" rowspan="1" colspan="1">02.01 Oral neoplasms</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.013</td><td align="center" rowspan="1" colspan="1">0.010</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.072</td></tr><tr><td align="left" rowspan="1" colspan="1">02.02 Digestive neoplasms</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.133</td><td align="center" rowspan="1" colspan="1">0.107</td><td align="center" rowspan="1" colspan="1">1.357</td><td align="center" rowspan="1" colspan="1">2.704</td></tr><tr><td align="left" rowspan="1" colspan="1">02.03 Respiratory neoplasms</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.096</td><td align="center" rowspan="1" colspan="1">0.084</td><td align="center" rowspan="1" colspan="1">0.879</td><td align="center" rowspan="1" colspan="1">2.384</td></tr><tr><td align="left" rowspan="1" colspan="1">02.04 Breast neoplasms</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.066</td><td align="center" rowspan="1" colspan="1">0.019</td><td align="center" rowspan="1" colspan="1">0.124</td></tr><tr><td align="left" rowspan="1" colspan="1">02.05, 02.06 Reproductive neoplasms M,F</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.014</td><td align="center" rowspan="1" colspan="1">0.084</td><td align="center" rowspan="1" colspan="1">0.133</td><td align="center" rowspan="1" colspan="1">0.730</td></tr><tr><td align="left" rowspan="1" colspan="1">02.99 Other and unspecified neoplasms</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.057</td><td align="center" rowspan="1" colspan="1">0.005</td><td align="center" rowspan="1" colspan="1">0.002</td><td align="center" rowspan="1" colspan="1">0.075</td><td align="center" rowspan="1" colspan="1">0.019</td><td align="center" rowspan="1" colspan="1">0.370</td><td align="center" rowspan="1" colspan="1">1.703</td></tr><tr><td align="left" rowspan="1" colspan="1">03.01 Severe anaemia</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.021</td><td align="center" rowspan="1" colspan="1">0.011</td></tr><tr><td align="left" rowspan="1" colspan="1">03.02 Severe malnutrition</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.321</td><td align="center" rowspan="1" colspan="1">0.214</td><td align="center" rowspan="1" colspan="1">0.005</td><td align="center" rowspan="1" colspan="1">0.005</td><td align="center" rowspan="1" colspan="1">0.002</td><td align="center" rowspan="1" colspan="1">0.037</td><td align="center" rowspan="1" colspan="1">0.361</td></tr><tr><td align="left" rowspan="1" colspan="1">03.03 Diabetes mellitus</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.030</td><td align="center" rowspan="1" colspan="1">0.013</td><td align="center" rowspan="1" colspan="1">0.008</td><td align="center" rowspan="1" colspan="1">0.065</td><td align="center" rowspan="1" colspan="1">0.050</td><td align="center" rowspan="1" colspan="1">0.757</td><td align="center" rowspan="1" colspan="1">3.249</td></tr><tr><td align="left" rowspan="1" colspan="1">04.01 Acute cardiac disease</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.023</td><td align="center" rowspan="1" colspan="1">0.006</td><td align="center" rowspan="1" colspan="1">0.204</td><td align="center" rowspan="1" colspan="1">0.355</td></tr><tr><td align="left" rowspan="1" colspan="1">04.02 Stroke</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.003</td><td align="center" rowspan="1" colspan="1">0.072</td><td align="center" rowspan="1" colspan="1">0.079</td><td align="center" rowspan="1" colspan="1">1.063</td><td align="center" rowspan="1" colspan="1">4.966</td></tr><tr><td align="left" rowspan="1" colspan="1">04.99 Other and unspecified cardiac dis</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.008</td><td align="center" rowspan="1" colspan="1">0.002</td><td align="center" rowspan="1" colspan="1">0.056</td><td align="center" rowspan="1" colspan="1">0.074</td><td align="center" rowspan="1" colspan="1">1.071</td><td align="center" rowspan="1" colspan="1">4.688</td></tr><tr><td align="left" rowspan="1" colspan="1">05.01 Chronic obstructive pulmonary dis</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.076</td><td align="center" rowspan="1" colspan="1">0.051</td><td align="center" rowspan="1" colspan="1">0.645</td><td align="center" rowspan="1" colspan="1">3.162</td></tr><tr><td align="left" rowspan="1" colspan="1">05.02 Asthma</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.090</td><td align="center" rowspan="1" colspan="1">0.034</td><td align="center" rowspan="1" colspan="1">0.058</td><td align="center" rowspan="1" colspan="1">0.110</td><td align="center" rowspan="1" colspan="1">0.626</td><td align="center" rowspan="1" colspan="1">1.444</td></tr><tr><td align="left" rowspan="1" colspan="1">06.01 Acute abdomen</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.375</td><td align="center" rowspan="1" colspan="1">0.083</td><td align="center" rowspan="1" colspan="1">0.010</td><td align="center" rowspan="1" colspan="1">0.045</td><td align="center" rowspan="1" colspan="1">0.054</td><td align="center" rowspan="1" colspan="1">0.357</td><td align="center" rowspan="1" colspan="1">0.913</td></tr><tr><td align="left" rowspan="1" colspan="1">06.02 Liver cirrhosis</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.043</td><td align="center" rowspan="1" colspan="1">0.009</td><td align="center" rowspan="1" colspan="1">0.019</td><td align="center" rowspan="1" colspan="1">0.016</td><td align="center" rowspan="1" colspan="1">0.138</td><td align="center" rowspan="1" colspan="1">0.327</td></tr><tr><td align="left" rowspan="1" colspan="1">07.01 Renal failure</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.016</td><td align="center" rowspan="1" colspan="1">0.006</td><td align="center" rowspan="1" colspan="1">0.004</td><td align="center" rowspan="1" colspan="1">0.013</td><td align="center" rowspan="1" colspan="1">0.003</td><td align="center" rowspan="1" colspan="1">0.070</td><td align="center" rowspan="1" colspan="1">0.208</td></tr><tr><td align="left" rowspan="1" colspan="1">08.01 Epilepsy</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.009</td><td align="center" rowspan="1" colspan="1">0.022</td><td align="center" rowspan="1" colspan="1">0.025</td><td align="center" rowspan="1" colspan="1">0.019</td><td align="center" rowspan="1" colspan="1">0.087</td><td align="center" rowspan="1" colspan="1">0.162</td></tr><tr><td align="left" rowspan="1" colspan="1">98 Other and unspecified NCD</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.036</td><td align="center" rowspan="1" colspan="1">0.026</td><td align="center" rowspan="1" colspan="1">0.169</td><td align="center" rowspan="1" colspan="1">1.204</td></tr><tr><td align="left" rowspan="1" colspan="1">10.06 Congenital malformation</td><td align="center" rowspan="1" colspan="1">0.753</td><td align="center" rowspan="1" colspan="1">0.294</td><td align="center" rowspan="1" colspan="1">0.015</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">10.01 Prematurity</td><td align="center" rowspan="1" colspan="1">12.115</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">10.02 Birth asphyxia</td><td align="center" rowspan="1" colspan="1">21.428</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">10.03 Neonatal pneumonia</td><td align="center" rowspan="1" colspan="1">42.198</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">10.04 Neonatal sepsis</td><td align="center" rowspan="1" colspan="1">5.590</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">10.99 Other and unspecified neonatal CoD</td><td align="center" rowspan="1" colspan="1">11.196</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">12.01 Road traffic accident</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.128</td><td align="center" rowspan="1" colspan="1">0.116</td><td align="center" rowspan="1" colspan="1">0.073</td><td align="center" rowspan="1" colspan="1">0.385</td><td align="center" rowspan="1" colspan="1">0.094</td><td align="center" rowspan="1" colspan="1">0.321</td><td align="center" rowspan="1" colspan="1">0.312</td></tr><tr><td align="left" rowspan="1" colspan="1">12.04 Accid drowning and submersion</td><td align="center" rowspan="1" colspan="1">0.384</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.015</td><td align="center" rowspan="1" colspan="1">0.036</td><td align="center" rowspan="1" colspan="1">0.011</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.015</td><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">12.05 Accid expos to smoke, fire & flame</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.073</td><td align="center" rowspan="1" colspan="1">0.028</td><td align="center" rowspan="1" colspan="1">0.015</td><td align="center" rowspan="1" colspan="1">0.013</td><td align="center" rowspan="1" colspan="1">0.014</td><td align="center" rowspan="1" colspan="1">0.08</td><td align="center" rowspan="1" colspan="1">0.175</td></tr><tr><td align="left" rowspan="1" colspan="1">12.07 Accid poisoning and noxious subs</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.023</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.009</td><td align="center" rowspan="1" colspan="1">0.001</td><td align="center" rowspan="1" colspan="1">0.003</td><td align="center" rowspan="1" colspan="1">0.016</td></tr><tr><td align="left" rowspan="1" colspan="1">12.08 Intentional self-harm</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.014</td><td align="center" rowspan="1" colspan="1">0.103</td><td align="center" rowspan="1" colspan="1">0.122</td><td align="center" rowspan="1" colspan="1">0.174</td><td align="center" rowspan="1" colspan="1">0.115</td></tr><tr><td align="left" rowspan="1" colspan="1">12.09 Assault</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.077</td><td align="center" rowspan="1" colspan="1">0.008</td><td align="center" rowspan="1" colspan="1">0.010</td><td align="center" rowspan="1" colspan="1">0.479</td><td align="center" rowspan="1" colspan="1">0.119</td><td align="center" rowspan="1" colspan="1">0.533</td><td align="center" rowspan="1" colspan="1">0.345</td></tr><tr><td align="left" rowspan="1" colspan="1">12.99 Other and unspecified external CoD</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.019</td><td align="center" rowspan="1" colspan="1">0.013</td><td align="center" rowspan="1" colspan="1">0.005</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.047</td><td align="center" rowspan="1" colspan="1">0.138</td></tr><tr><td align="left" rowspan="1" colspan="1">09.01 Ectopic pregnancy</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.002</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">09.02 Abortion-related death</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.001</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">09.03 Pregnancy-induced hypertension</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.015</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">09.04 Obstetric haemorrhage</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.053</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">09.05 Obstructed labour</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.004</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">09.06 Pregnancy-related sepsis</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.013</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">09.07 Anaemia of pregnancy</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.013</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">09.99 Other and unspecified maternal CoD</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.012</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">99 Indeterminate</td><td align="center" rowspan="1" colspan="1">30.952</td><td align="center" rowspan="1" colspan="1">6.298</td><td align="center" rowspan="1" colspan="1">1.241</td><td align="center" rowspan="1" colspan="1">0.252</td><td align="center" rowspan="1" colspan="1">1.244</td><td align="center" rowspan="1" colspan="1">0.919</td><td align="center" rowspan="1" colspan="1">4.855</td><td align="center" rowspan="1" colspan="1">12.914</td></tr><tr><td align="left" rowspan="1" colspan="1">All causes</td><td align="center" rowspan="1" colspan="1">126.465</td><td align="center" rowspan="1" colspan="1">29.906</td><td align="center" rowspan="1" colspan="1">6.177</td><td align="center" rowspan="1" colspan="1">1.033</td><td align="center" rowspan="1" colspan="1">6.269</td><td align="center" rowspan="1" colspan="1">6.422</td><td align="center" rowspan="1" colspan="1">24.723</td><td align="center" rowspan="1" colspan="1">56.808</td></tr></tbody></table></table-wrap></sec><sec sec-type="discussion" id="S0004"><title>Discussion</title><p>Mortality findings at the Agincourt HDSS for the overall period 1992–2011 reflect, as expected, many facets from interim publications. This paper provides a 20-year retrospective picture of a community in which mortality patterns changed drastically over time as a consequence of the HIV epidemic, as is clearly visible in <xref ref-type="fig" rid="F0001">Fig. 1</xref>. Few epidemics in human history have caused a sudden doubling in all-cause mortality in a particular population, sustained over a decade. There is still some way to go until mortality rates return to near pre-epidemic levels, despite encouraging recent decreases.</p><p>Attributing individual deaths unambiguously to HIV, in the absence of biomedical information, is not simple. Another study showed that the InterVA-4 model achieved a high level of specificity for HIV/AIDS related mortality in relation to sero-status (<xref rid="CIT0044" ref-type="bibr">44</xref>). However, it was also clear that, given higher cause-specific mortality rates for other causes among HIV positives, substantial proportions of HIV-related mortality are likely not to be attributed as such. This is seen in the Agincourt population, with substantially higher rates of TB, other infections, and NCDs during the peak incidence of HIV/AIDS deaths. There may also be some concomitant increases in NCD mortality rates due to other risk factors such as smoking and obesity, but the INDEPTH multisite analysis of adult NCD mortality showed significant correlations between HIV and NCD mortality, over a wide range of rates (<xref rid="CIT0048" ref-type="bibr">48</xref>).</p><p>
<xref ref-type="fig" rid="F0002">Figure 2</xref> details the effects of the HIV epidemic on each age group. Although in infancy HIV/AIDS as a specific cause was not the major cause of death, it is clear that other infections were mainly responsible for the increase in infant deaths due to the peak of the overall epidemic. It must be presumed therefore that these additional infant deaths due to other infections were associated with HIV. In the 1–4 year age group, the major increases appear to be more clearly associated with clinically typical HIV/AIDS, and hence a large part of the additional mortality was assigned as such. The mortality peaks both for the infant and 1–4 year age groups appear to slightly precede the peaks in older age groups. On the assumption that most of the childhood HIV/AIDS deaths would follow perinatally acquired infections, this may reflect young children developing disease fairly rapidly and dying from HIV-related causes before their mothers also went on to die, particularly at the stage before prevention of mother to child transmission (PMTCT) and adult anti-retroviral treatment (ART) programmes were well established in South Africa. It is not clear what would have caused the two peaks in the infant and child mortality patterns, but because they occurred during the time of peak HIV/AIDS mortality, they could have resulted from inconsistencies in the provision of PMTCT. For the 5–14 year age group, the peak of HIV-related mortality was later than in other age groups. HIV still accounted for a substantial increase in the relatively low overall mortality rates in this age group, presumably largely reflecting perinatal infections some years earlier in a sub-group of children who survived for several years. A study of mortality in young people found that HIV and TB combined was the third ranking cause of death globally in the 10–14 age group (<xref rid="CIT0049" ref-type="bibr">49</xref>), and HIV/AIDS was the leading cause of adolescent hospitalisation in Zimbabwe (<xref rid="CIT0050" ref-type="bibr">50</xref>).</p><p>Among adults, the 15–49 year age group had the largest proportion of HIV/AIDS deaths, though the cause-specific rate was lower than in the 50–64 year age group. In both of those age groups, rates of TB and NCDs increased substantially with the increase in HIV/AIDS, but there was no sign of NCDs decreasing in later years as HIV/AIDS and TB decreased. The 65+ year age group was the least affected by HIV/AIDS, as might be expected, although the relatively small proportion of HIV/AIDS deaths in this age group even during the epidemic peak amounted to an appreciable mortality rate, given the high overall mortality in this age group. As would be expected, NCDs accounted for the single largest category in this age group, and also increased over time.</p><p>Despite improvement since 2007, and contrary to global trends, infant and under-5 mortality rates (<xref ref-type="fig" rid="F0002">Fig. 2</xref>) remained substantially higher in 2011 than they were 20 years earlier. There is still some way to go to achieving reductions in these key parameters, not only in relation to HIV/AIDS, but also in terms of further improvements in the provision of maternal and perinatal health care. Inevitably the major political changes in South Africa over the same period are likely to have also affected mortality rates. However, from an analytical perspective, given the overwhelming impact of the HIV epidemic, it is hard to distinguish the effects of socio-political developments on mortality.</p><p>From a methodological perspective, despite some shortcomings associated with the use of proxy respondents (common to all VA methods), and that vital events are updated annually in Agincourt resulting in longer recall periods and potentially underestimating perinatal and infant mortality (<xref rid="CIT0001" ref-type="bibr">1</xref>), the evidence is very clear that the surveillance operations mounted in the Agincourt HDSS have succeeded to a large extent in identifying and registering deaths, and subsequently determining cause of death. The use of the WHO 2012 VA standard and the InterVA-4 model, even as applied here retrospectively to VA material that was not originally envisaged to be computer coded, has resulted in a plausible picture of a major epidemic. This was possible even though the methods had no means of ‘knowing’ about the development of the epidemic. This clearly demonstrates that it is feasible to use a general model for cause of death assignment, which does not need to be fed with local knowledge of mortality patterns and changes.</p><p>The analyses in this paper were restricted by the available data, and conformed to WHO-defined age groups and cause categories. We anticipate forthcoming papers utilizing advanced statistical analysis techniques and an extended time period to capture more fully the effects of the public sector rollout of ART and also assess the impacts of the seismic political and social changes following the abolition of racial segregation (apartheid), including the changing exposures associated with evolving patterns of migration and livelihoods, and the first universal elections in South Africa in 1994.</p></sec><sec sec-type="conclusions" id="S0005"><title>Conclusions</title><p>The Agincourt population has experienced a major mortality shock over the past two decades, from which it will take time to recover. The basic mortality pattern is consistent with the generalised pattern of mortality from an unfinished burden of communicable and nutritional diseases, HIV/AIDS, NCD, and violence and injuries observed elsewhere in South Africa (<xref rid="CIT0051" ref-type="bibr">51</xref>–<xref rid="CIT0054" ref-type="bibr">54</xref>), but the detailed individual surveillance behind these analyses allows finer-grained analyses of specific causes, age-related risks, and trends over time. The mortality trends presented here contribute to the understanding of South Africa's progress towards the achievement of some of the health-related millennium development goals. Most important, the trends highlight the importance of monitoring the evolution of NCD conditions and their risk factors and the need for South Africa to focus on their prevention, control, and treatment while continuing to strengthen HIV/AIDS prevention and treatment programmes in order to achieve further reductions in mortality rates.</p></sec> |
Trends in non-communicable disease mortality among adult residents in Nairobi's slums, 2003–2011: applying InterVA-4 to verbal autopsy data | <sec id="st1"><title>Background</title><p>About 80% of deaths from non-communicable diseases (NCDs) occur in developing countries such as Kenya. However, not much is known about the burden of NCDs in slums, which account for about 60% of the residences of the urban population in Kenya. This study examines trends in NCD mortality from two slum settings in Nairobi.</p></sec><sec id="st2"><title>Design</title><p>We use verbal autopsy data on 1954 deaths among adults aged 35 years and older who were registered in the Nairobi Urban Health and Demographic Surveillance System between 2003 and 2011. InterVA-4, a computer-based program, was used to assign causes of death for each case. Results are presented as annualized cause-specific mortality rates (CSMRs) and cause-specific mortality fractions (CSMFs) by sex.</p></sec><sec id="st3"><title>Results</title><p>The CSMRs for NCDs did not appear to change significantly over time for both males and females. Among males, cardiovascular diseases (CVDs) and neoplasms were the leading NCDs – contributing CSMFs of 8 and 5%, respectively, on average over time. Among females, CVDs contributed a CSMF of 14% on average over time, while neoplasms contributed 8%. Communicable diseases and related conditions remained the leading causes of death, contributing a CSMF of over 50% on average in males and females over time.</p></sec><sec id="st4"><title>Conclusions</title><p>Our findings are consistent with the Global Burden of Disease 2010 study which shows that communicable diseases remain the dominant cause of death in Africa, although NCDs were still significant contributors to mortality. We recommend an integrated approach towards disease prevention that focuses on health systems strengthening in resource-limited settings such as slums.</p></sec> | <contrib contrib-type="author"><name><surname>Oti</surname><given-names>Samuel O.</given-names></name><xref ref-type="aff" rid="AF0001">1</xref><xref ref-type="aff" rid="AF0002">2</xref><xref ref-type="aff" rid="AF0003">3</xref><xref ref-type="aff" rid="AF0004">4</xref><xref ref-type="corresp" rid="cor1">*</xref></contrib><contrib contrib-type="author"><name><surname>van de Vijver</surname><given-names>Steven</given-names></name><xref ref-type="aff" rid="AF0001">1</xref><xref ref-type="aff" rid="AF0002">2</xref><xref ref-type="aff" rid="AF0003">3</xref></contrib><contrib contrib-type="author"><name><surname>Kyobutungi</surname><given-names>Catherine</given-names></name><xref ref-type="aff" rid="AF0001">1</xref><xref ref-type="aff" rid="AF0004">4</xref></contrib> | Global Health Action | <p>Non-communicable diseases (NCDs) are traditionally considered to be diseases of affluent societies. However, global projections show that the largest increase in deaths from NCDs will occur in low-resource settings where, already, 80% of global NCD deaths occur (<xref rid="CIT0001" ref-type="bibr">1</xref>, <xref rid="CIT0002" ref-type="bibr">2</xref>). Specifically, the evidence indicates that four major NCDs – cardiovascular disease (CVD), cancer, diabetes, and chronic respiratory disease – together make the largest contribution to the burden of NCDs globally and in low- and middle-income countries (LMICs) (<xref rid="CIT0002" ref-type="bibr">2</xref>). These trends have been strongly linked with high rates of urbanization and changes in individual and societal lifestyle in LMICs, such as an increase in tobacco use, excessive alcohol consumption, reduced physical activity, and adoption of ‘Western’ diets that are high in salt, refined sugar, and unhealthy fats and oils (<xref rid="CIT0003" ref-type="bibr">3</xref>). Population ageing is also believed to be associated with increased risk of NCDs in LMICs (<xref rid="CIT0004" ref-type="bibr">4</xref>).</p><p>The rising burden of NCDs is of particular concern among poor communities partly because of lack of awareness and limited access to quality health care. The poor are often affected disproportionately by NCDs due to having only limited access to preventive and curative services, and having to spend a higher proportion of their income on health care costs for these lifelong conditions. This has the potential to further widen health inequalities within and between countries (<xref rid="CIT0005" ref-type="bibr">5</xref>). The urban poor, resident in vast slums across many LMIC cities (<xref rid="CIT0006" ref-type="bibr">6</xref>), are most vulnerable to NCDs. This is because of high exposure to risk factors like poor diets, excessive alcohol consumption, tobacco use, stress and environmental pollution, and limited access to screening, preventive, and curative services for NCDs (<xref rid="CIT0007" ref-type="bibr">7</xref>–<xref rid="CIT0010" ref-type="bibr">10</xref>). Specifically, there is some evidence that the levels of risk factors for common NCDs like smoking and reduced physical activity are relatively higher in the slums in LMICs compared to the rural areas (<xref rid="CIT0010" ref-type="bibr">10</xref>). Considering that the majority of urban populations in LMICs reside in slums or slum-like conditions (<xref rid="CIT0006" ref-type="bibr">6</xref>, <xref rid="CIT0011" ref-type="bibr">11</xref>, <xref rid="CIT0012" ref-type="bibr">12</xref>), the potential impact of an NCD epidemic on these populations cannot be ignored. Unfortunately, the public health systems in most LMICs are not prepared to deal with the rising burden of NCDs, even though cost-effective interventions for NCD prevention and control are described (<xref rid="CIT0013" ref-type="bibr">13</xref>–<xref rid="CIT0015" ref-type="bibr">15</xref>). In order to ensure that NCDs receive attention in the allocation of limited resources for health care provision in most LMICs, it is important to have robust evidence on the burden of NCDs in such settings.</p><p>One way to quantify the burden of the NCDs is through population-level data on mortality attributable to NCDs. Unfortunately, most of what is known about NCDs in LMICs is based on statistical estimates since most of these countries lack the complete vital registration–type data needed to generate actual population-level figures such as national NCD mortality estimates (<xref rid="CIT0016" ref-type="bibr">16</xref>–<xref rid="CIT0018" ref-type="bibr">18</xref>). Statistical estimates of mortality such as those based largely on hospital records may be inaccurate as they will fail to capture deaths outside a health facility – which account for up to 60% of deaths in a country like Kenya (<xref rid="CIT0019" ref-type="bibr">19</xref>). In the absence of vital registration systems and data on causes of death, Health and Demographic Surveillance Systems (HDSS) provide a useful platform for contributing to the understanding of the population-level mortality. Typically, HDSS monitor and track demographic and health indicators in a population within a defined geographic area (<xref rid="CIT0020" ref-type="bibr">20</xref>). HDSS also typically generate cause-of-death data using a process known as <italic>verbal autopsy</italic> (VA) – in which retrospective interviews are conducted with primary caregivers of deceased persons to determine the circumstances surrounding deaths (<xref rid="CIT0021" ref-type="bibr">21</xref>). These interviews can be analysed to determine the most likely causes of death in the population under surveillance to inform health planning (<xref rid="CIT0022" ref-type="bibr">22</xref>).</p><p>This article is based on cause-of-death data generated from VA in two slums covered by the Nairobi Urban Health and Demographic Surveillance System (NUHDSS) in Kenya. Specifically, we examine trends in NCD mortality in the NUHDSS from 2003 to 2011 – the earliest and latest years, respectively, for which VA data are complete.</p><sec sec-type="methods" id="S0002"><title>Methods</title><sec id="S0002-S20001"><title>Study area and population</title><p>We used VA data from deaths that occurred among adults aged 35 years and older in the NUHDSS between 2003 and 2011. Details about the operation of the NUHDSS have been described elsewhere (<xref rid="CIT0023" ref-type="bibr">23</xref>). In brief, the NUHDSS covers the two slums of Viwandani and Korogocho, both located less than 10 km from Nairobi, the capital of Kenya. In total, the population under surveillance in both slums is about 72,000 people, living in close to 28,500 households. We focus on deaths in adults aged 35 years and older due to the fact that the largest proportion of deaths due to NCDs is typically expected in this age group, and based on our analysis (results not shown) of younger age groups which showed very few NCD deaths. Moreover, slum populations like ours tend to have fewer older people compared to the INDEPTH 2013 standard (<xref rid="CIT0024" ref-type="bibr">24</xref>). The data used in this analysis can be accessed via the INDEPTH Network Data Repository (<xref rid="CIT0025" ref-type="bibr">25</xref>).</p></sec><sec id="S0002-S20002"><title>Cause-of-death data in the NUHDSS</title><p>Cause-of-death data using VA are generated in the NUHDSS for the two slum areas under surveillance, and full details of this process have also been published elsewhere (<xref rid="CIT0026" ref-type="bibr">26</xref>, <xref rid="CIT0027" ref-type="bibr">27</xref>). In brief, trained interviewers visit a household, on average within 3 months after a death has occurred, to conduct a VA interview with a credible respondent – usually a spouse or other close family member who would be aware of the circumstances surrounding the death. The interview is conducted using a semi-structured questionnaire that mostly inquires about probable symptoms and signs that the deceased may have portrayed before death.</p></sec><sec id="S0002-S20003"><title>Interpretation of VA data – the InterVA model</title><p>In order to generate causes of death, the data from completed VA questionnaires are entered into InterVA-4 – a computer program that applies probabilistic modelling to arrive at possible causes of death. We use InterVA because it is a freely accessible (open source) and widely validated program for cause-of-death interpretation. Full details of the InterVA model, including its use in interpreting cause-of-death data from HDSS across the developing world, have also been described in previous publications (<xref rid="CIT0028" ref-type="bibr">28</xref>–<xref rid="CIT0038" ref-type="bibr">38</xref>). Briefly, the data in the completed VA questionnaires were captured electronically into an SQL database, converted into a comma-separated value (.csv) file, and imported into STATA 12 (StataCorp. 2011. <italic>Stata Statistical Software: Release 12</italic>. College Station, TX: StataCorp LP). Using a STATA script, the data in the .csv file were converted into the input indicators required by the InterVA probabilistic model. The resultant input indicator file, also in .csv format, was run in InterVA-4's batch mode which in turn generates likely causes of death. A maximum of three likely causes of death per case with a likelihood value between 0 and 1 was produced by InterVA-4 for each cause. If the likelihood cause-of-death values for a particular death did not sum to 1, the difference between the sum of the likelihood values for probable causes of death and 1 were allocated to the ‘indeterminate’ cause. As recommended in the InterVA-4 user guide (<xref rid="CIT0039" ref-type="bibr">39</xref>), all identified causes were considered proportionate to their likelihood values in the mortality rate calculations In other words, to get the total number of deaths due to each cause, we sum the three likelihood values generated by InterVA and then divide this by the person years (PYs) (see below) to arrive at the cause-specific mortality rate (CSMR). The output from InterVA-4 was then imported into STATA for analysis.</p></sec><sec id="S0002-S20004"><title>Statistical approach</title><p>All analysis was performed using STATA 13 (StataCorp. 2013. <italic>Stata Statistical Software: Release 13</italic>. College Station, TX: StataCorp LP). Deaths and observed PYs were aggregated annually for all individuals in the study population for the period from 1 January 2003 to 31 December 2011. Individuals contributed PYs as long as they were living in the NUHDSS area. Residents stopped contributing time if they out-migrated and resumed contributing if they re-entered the NUHDSS area. We classified cause of death by three main groups: Group I – communicable, maternal, perinatal, and nutritional conditions; Group II – NCDs; and Group III – injuries, according to the Global Burden of Disease classification (<xref rid="CIT0040" ref-type="bibr">40</xref>). NCD-related deaths are further sub-categorized into two major groups – neoplasms and CVDs. Due to few numbers, all other NCDs are classified as ‘Other NCD’. Those cases for which no cause of death could be determined by InterVA-4 were classified as ‘Indeterminate’. Annual mortality rates, expressed as deaths per 10,000 PYs, were then calculated for each of the above cause-of-death categories by gender and age group. Results are presented as annualized group- and/or CSMRs and cause-specific mortality fractions (CSMFs) by sex and age group. We then apply the Mann–Kendall statistical test (<xref rid="CIT0041" ref-type="bibr">41</xref>, <xref rid="CIT0042" ref-type="bibr">42</xref>) to the annualized group-specific mortality data. This test is a non-parametric test for identifying trends in time series data. One benefit of this test is that the data need not conform to any particular distribution. That is, no assumption of normality is required. Typically, the test is used to determine whether the central or median value of the random response variable of interest changes over time. We report the results of the Mann–Kendall statistic, S, for each annualized group-specific mortality trend line. Let <italic>x</italic><sub>1</sub>, <italic>x</italic>
<sub>2</sub>, … <italic>x</italic>
<sub><italic>n</italic></sub> represent <italic>n</italic> data points where <italic>x</italic>
<sub>j</sub> represents the data point at time <italic>j</italic>. Then, the basic specification for S is given by:<disp-formula id="UFD1"><mml:math id="UM1"><mml:mrow><mml:mi>S</mml:mi><mml:mo>=</mml:mo><mml:munderover><mml:mo>∑</mml:mo><mml:mrow><mml:mi>k</mml:mi><mml:mo>=</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mrow><mml:mi>n</mml:mi><mml:mo>-</mml:mo><mml:mn>1</mml:mn></mml:mrow></mml:munderover><mml:mrow><mml:munderover><mml:mo>∑</mml:mo><mml:mrow><mml:mi>j</mml:mi><mml:mo>=</mml:mo><mml:mi>k</mml:mi><mml:mo>+</mml:mo><mml:mn>1</mml:mn></mml:mrow><mml:mi>n</mml:mi></mml:munderover><mml:mrow><mml:mi>s</mml:mi><mml:mi>i</mml:mi><mml:mi>g</mml:mi><mml:mi>n</mml:mi><mml:mo stretchy="false">(</mml:mo><mml:msub><mml:mi>x</mml:mi><mml:mi>j</mml:mi></mml:msub><mml:mo>-</mml:mo><mml:msub><mml:mi>x</mml:mi><mml:mi>k</mml:mi></mml:msub><mml:mo stretchy="false">)</mml:mo></mml:mrow></mml:mrow></mml:mrow></mml:math></disp-formula> where
<disp-formula id="UFD2"><mml:math id="UM2"><mml:mrow><mml:mtable><mml:mtr><mml:mtd><mml:mrow><mml:mi>s</mml:mi><mml:mi>i</mml:mi><mml:mi>g</mml:mi><mml:mi>n</mml:mi><mml:mrow><mml:mo stretchy="true">(</mml:mo><mml:mrow><mml:msub><mml:mi>x</mml:mi><mml:mi>j</mml:mi></mml:msub><mml:mo>-</mml:mo><mml:msub><mml:mi>x</mml:mi><mml:mi>k</mml:mi></mml:msub></mml:mrow><mml:mo stretchy="true">)</mml:mo></mml:mrow><mml:mo>=</mml:mo><mml:mn>1</mml:mn><mml:mtext> if</mml:mtext><mml:mrow><mml:mo stretchy="true">(</mml:mo><mml:mrow><mml:msub><mml:mi>x</mml:mi><mml:mi>j</mml:mi></mml:msub><mml:mo>-</mml:mo><mml:msub><mml:mi>x</mml:mi><mml:mi>k</mml:mi></mml:msub></mml:mrow><mml:mo stretchy="true">)</mml:mo></mml:mrow><mml:mo>></mml:mo><mml:mn>0</mml:mn></mml:mrow></mml:mtd></mml:mtr><mml:mtr><mml:mtd><mml:mrow><mml:mo>=</mml:mo><mml:mn>0</mml:mn><mml:mtext> if</mml:mtext><mml:mrow><mml:mo stretchy="true">(</mml:mo><mml:mrow><mml:msub><mml:mi>x</mml:mi><mml:mi>j</mml:mi></mml:msub><mml:mo>-</mml:mo><mml:msub><mml:mi>x</mml:mi><mml:mi>k</mml:mi></mml:msub></mml:mrow><mml:mo stretchy="true">)</mml:mo></mml:mrow><mml:mo>=</mml:mo><mml:mn>0</mml:mn></mml:mrow></mml:mtd></mml:mtr><mml:mtr><mml:mtd><mml:mrow><mml:mo>=</mml:mo><mml:mo>-</mml:mo><mml:mn>1</mml:mn><mml:mtext> if</mml:mtext><mml:mrow><mml:mo stretchy="true">(</mml:mo><mml:mrow><mml:msub><mml:mi>x</mml:mi><mml:mi>j</mml:mi></mml:msub><mml:mo>-</mml:mo><mml:msub><mml:mi>x</mml:mi><mml:mi>k</mml:mi></mml:msub></mml:mrow><mml:mo stretchy="true">)</mml:mo></mml:mrow><mml:mo><</mml:mo><mml:mn>0</mml:mn></mml:mrow></mml:mtd></mml:mtr></mml:mtable></mml:mrow></mml:math></disp-formula>
</p><p>The variance of S is calculated and used to compute a normalized test statistic, <italic>Z</italic>
<sub>S</sub>. Finally, the probability of <italic>Z</italic>
<sub>S</sub> is computed at a 95% significance level. The trend is said to be decreasing if <italic>Z</italic>
<sub>S</sub> is negative and the computed probability is greater than the level of significance. The trend is said to be increasing if the <italic>Z</italic>
<sub>S</sub> is positive and the computed probability is greater than the level of significance. If the computed probability is less than the level of significance, there is no trend.</p></sec><sec id="S0002-S20005"><title>Ethical considerations</title><p>We utilized data that are routinely collected by the NUHDSS. In order to operate the NUHDSS, APHRC applied for and received approval from the Kenya Medical Research Institution's Ethics Review Committee (KEMRI/ERC).</p></sec></sec><sec sec-type="results" id="S0003"><title>Results</title><p>Between 1 January 2003 and 31 December 2011, there were 1954 deaths among the 132,973 PYs observed in adults aged 35 years and older in the NUHDSS. Of these deaths, 1,217 (62%) occurred in men and 737 (38%) occurred in females. <xref ref-type="table" rid="T0001">Table 1</xref> shows the age–gender distribution of deaths by year. <xref ref-type="fig" rid="F0001">Figure 1</xref> shows trends in the CSMRs per 10,000 PYs by major cause-of-death group among males and females, respectively. Among males, NCDs (group II) show the second highest levels of CSMRs (after group I – communicable, maternal, perinatal, and nutritional conditions) over the entire study period. However, the trend in CSMRs for NCDs over the study period does not show any significant upward or downward trajectory, with values fluctuating between 4.5 and 23 deaths per 10,000 PYs at various non-regular intervals over the study period. This is confirmed by a non-significant normalized Mann–Kendall statistic (<italic>Z</italic>
<sub>S</sub>=0.73). Overall, group I showed the highest overall CSMR, whereas group III (comprising injuries) showed the lowest levels of CSMR over time – with the former showing a significant downward trend (<italic>Z</italic>
<sub>S</sub>=−2.19) and the latter showing a significant upward trend (<italic>Z</italic>
<sub>S</sub>=1.98) over time.</p><fig id="F0001" position="float"><label>Fig. 1</label><caption><p>Trends in cause-specific mortality rates among adults aged 35 years and older by major cause-of-death groups by year per 10,000 person years, 2003–2011: (a) males; and (b) females.</p><p>Group I – Communicable, maternal, perinatal, and nutritional conditions; Group II – non-communicable diseases; and Group III – injuries.</p></caption><graphic xlink:href="GHA-7-25533-g001"/></fig><table-wrap id="T0001" position="float"><label>Table 1</label><caption><p>Age–gender distribution of deaths by year among adults aged 35 years and older, 2003–2011</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1"/><th align="center" colspan="2" rowspan="1">Males (<italic>N</italic>=1,217)</th><th align="center" colspan="2" rowspan="1">Females (<italic>N</italic>=737)</th></tr><tr><th align="left" rowspan="1" colspan="1"/><th colspan="2" rowspan="1">
<hr/>
</th><th colspan="2" rowspan="1">
<hr/>
</th></tr><tr><th align="left" rowspan="1" colspan="1"/><th align="center" rowspan="1" colspan="1"><50 years</th><th align="center" rowspan="1" colspan="1">50+ years</th><th align="center" rowspan="1" colspan="1"><50 years</th><th align="center" rowspan="1" colspan="1">50+ years</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">Year</td><td align="center" rowspan="1" colspan="1">
<italic>n</italic> (%)</td><td align="center" rowspan="1" colspan="1">
<italic>n</italic> (%)</td><td align="center" rowspan="1" colspan="1">
<italic>n</italic> (%)</td><td align="center" rowspan="1" colspan="1">
<italic>n</italic> (%)</td></tr><tr><td align="left" rowspan="1" colspan="1">2003</td><td align="center" rowspan="1" colspan="1">80 (67)</td><td align="center" rowspan="1" colspan="1">39 (33)</td><td align="center" rowspan="1" colspan="1">75 (68)</td><td align="center" rowspan="1" colspan="1">35 (32)</td></tr><tr><td align="left" rowspan="1" colspan="1">2004</td><td align="center" rowspan="1" colspan="1">86 (64)</td><td align="center" rowspan="1" colspan="1">49 (36)</td><td align="center" rowspan="1" colspan="1">48 (66)</td><td align="center" rowspan="1" colspan="1">25 (34)</td></tr><tr><td align="left" rowspan="1" colspan="1">2005</td><td align="center" rowspan="1" colspan="1">69 (59)</td><td align="center" rowspan="1" colspan="1">47 (41)</td><td align="center" rowspan="1" colspan="1">31 (44)</td><td align="center" rowspan="1" colspan="1">40 (56)</td></tr><tr><td align="left" rowspan="1" colspan="1">2006</td><td align="center" rowspan="1" colspan="1">65 (53)</td><td align="center" rowspan="1" colspan="1">57 (47)</td><td align="center" rowspan="1" colspan="1">43 (65)</td><td align="center" rowspan="1" colspan="1">23 (35)</td></tr><tr><td align="left" rowspan="1" colspan="1">2007</td><td align="center" rowspan="1" colspan="1">81 (57)</td><td align="center" rowspan="1" colspan="1">61 (43)</td><td align="center" rowspan="1" colspan="1">30 (43)</td><td align="center" rowspan="1" colspan="1">40 (57)</td></tr><tr><td align="left" rowspan="1" colspan="1">2008</td><td align="center" rowspan="1" colspan="1">82 (65)</td><td align="center" rowspan="1" colspan="1">45 (35)</td><td align="center" rowspan="1" colspan="1">51 (73)</td><td align="center" rowspan="1" colspan="1">19 (27)</td></tr><tr><td align="left" rowspan="1" colspan="1">2009</td><td align="center" rowspan="1" colspan="1">63 (62)</td><td align="center" rowspan="1" colspan="1">39 (38)</td><td align="center" rowspan="1" colspan="1">33 (55)</td><td align="center" rowspan="1" colspan="1">28 (46)</td></tr><tr><td align="left" rowspan="1" colspan="1">2010</td><td align="center" rowspan="1" colspan="1">111 (63)</td><td align="center" rowspan="1" colspan="1">66 (37)</td><td align="center" rowspan="1" colspan="1">55 (52)</td><td align="center" rowspan="1" colspan="1">51 (48)</td></tr><tr><td align="left" rowspan="1" colspan="1">2011</td><td align="center" rowspan="1" colspan="1">118 (67)</td><td align="center" rowspan="1" colspan="1">59 (33)</td><td align="center" rowspan="1" colspan="1">70 (64)</td><td align="center" rowspan="1" colspan="1">40 (36)</td></tr></tbody></table></table-wrap><p>Similarly, among females, CSMRs for NCDs did not show any significant trends. For example, at the start of 2003, there were about 17 NCD deaths per 10,000 PYs. The CSMR for NCDs then peaks at 56 deaths per 10,000 PY in 2005, drops to 26.5 deaths per 10,000 PYs in 2009, and finally rises to 37 deaths per 10,000 PYs in 2011. The CSMR for group I did, however, show a general downward trend from 124 deaths per 10,000 PYs in 2003 to 51 deaths per 10,000 PYs in 2011. Unlike males, however, injuries had the lowest CSMRs over the study period but did not show any clear pattern in trends – varying from 4.5 deaths per 10,000 PYs in 2003, down to 2.3 per 10,000 PYs in 2005, and peaking at 14 per 10,000 PYs in 2011. The normalized Mann–Kendall statistics for group I, II, and III causes did not indicate any significant trends (<italic>Z</italic><sub>S</sub>=1.77, 0.52, and 0.73, respectively).</p><p>
<xref ref-type="table" rid="T0002">Table 2</xref> shows the CSMFs for the broad groups of NCDs – neoplasms, CVDs, and other NCDs (see also <xref ref-type="fig" rid="F0002">Figure 2</xref>). Again, no clear patterns can be observed over time in CSMFs contributed by neoplasms, CVDs, or other NCDs among either males or females. What is clear is that, over time, group I conditions are consistently the most dominant contributor (54% on average) to mortality in both males and females. Among males, however, injuries are the next largest contributor – 12% on average over time. CVDs, neoplasms, and other NCDs contribute 8, 5, and 6%, respectively, of the CSMFs on average over time. Among females, CVDs are the second highest contributor to CSMF, accounting for 14% of mortality on average over time. This is followed by neoplasms (8%), other NCDs (5%), and injuries (4%).</p><fig id="F0002" position="float"><label>Fig. 2</label><caption><p>Trends in cause-specific mortality fractions among adults aged 35 years and older by year, 2003–2011: (a) males; and (b) females.</p></caption><graphic xlink:href="GHA-7-25533-g002"/></fig><table-wrap id="T0002" position="float"><label>Table 2</label><caption><p>Cause-specific mortality fractions among adult males and females aged 35 years and older, 2003–2011</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1"/><th align="center" colspan="9" rowspan="1">Year of death</th></tr><tr><th align="left" rowspan="1" colspan="1"/><th colspan="9" rowspan="1">
<hr/>
</th></tr><tr><th align="left" rowspan="1" colspan="1"/><th align="center" rowspan="1" colspan="1">2003</th><th align="center" rowspan="1" colspan="1">2004</th><th align="center" rowspan="1" colspan="1">2005</th><th align="center" rowspan="1" colspan="1">2006</th><th align="center" rowspan="1" colspan="1">2007</th><th align="center" rowspan="1" colspan="1">2008</th><th align="center" rowspan="1" colspan="1">2009</th><th align="center" rowspan="1" colspan="1">2010</th><th align="center" rowspan="1" colspan="1">2011</th></tr></thead><tbody><tr><td colspan="10" align="left" rowspan="1">Males</td></tr><tr><td align="left" rowspan="1" colspan="1"> Communicable, maternal, or neonatal</td><td align="center" rowspan="1" colspan="1">63.5</td><td align="center" rowspan="1" colspan="1">56.1</td><td align="center" rowspan="1" colspan="1">61.4</td><td align="center" rowspan="1" colspan="1">64.4</td><td align="center" rowspan="1" colspan="1">48.0</td><td align="center" rowspan="1" colspan="1">51.2</td><td align="center" rowspan="1" colspan="1">53.3</td><td align="center" rowspan="1" colspan="1">49.2</td><td align="center" rowspan="1" colspan="1">46.8</td></tr><tr><td align="left" rowspan="1" colspan="1"> Neoplasms</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">2.4</td><td align="center" rowspan="1" colspan="1">11.0</td><td align="center" rowspan="1" colspan="1">6.3</td><td align="center" rowspan="1" colspan="1">6.7</td><td align="center" rowspan="1" colspan="1">5.7</td><td align="center" rowspan="1" colspan="1">4.9</td><td align="center" rowspan="1" colspan="1">8.4</td><td align="center" rowspan="1" colspan="1">1.8</td></tr><tr><td align="left" rowspan="1" colspan="1"> Other NCDs</td><td align="center" rowspan="1" colspan="1">3.4</td><td align="center" rowspan="1" colspan="1">4.3</td><td align="center" rowspan="1" colspan="1">6.5</td><td align="center" rowspan="1" colspan="1">7.0</td><td align="center" rowspan="1" colspan="1">9.3</td><td align="center" rowspan="1" colspan="1">4.1</td><td align="center" rowspan="1" colspan="1">8.4</td><td align="center" rowspan="1" colspan="1">7.4</td><td align="center" rowspan="1" colspan="1">5.0</td></tr><tr><td align="left" rowspan="1" colspan="1"> CVDs</td><td align="center" rowspan="1" colspan="1">1.7</td><td align="center" rowspan="1" colspan="1">5.9</td><td align="center" rowspan="1" colspan="1">8.3</td><td align="center" rowspan="1" colspan="1">6.1</td><td align="center" rowspan="1" colspan="1">7.4</td><td align="center" rowspan="1" colspan="1">11.0</td><td align="center" rowspan="1" colspan="1">9.4</td><td align="center" rowspan="1" colspan="1">8.8</td><td align="center" rowspan="1" colspan="1">9.8</td></tr><tr><td align="left" rowspan="1" colspan="1"> Injuries</td><td align="center" rowspan="1" colspan="1">8.4</td><td align="center" rowspan="1" colspan="1">9.9</td><td align="center" rowspan="1" colspan="1">5.2</td><td align="center" rowspan="1" colspan="1">7.3</td><td align="center" rowspan="1" colspan="1">11.6</td><td align="center" rowspan="1" colspan="1">16.9</td><td align="center" rowspan="1" colspan="1">11.7</td><td align="center" rowspan="1" colspan="1">11.4</td><td align="center" rowspan="1" colspan="1">22.7</td></tr><tr><td align="left" rowspan="1" colspan="1"> Indeterminate</td><td align="center" rowspan="1" colspan="1">23.0</td><td align="center" rowspan="1" colspan="1">21.5</td><td align="center" rowspan="1" colspan="1">7.6</td><td align="center" rowspan="1" colspan="1">8.9</td><td align="center" rowspan="1" colspan="1">17.1</td><td align="center" rowspan="1" colspan="1">11.1</td><td align="center" rowspan="1" colspan="1">12.3</td><td align="center" rowspan="1" colspan="1">14.8</td><td align="center" rowspan="1" colspan="1">13.8</td></tr><tr><td align="left" rowspan="1" colspan="1"> Total</td><td align="center" rowspan="1" colspan="1">100.0</td><td align="center" rowspan="1" colspan="1">100.0</td><td align="center" rowspan="1" colspan="1">100.0</td><td align="center" rowspan="1" colspan="1">100.0</td><td align="center" rowspan="1" colspan="1">100.0</td><td align="center" rowspan="1" colspan="1">100.0</td><td align="center" rowspan="1" colspan="1">100.0</td><td align="center" rowspan="1" colspan="1">100.0</td><td align="center" rowspan="1" colspan="1">100.0</td></tr><tr><td colspan="10" align="left" rowspan="1">Females</td></tr><tr><td align="left" rowspan="1" colspan="1"> Communicable, maternal, or neonatal</td><td align="center" rowspan="1" colspan="1">69.7</td><td align="center" rowspan="1" colspan="1">68.1</td><td align="center" rowspan="1" colspan="1">47.2</td><td align="center" rowspan="1" colspan="1">59.8</td><td align="center" rowspan="1" colspan="1">48.9</td><td align="center" rowspan="1" colspan="1">65.6</td><td align="center" rowspan="1" colspan="1">56.2</td><td align="center" rowspan="1" colspan="1">47.9</td><td align="center" rowspan="1" colspan="1">45.8</td></tr><tr><td align="left" rowspan="1" colspan="1"> Neoplasms</td><td align="center" rowspan="1" colspan="1">0.8</td><td align="center" rowspan="1" colspan="1">2.0</td><td align="center" rowspan="1" colspan="1">14.5</td><td align="center" rowspan="1" colspan="1">6.3</td><td align="center" rowspan="1" colspan="1">16.3</td><td align="center" rowspan="1" colspan="1">10.0</td><td align="center" rowspan="1" colspan="1">5.0</td><td align="center" rowspan="1" colspan="1">11.9</td><td align="center" rowspan="1" colspan="1">8.4</td></tr><tr><td align="left" rowspan="1" colspan="1"> Other NCDs</td><td align="center" rowspan="1" colspan="1">5.5</td><td align="center" rowspan="1" colspan="1">3.5</td><td align="center" rowspan="1" colspan="1">3.9</td><td align="center" rowspan="1" colspan="1">8.1</td><td align="center" rowspan="1" colspan="1">3.7</td><td align="center" rowspan="1" colspan="1">5.3</td><td align="center" rowspan="1" colspan="1">6.3</td><td align="center" rowspan="1" colspan="1">5.3</td><td align="center" rowspan="1" colspan="1">6.6</td></tr><tr><td align="left" rowspan="1" colspan="1"> CVDs</td><td align="center" rowspan="1" colspan="1">3.1</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">28.3</td><td align="center" rowspan="1" colspan="1">16.9</td><td align="center" rowspan="1" colspan="1">19.0</td><td align="center" rowspan="1" colspan="1">10.6</td><td align="center" rowspan="1" colspan="1">23.4</td><td align="center" rowspan="1" colspan="1">10.2</td><td align="center" rowspan="1" colspan="1">18.5</td></tr><tr><td align="left" rowspan="1" colspan="1"> Injuries</td><td align="center" rowspan="1" colspan="1">2.5</td><td align="center" rowspan="1" colspan="1">4.2</td><td align="center" rowspan="1" colspan="1">1.9</td><td align="center" rowspan="1" colspan="1">2.7</td><td align="center" rowspan="1" colspan="1">5.0</td><td align="center" rowspan="1" colspan="1">3.9</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">4.7</td><td align="center" rowspan="1" colspan="1">12.6</td></tr><tr><td align="left" rowspan="1" colspan="1"> Indeterminate</td><td align="center" rowspan="1" colspan="1">18.4</td><td align="center" rowspan="1" colspan="1">22.2</td><td align="center" rowspan="1" colspan="1">4.2</td><td align="center" rowspan="1" colspan="1">6.2</td><td align="center" rowspan="1" colspan="1">7.1</td><td align="center" rowspan="1" colspan="1">4.6</td><td align="center" rowspan="1" colspan="1">9.1</td><td align="center" rowspan="1" colspan="1">20.0</td><td align="center" rowspan="1" colspan="1">8.2</td></tr><tr><td align="left" rowspan="1" colspan="1"> Total</td><td align="center" rowspan="1" colspan="1">100.0</td><td align="center" rowspan="1" colspan="1">100.0</td><td align="center" rowspan="1" colspan="1">100.0</td><td align="center" rowspan="1" colspan="1">100.0</td><td align="center" rowspan="1" colspan="1">100.0</td><td align="center" rowspan="1" colspan="1">100.0</td><td align="center" rowspan="1" colspan="1">100.0</td><td align="center" rowspan="1" colspan="1">100.0</td><td align="center" rowspan="1" colspan="1">100.0</td></tr></tbody></table><table-wrap-foot><fn><p>NCD=non-communicable diseases; CVD=cardiovascular diseases.</p></fn></table-wrap-foot></table-wrap></sec><sec sec-type="discussion" id="S0004"><title>Discussion</title><p>We set out to demonstrate trends in NCD deaths among adults aged 35 years and older between 2003 and 2011 in the NUHDSS. The NUHDSS covers two urban slum settlements in Nairobi. As far as we know, no similar study exists that focuses on trends in the burden of NCDs in slums in Kenya or other parts of sub-Saharan Africa. Previous mortality studies in the slums of Kenya have looked at the burden of disease in general rather than in trends, or have focused on injuries (<xref rid="CIT0026" ref-type="bibr">26</xref>, <xref rid="CIT0043" ref-type="bibr">43</xref>).</p><p>We did not find any clear trends in NCD mortality over time. However, we observed that, comparatively, trends in NCD mortality among males and females had remained stable over time. Group I conditions showed a declining trend, particularly among women. The declining trends in communicable disease, which constitute the bulk of Group I causes, mortality among adults are largely driven by reductions in AIDS and tuberculosis mortality over time (<xref rid="CIT0044" ref-type="bibr">44</xref>). This most likely reflects the huge investments in HIV prevention, treatment, and control programs in Kenya and other developing countries over the past decade or so (<xref rid="CIT0045" ref-type="bibr">45</xref>). Injuries did not show any significant changes over time, although males appeared to have a higher burden than females. This may be explained by the known fact that males tend to engage in high-risk behaviour such as violent crime, particularly in the slum context (<xref rid="CIT0026" ref-type="bibr">26</xref>, <xref rid="CIT0043" ref-type="bibr">43</xref>).</p><p>Global projections show that NCD mortality will continue to increase in developing countries while communicable diseases will decrease. Concerns about these projections have led to unified global calls to action to combat NCDs, including the UN Summit on NCDs that was held in New York in September 2011 and the World Health Assembly Resolution on preventing and controlling NCDs in May 2012. However, rather than institute vertical programs to tackle the burden of NCDs, there have been calls for an integrated approach that aims at strengthening health systems in developing countries (<xref rid="CIT0013" ref-type="bibr">13</xref>, <xref rid="CIT0046" ref-type="bibr">46</xref>). We believe that these calls are logical and relevant in our setting, where resources are scarce and need to be maximized. Moreover, we have evidence that certain risk factors for NCDs are on the rise in slum settings in Africa (<xref rid="CIT0007" ref-type="bibr">7</xref>, <xref rid="CIT0010" ref-type="bibr">10</xref>). Therefore, the time to act is now.</p><p>Our study has a number of limitations. We could not explore age differentials due to the overall small numbers of NCD deaths. Also, again due to small numbers of NCD deaths, we could not meaningfully further disaggregate NCD deaths into more specific mortality causes beyond the three broad groups of neoplasms, CVDs, and other NCDs. Finally, the proportion of indeterminate causes contributed to between 4 and 23% of the CSMFs over time. Specifically, the years 2003 and 2004 yielded the highest proportion of indeterminate causes. It is difficult to explain this as InterVA-4 was applied uniformly to the data set across all years, and the similar data collection tools were used over the study period. However, this may be suggestive of data inconsistencies in the affected years.</p></sec><sec sec-type="conclusions" id="S0005"><title>Conclusions</title><p>In conclusion, our findings are consistent with the recent Global Burden of Disease 2013 study which shows that Group I conditions remain the dominant cause of death in Africa, although NCDs and injuries still played a significant role over time (<xref rid="CIT0047" ref-type="bibr">47</xref>). We recommend an integrated approach towards disease prevention that focuses on health systems strengthening in resource-limited settings such as ours.</p></sec> |
Taspase1 cleaves MLL1 to activate cyclin E for HER2/neu breast tumorigenesis | <p>Taspase1, a highly conserved threonine protease, cleaves nuclear transcriptional regulators mixed-lineage leukemia (MLL, MLL1), MLL2, TFIIA, and ALF to orchestrate a wide variety of biological processes. <italic>In vitro</italic> studies thus far demonstrated that Taspase1 plays important roles in the proliferation of various cancer cell lines, including HER2-positive breast cancer cells. To investigate the role of Taspase1 in breast tumorigenesis <italic>in vivo</italic>, we deleted Taspase1 from mouse mammary glands by generating <italic>MMTV-neu;MMTV-cre;Tasp1</italic><sup><italic>F</italic>/−</sup> mice. We demonstrate that initiation of <italic>MMTV-neu</italic>- but not <italic>MMTV-wnt-driven</italic> breast cancer is blocked in the absence of Taspase1. Importantly, <italic>Taspase1</italic> loss alone neither impacts normal development nor pregnancy physiology of the mammary gland. In mammary glands <italic>Taspase1</italic> deficiency abrogates <italic>MMTV-neu</italic>-induced cyclins E and A expression, thereby preventing tumorigenesis. The mechanisms were explored in HER2-positive breast cancer cell line BT474 and HER2-transformed MCF10A cells and validated using knockdown-resistant Taspase1. As Taspase1 was shown to cleave MLL which forms complexes with E2F transcription factors to regulate <italic>Cyclins E</italic>, <italic>A</italic>, and <italic>B</italic> expression in mouse embryonic fibroblasts (MEFs), we investigated whether the cleavage of MLL by Taspase1 constitutes an essential <italic>in vivo</italic> axis for HER2/neu-induced mammary tumorigenesis. To this end, we generated <italic>MMTV-neu;MLL<sup>nc/nc</sup></italic> transgenic mice that carry homozygous non-cleavable <italic>MLL</italic> alleles. Remarkably, these mice are also protected from HER2/neu-driven breast tumorigenesis. Hence, MLL is the primary Taspase1 substrate whose cleavage is required for <italic>MMTV-neu</italic>-induced tumor formation. As Taspase1 plays critical roles in breast cancer pathology, it may serve as a therapeutic target for HER2-positive human breast cancer.</p> | <contrib contrib-type="author"><name><surname>Dong</surname><given-names>Yiyu</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="author-notes" rid="note1"><sup>*</sup></xref></contrib><contrib contrib-type="author"><name><surname>Van Tine</surname><given-names>Brian A</given-names></name><xref ref-type="aff" rid="aff5">5</xref><xref ref-type="author-notes" rid="note1"><sup>*</sup></xref></contrib><contrib contrib-type="author"><name><surname>Oyama</surname><given-names>Toshinao</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Wang</surname><given-names>Patricia I</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Cheng</surname><given-names>Emily H</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Hsieh</surname><given-names>James J</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="aff" rid="aff3">3</xref><xref ref-type="aff" rid="aff4">4</xref><xref ref-type="corresp" rid="caf1">*</xref></contrib><aff id="aff1"><label>1</label><institution>Human Oncology & Pathogenesis Program, Memorial Sloan-Kettering Cancer Center</institution>, New York, NY 10065, <country>USA</country></aff><aff id="aff2"><label>2</label><institution>Department of Pathology, Memorial Sloan-Kettering Cancer Center</institution>, New York, NY 10065, <country>USA</country></aff><aff id="aff3"><label>3</label><institution>Department of Medicine, Memorial Sloan-Kettering Cancer Center</institution>, New York, NY 10065, <country>USA</country></aff><aff id="aff4"><label>4</label><institution>Department of Medicine, Weill Cornell Medical College</institution>, New York, NY 10021, <country>USA</country></aff><aff id="aff5"><label>5</label><institution>Department of Internal Medicine, Washington University</institution>, St Louis, MO 63110, <country>USA</country></aff> | Cell Research | <sec sec-type="intro"><title>Introduction</title><p>Human breast cancer is a heterogeneous disease comprised of three major subgroups, each encompassing unique molecular signatures, prognoses, and responses to therapies<sup><xref ref-type="bibr" rid="bib1">1</xref></sup>. HER2 is a member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, which includes EGFR, HER2, HER3, and HER4<sup><xref ref-type="bibr" rid="bib2">2</xref>,<xref ref-type="bibr" rid="bib3">3</xref></sup>. Homo- or hetero-dimerization of these receptors results in the phosphorylation of residues in the intracellular domain and the consequent recruitment of adapter molecules responsible for the initiation of several signaling pathways involved in cell proliferation and survival. HER2-amplified/overexpressed breast cancer is one of the aforementioned subgroups. It accounts for approximately 20% - 30% of all breast cancer cases, and it is characterized by an aggressive phenotype and poor overall survival<sup><xref ref-type="bibr" rid="bib4">4</xref>,<xref ref-type="bibr" rid="bib5">5</xref>,<xref ref-type="bibr" rid="bib6">6</xref></sup>. Despite therapeutic advances brought forth by anti-HER2 agents including monoclonal antibodies such as trastuzumab (Herceptin) and small molecule tyrosine kinase inhibitors, patients with advanced HER2-positive breast cancer frequently experience disease progression and/or recurrence<sup><xref ref-type="bibr" rid="bib7">7</xref>,<xref ref-type="bibr" rid="bib8">8</xref>,<xref ref-type="bibr" rid="bib9">9</xref></sup>.</p><p>One of the hallmarks of cancer is dysregulated proliferation<sup><xref ref-type="bibr" rid="bib10">10</xref></sup>. Unsurprisingly, molecular characterization of human tumors reveals that key cell cycle regulators are frequently dysregulated<sup><xref ref-type="bibr" rid="bib11">11</xref></sup>. During cell cycle progression, cyclin-dependent kinases (CDKs) and cyclins constitute the central regulatory apparatus. In mammalian cells, kinase subunits (CDK4, CDK6, CDK2, and CDC2) are expressed alongside cyclins (cyclin D, E, A, and B) sequentially as the cells progress from G1 through mitosis. CDK4 and CDK6 form complexes with one of several D-type cyclins and function early in G1 phase, probably in response to growth factors. CDK2 forms complexes with cyclins E or A and functions in the G1/S phase transition and S phase DNA replication<sup><xref ref-type="bibr" rid="bib12">12</xref>,<xref ref-type="bibr" rid="bib13">13</xref>,<xref ref-type="bibr" rid="bib14">14</xref></sup>. A major molecular consequence of HER2 up-regulation is the increased expression of G<sub>1</sub>-to-S cell cycle regulatory proteins cyclins D and E, which leads to aberrant cell proliferation<sup><xref ref-type="bibr" rid="bib7">7</xref>,<xref ref-type="bibr" rid="bib12">12</xref>,<xref ref-type="bibr" rid="bib13">13</xref>,<xref ref-type="bibr" rid="bib14">14</xref>,<xref ref-type="bibr" rid="bib15">15</xref>,<xref ref-type="bibr" rid="bib16">16</xref></sup>. High cyclin E expression is a marker that correlates strongly with poor outcome in patients with breast cancer. Since <italic>Cyclin E</italic> amplification/overexpression leads to trastuzumab resistance, disrupting <italic>Cyclin E</italic> expression could have therapeutic importance for HER2-positive breast cancers<sup><xref ref-type="bibr" rid="bib17">17</xref>,<xref ref-type="bibr" rid="bib18">18</xref></sup>.</p><p>Taspase1 was originally purified as the protease that cleaves MLL (the <underline>M</underline>ixed-<underline>L</underline>ineage <underline>L</underline>eukemia protein; also known as MLL1) for proper regulation of <italic>HOX</italic> gene expression<sup><xref ref-type="bibr" rid="bib19">19</xref>,<xref ref-type="bibr" rid="bib20">20</xref></sup>. Other genetically and biochemically proven Taspase1 substrates include MLL2 (also known as MLL4), TFIIAα-β, ALFα-β (TFII<underline>A-L</underline>ike <underline>F</underline>actor) and <italic>Drosophila</italic> HCF-1 (<underline>H</underline>ost <underline>C</underline>ell <underline>F</underline>actor 1)<sup><xref ref-type="bibr" rid="bib20">20</xref>,<xref ref-type="bibr" rid="bib21">21</xref>,<xref ref-type="bibr" rid="bib22">22</xref>,<xref ref-type="bibr" rid="bib23">23</xref>,<xref ref-type="bibr" rid="bib24">24</xref></sup>. Interestingly, all confirmed Taspase1 substrates are nuclear transcription factors that play important roles in gene regulation. <italic>Taspase1</italic> encodes a highly conserved 50 kDa α-β proenzyme, which undergoes intramolecular autoproteolysis, producing the mature α28/β22 heterodimeric enzyme that displays an overall α/β/β/α structure<sup><xref ref-type="bibr" rid="bib20">20</xref>,<xref ref-type="bibr" rid="bib25">25</xref></sup>. A complete genetic knockout of <italic>Taspase1</italic> in mice resulted in profound early postnatal lethality and the few surviving <italic>Taspase1</italic><sup>−/−</sup> mice universally exhibited small body sizes and homeotic transformations at the axial skeleton<sup><xref ref-type="bibr" rid="bib22">22</xref></sup>. <italic>Taspaes1</italic><sup>−/−</sup> mouse embryonic fibroblasts (MEFs) displayed cell cycle progression defects with downregulation of <italic>cyclins E</italic>, <italic>A</italic>, and <italic>B</italic> and upregulation of <italic>CDKIs</italic> (cyclin-dependent kinase inhibitors) <italic>p16</italic>, <italic>p21</italic> and <italic>p27</italic><sup><xref ref-type="bibr" rid="bib22">22</xref></sup>. Importantly, <italic>Taspase1</italic><sup>−/−</sup> MEFs were resistant to oncogenic transformation <italic>in vitro</italic><sup><xref ref-type="bibr" rid="bib26">26</xref></sup>. In MEFs, Taspase1 cleaves MLL that interacts with E2Fs, core transcription factors of the mammalian cell cycle, to activate select <italic>Cyclin</italic> genes<sup><xref ref-type="bibr" rid="bib22">22</xref>,<xref ref-type="bibr" rid="bib27">27</xref>,<xref ref-type="bibr" rid="bib28">28</xref></sup>. How Taspase1 regulates <italic>CDKIs</italic>, however, remains unclear. Importantly, Taspase1 shows a high level of expression in most human cancer cell lines<sup><xref ref-type="bibr" rid="bib22">22</xref></sup>, and knockdown of Taspase1 in many cancer cell lines impairs cancer cell proliferation and even sensitizes brain cancer and melanoma cells to anoikis<sup><xref ref-type="bibr" rid="bib26">26</xref></sup>.</p><p>Encouragingly, despite the fact that Taspase1 plays an important role in mammalian embryogenesis, acute genetic deletion of Taspase1 in adult mice does not confer discernible toxicities on the mice, which suggests a wide therapeutic index for Taspase1 inhibition in adult cancer patients<sup><xref ref-type="bibr" rid="bib29">29</xref></sup>. Moreover, pharmacological inhibition of Taspase1 has been attempted<sup><xref ref-type="bibr" rid="bib29">29</xref>,<xref ref-type="bibr" rid="bib30">30</xref></sup>, and a primitive small molecular Taspase1 inhibitor (TASPIN) showed effects on U251 brain tumor xenografts and HER2-driven mouse breast cancers<sup><xref ref-type="bibr" rid="bib29">29</xref></sup>. The latter findings prompted us to hypothesize that Taspase1 could play a critical role in HER2-positive breast cancer and that Taspase1 inhibitors may be developed as a safe treatment option for Taspase1-dependent cancers.</p><p>Here, we report the data from preclinical experiments that we conducted by constructing genetically well-defined mouse models, demonstrating that Taspase1 ablation blocks MMTV-neu-driven breast cancer initiation <italic>in vivo</italic>. We further pinpoint that MLL is the key Taspase1 substrate whose cleavage is required for <italic>MMTV-neu</italic>-induced tumor formation. The cleavage of MLL by Taspase1 enables HER2/neu-induced overexpression of <italic>Cyclins E</italic> and <italic>A</italic>, presenting an essential <italic>in vivo</italic> genetic network conferring breast tumorigenesis.</p></sec><sec sec-type="results"><title>Results</title><sec><title>Taspase1 deficiency disrupts the expression of cyclins and proliferation of HER2<sup>+</sup> breast cancer cells</title><p>To determine whether Taspase1 is required for HER2-positive breast cancer cell proliferation, we conducted genetic knockdown experiments in two HER2-overexpressing breast cancer cell lines, BT474 and HCC1419. Taspase1 deficiency significantly reduced the cell number in both cell lines (<xref ref-type="fig" rid="fig1">Figure 1A</xref>). Cell death assay confirmed that there is no significant difference in cell death between the Taspase1 knockdown cells and the control in either cell line (<xref ref-type="fig" rid="fig1">Figure 1B</xref>). On the other hand, cell cycle analysis showed that Taspase1 knockdown significantly decreased the S phase population in both cell lines (<xref ref-type="fig" rid="fig1">Figure 1C</xref>). These data suggest that Taspase1 regulates HER2-positive breast cancer cell proliferation through promoting cell cycle progression.</p><p>We next investigated the underlying mechanisms by which Taspase1 regulates cell division. Key regulators of the mammalian cell cycle machinery include E2Fs, Rbs, cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors (CDKIs), which form complex positive and negative epistatic regulatory loops to ensure accurate cell cycle progression. In MEFs, following the cleavage by Taspase1, MLL<sup>N320/C180</sup> targets to <italic>Cyclins E</italic> and <italic>A</italic> promoters through interaction with E2Fs to methylate histone H3 at K4, thereby transactivating <italic>Cyclins E</italic> and <italic>A</italic> for cell proliferation<sup><xref ref-type="bibr" rid="bib22">22</xref>,<xref ref-type="bibr" rid="bib28">28</xref></sup>. To gain mechanistic insight into how Taspase1 regulates HER2-positive breast cancer cell proliferation, we examined the expression of several key cell cycle regulators. Western blot analyses of Taspase1-knockdown BT474 and HCC1419 cells revealed a significant decrease in cyclins E2 and A, but not D1 (<xref ref-type="fig" rid="fig1">Figure 1D</xref>), consistent with our prior results obtained in MEFs<sup><xref ref-type="bibr" rid="bib22">22</xref></sup>. Altogether, these results indicate that in HER2-positive breast cancer cells Taspase1 assures the proper accumulation of cyclins E and A for proliferation.</p><p>The ability of cancer cells to form colonies on soft agar is a stringent <italic>in vitro</italic> surrogate of <italic>in vivo</italic> tumorigenicity. Soft agar assays assess the capacity of tumor cells to not only proliferate but also resist anoikis under three-dimensional culture conditions that imitate the <italic>in vivo</italic> tumor growth environment. We determined the degree to which Taspase1 is required for the colony formation capability of HER2-positive breast cancer cells on soft agar. Knockdown of Taspase1 (sh-T1) in BT474 cells severely compromised their ability to grow as colonies on soft agar (<xref ref-type="fig" rid="fig1">Figure 1E</xref>). To validate the specific requirement of Taspase1 for cancer cell growth on soft agar, we engineered a sh-T1 knockdown resistant version of Taspase1 (RT1). Retroviral delivery of RT1 rescued the ability of Taspase1-knockdown (sh-T1) BT474 cells to form colonies (<xref ref-type="fig" rid="fig1">Figure 1E</xref>). Western blot analysis confirmed the successful knockdown of Taspase1 in BT474 cells by sh-T1 and the resulting reduced protein levels of cyclins E2 and A (<xref ref-type="fig" rid="fig1">Figure 1F</xref>), and the resistance of RT1 to sh-T1 and the restoration of the protein levels of cyclins E2 and A in RT1-reconstituted sh-T1 BT474 cells (<xref ref-type="fig" rid="fig1">Figure 1F</xref>). We further interrogated the mechanisms by which Taspase1 sustains cyclins levels. Quantitative real-time PCR (qRT-PCR) assays revealed that the mRNA levels of <italic>Cyclins E1, E2</italic>, and <italic>A</italic> were reduced in sh-T1 BT474 cells and were restored to baseline in RT1 sh-T1 BT474 cells (<xref ref-type="fig" rid="fig1">Figure 1G</xref>). Similar results were obtained utilizing HER2-transformed MCF10A cells (<xref ref-type="supplementary-material" rid="sup1">Supplementary information, Figure S1A and S1B</xref>). Altogether, these results indicate that Taspase1 controls the cell division cycle of HER2-positive breast cancer cells largely by conferring proper transcription of the <italic>Cyclins E</italic> and <italic>A</italic> genes upon aberrant receptor tyrosine kinase signaling.</p></sec><sec><title>Deletion of Taspase1 in mouse mammary glands blocks <italic>MMTV-neu</italic>-driven breast cancer formation</title><p>To determine whether Taspase1 is required for breast tumorigenesis <italic>in vivo</italic>, we generated <italic>MMTV-neu;MMTV-cre;Tasp1</italic><sup><italic>F</italic>/−</sup> mice by employing the widely adapted <italic>MMTV-neu</italic> mouse model<sup><xref ref-type="bibr" rid="bib15">15</xref>,<xref ref-type="bibr" rid="bib31">31</xref></sup>. <italic>MMTV-neu;MMTV-cre;Tasp1</italic><sup>+/+</sup> female mice were generated as positive controls and monitored for breast cancer formation. All of our <italic>MMTV-neu;MMTV-cre;Tasp1</italic><sup>+/+</sup> virgin female mice (<italic>n</italic> = 30) developed breast tumors between 30 and 50 weeks of age (<xref ref-type="fig" rid="fig2">Figure 2A, 2B</xref>), as did their counterparts in previously published studies<sup><xref ref-type="bibr" rid="bib15">15</xref>,<xref ref-type="bibr" rid="bib31">31</xref></sup>. In stark contrast, 26 of 30 <italic>MMTV-neu;MMTV-cre;Tasp1</italic><sup><italic>F</italic>/−</sup> females remained breast cancer free at 60 weeks of age (<xref ref-type="fig" rid="fig2">Figure 2A</xref>). Notably, western blot analysis of 12 week-old mammary glands detected similar levels of HER2/neu protein expression in both <italic>MMTV-neu;MMTV-cre;Tasp1</italic><sup>+/+</sup> and <italic>MMTV-neu;MMTV-cre;Tasp1</italic><sup><italic>F</italic>/−</sup> female virgin mice (<xref ref-type="supplementary-material" rid="sup1">Supplementary information, Figure S2</xref>). Interestingly, Taspase1 ablation did not block the tumor initiation in <italic>MMTV-Wnt;MMTV-cre;Tasp1</italic><sup><italic>F</italic>/−</sup> female mice, which suggests the specificity of Taspase1 function in HER2-driven breast cancer (<xref ref-type="fig" rid="fig2">Figure 2C-2E</xref>). Altogether, these <italic>in vivo</italic> data in conjunction with our <italic>in vitro</italic> assays unequivocally establish an essential role for Taspase1 in <italic>HER2/neu</italic>-induced breast tumorigenesis in mice and probably in humans.</p></sec><sec><title>Genetic deletion of Taspase1 impacts neither normal development nor physiological proliferation of mouse mammary glands</title><p>We next investigated the mechanisms by which loss of Taspase1 in the mammary gland protects <italic>MMTV-neu</italic> female mice from developing breast cancer by determining whether Taspase1 ablation simply disrupts mammary gland development and/or response to physiological proliferation signals such as pregnancy. To this end, we compared whole mounts of dissected, carmine-stained mammary glands from <italic>MMTV-cre;Tasp1</italic><sup>+/+</sup> and <italic>MMTV-cre;Tasp1</italic><sup><italic>F</italic>/−</sup> female mice, prepared at 6 weeks of age, day 13 of pregnancy, and day 1 of lactation. No macroscopic differences were detected between <italic>MMTV-cre;Tasp1</italic><sup>+/+</sup> and <italic>MMTV-cre;Tasp1</italic><sup><italic>F</italic>/−</sup> mice at any of these pre-specified developmental and physiological states (<xref ref-type="fig" rid="fig3">Figure 3</xref> and <xref ref-type="supplementary-material" rid="sup1">Supplementary information, Figure S3</xref>). Thus, ablation of Taspase1 in mouse mammary tissues using <italic>MMTV-cre</italic> has no effect on normal development or physiological proliferation of mammary glands. Of note, the importance of Taspase1 in breast tumorigenesis (<xref ref-type="fig" rid="fig2">Figure 2A</xref>) and its dispensability in normal mammary gland physiology (<xref ref-type="fig" rid="fig3">Figure 3</xref> and <xref ref-type="supplementary-material" rid="sup1">Supplementary information, Figure S3</xref>) are reminiscent of those reported with <italic>Cyclin D1</italic> knockout mice<sup><xref ref-type="bibr" rid="bib15">15</xref></sup>.</p></sec><sec><title>Taspase1 deficiency prevents tumor formation of <italic>MMTV-neu</italic> mammary glands</title><p>We analyzed mammary glands of transgenic mice to further dissect the mechanisms by which Taspase1 enables <italic>MMTV-neu-</italic> induced breast tumorigenesis. Whole mounts were performed using mammary glands of 12-week-old wild-type, <italic>MMTV-neu;MMTV-cre;Tasp1</italic><sup>+/+</sup>, and <italic>MMTV-neu;MMTV-cre;Tasp1</italic><sup><italic>F</italic>/−</sup> female mice to evaluate glandular structures. In comparison to those of wild-type control mice, mammary glands of <italic>MMTV-neu;MMTV-cre;Tasp1</italic><sup>+/+</sup> mice displayed increased densities in ducts and end buds (<xref ref-type="fig" rid="fig4">Figure 4A</xref>), similar to what was previously documented for this mouse strain<sup><xref ref-type="bibr" rid="bib31">31</xref></sup>. Interestingly, this overproliferation phenotype induced by the <italic>MMTV-neu</italic> transgene was not observed in the mammary glands of <italic>MMTV-neu;MMTV-cre;Tasp1</italic><sup><italic>F</italic>/−</sup> mice (<xref ref-type="fig" rid="fig4">Figure 4A</xref>). Immunohistochemistry for histone H3 serine 10 phosphorylation (pH3S10) which is a cell proliferation marker, showed that mammary glands of 12-week-old <italic>MMTV-neu;MMTV-cre;Tasp1</italic><sup>+/+</sup> mice had greater than 10-fold more pH3S10 positive cells than those of Wild-type and <italic>MMTV-neu;MMTV-cre;Tasp1</italic><sup><italic>F</italic>/−</sup> mice (<xref ref-type="fig" rid="fig4">Figure 4B</xref>). Altogether, these genetic data indicate that Taspase1 is required for the <italic>MMTV-neu</italic>-driven aberrant gland proliferation and thus breast carcinogenesis in mice.</p><p><italic>MMTV-neu</italic> mice can develop multiple microscopic mammary gland tumors at as early as 14 weeks of age<sup><xref ref-type="bibr" rid="bib31">31</xref></sup>. Accordingly, we examined mammary glands of 20-week-old wild-type, <italic>MMTV-neu;MMTV-cre;Tasp1</italic><sup>+/+</sup>, and <italic>MMTV-neu;MMTV-cre;Tasp1</italic><sup><italic>F</italic>/−</sup> female virgin mice by whole mounts. The mammary glands of <italic>MMTV-neu;MMTV-cre;Tasp1</italic><sup>+/+</sup> mice showed significant glandular proliferation and developed tumor foci of various sizes, whereas those of <italic>MMTV-neu;MMTV-cre;Tasp1</italic><sup><italic>F</italic>/−</sup> mice displayed neither overproliferation nor tumor foci, which are macroscopically indistinguishable from wild-type (<xref ref-type="fig" rid="fig4">Figure 4C</xref> and <xref ref-type="supplementary-material" rid="sup1">Supplementary information, Figure S4</xref>). Western blot analysis of 10-week-old wild-type, <italic>MMTV-neu;MMTV-cre;Tasp1</italic><sup>+/+</sup>, and <italic>MMTV-neu;MMTV-cre;Tasp1</italic><sup><italic>F</italic>/−</sup> mammary glands for cyclins E, A, and D, and CDKIs p16 and p27 detected consistent overexpression of cyclin E in <italic>MMTV-neu;MMTV-cre;Tasp1</italic><sup>+/+</sup> mice (<xref ref-type="fig" rid="fig4">Figure 4D</xref>). A higher level of Taspase1 expression was also detected in these <italic>MMTV-neu;MMTV-cre;Tasp1</italic><sup>+/+</sup> mammary glands, which is consistent with our prior observation that Taspase1 is commonly overexpressed in cancer cells<sup><xref ref-type="bibr" rid="bib26">26</xref></sup>. Moreover, examination of <italic>MMTV-neu;MMTV-cre;Tasp1</italic><sup>+/+</sup> breast tumors revealed that cyclins E, A, D, and Taspase1 were all expressed at high abundance (<xref ref-type="fig" rid="fig4">Figure 4D</xref>). The successful deletion of Taspase1 was observed in the <italic>MMTV-neu;MMTV-cre;Tasp1</italic><sup><italic>F</italic>/−</sup> mammary glands and remarkably, <italic>MMTV-neu</italic> was no longer able to induce cyclin E overexpression in these glands (<xref ref-type="fig" rid="fig4">Figure 4D</xref>). These data support the notion that overexpression of cyclin E and Taspase1 occurs early in <italic>MMTV-neu</italic>-driven breast tumorigenesis and precedes the overexpression of cyclins A and D.</p></sec><sec><title>Taspase1-mediated proteolytic cleavage of MLL is required for <italic>MMTV-neu</italic>-driven breast cancer formation but dispensable for mammary gland development</title><p>Our biochemical and genetic data thus far suggest that Taspase1 enables HER2/neu-driven breast tumorigenesis by permitting transcriptional activation of <italic>Cyclin E</italic> (<xref ref-type="fig" rid="fig1">Figures 1</xref>,<xref ref-type="fig" rid="fig2">2</xref>,<xref ref-type="fig" rid="fig3">3</xref>,<xref ref-type="fig" rid="fig4">4</xref>). Since Taspase1 cleaves nuclear factors MLL, MLL2, TFIIA, and ALF to regulate transcriptional programs, we asked the cleavage of which substrate(s) by Taspase1 is necessary for <italic>MMTV-neu</italic>-induced breast carcinogenesis. Of note, our prior biochemical studies in MEFs revealed that Taspase1 cleaves MLL to transactivate <italic>Cyclin</italic> E<sup><xref ref-type="bibr" rid="bib22">22</xref></sup>. However, MLL plays complex and context-dependent roles in cell cycle control. It can either positively or negatively regulate cell proliferation. For example, contrary to the positive regulation of cell proliferation that is more commonly observed, MLL was shown to negatively regulate pancreatic neuroendocrine cell proliferation. In this context MLL interacts with the tumor suppressor Menin to activate p27, a CDKI for cell cycle inhibition and thus prevent aberrant neoplastic proliferation<sup><xref ref-type="bibr" rid="bib32">32</xref></sup>. Furthermore, an independently generated non-cleavable MLL mouse model showed no overt proliferation defects in MEFs<sup><xref ref-type="bibr" rid="bib33">33</xref></sup>. Hence, it is of significance to address the role of MLL cleavage, if any, in <italic>MMTV-neu</italic>-driven breast tumorigenesis using genetic models. Our data suggested that specific ablation of Taspase1-mediated MLL cleavage could suppress <italic>MMTV-neu</italic>-driven breast cancer formation. We accordingly generated <italic>MMTV-neu;MLL<sup>nc/nc</sup></italic> transgenic mice that harbor homozygous Taspase1 non-cleavable (nc) alleles of <italic>MLL</italic>. Unlike <italic>Taspase1</italic><sup>−/−</sup> mice, which died prematurely, <italic>MLL<sup>nc/nc</sup></italic> mice were viable and fertile<sup><xref ref-type="bibr" rid="bib34">34</xref></sup>. Like <italic>MMTV-neu;MMTV-cre;Tasp1</italic><sup><italic>F</italic>/−</sup> mice (<xref ref-type="fig" rid="fig2">Figure 2A</xref>), most <italic>MMTV-neu;MLL<sup>nc/nc</sup></italic> females (27 of 30) were, remarkably, free of breast cancer at 60 weeks of age (<xref ref-type="fig" rid="fig5">Figure 5A</xref>). We then determined whether <italic>MLL<sup>nc/nc</sup></italic> females display any mammary gland defects. Whole mounts of <italic>MLL<sup>nc/nc</sup></italic> female mammary glands at 6 weeks of age, day 13 of pregnancy, and day 1 of lactation did not reveal any abnormalities (<xref ref-type="fig" rid="fig5">Figure 5B</xref>). Altogether, these results indicate that Taspase1-mediated MLL cleavage plays a critical role in <italic>MMTV-neu</italic>-induced breast carcinogenesis <italic>in vivo</italic>.</p></sec><sec><title>Non-cleavable MLL disrupts <italic>MMTV-neu</italic>-induced aberrant proliferation and cyclin E overexpression in mammary glands</title><p>We next investigated the molecular basis underlying <italic>MLL<sup>nc/nc</sup></italic> females' resistance to <italic>MMTV-neu</italic>-induced breast tumorigenesis by comparing the mammary glands of <italic>MMTV-neu;MLL<sup>nc/nc</sup></italic> female virgin mice to those of wild-type and <italic>MMTV-neu;MMTV-cre;Tasp1</italic><sup>+/+</sup> mice at 12 and 20 weeks of age. Consistently, the increased duct and end bud density induced by the <italic>MMTV-neu</italic> transgene was not observed in mammary glands of <italic>MMTV-neu;MLL<sup>nc/nc</sup></italic> mice (<xref ref-type="fig" rid="fig6">Figure 6A</xref>). We subsequently performed immunohistochemistry analysis for histone H3 serine 10 phosphorylation (pH3S10) on the mammary glands of 12-week-old <italic>MMTV-neu;MLL<sup>nc/nc</sup></italic> mice. Like <italic>MMTV-neu;MMTV-cre;Tasp1</italic><sup><italic>F</italic>/−</sup> and wild-type mice (<xref ref-type="fig" rid="fig4">Figure 4B</xref>), <italic>MMTV-neu;MLL<sup>nc/nc</sup></italic> mice had 10-fold fewer pH3S10+ cells in their mammary glands than <italic>MMTV-neu;MMTV-cre;Tasp1</italic><sup>+/+</sup> mice (<xref ref-type="fig" rid="fig6">Figure 6B</xref>). Finally, we examined the expression of cyclins E, A, D, and CDKIs p16 and p27 in the mammary glands of 10-week-old <italic>MMTV-neu;MLL<sup>nc/nc</sup></italic> mice. Non-cleavage of MLL suppressed <italic>MMTV-neu</italic>-induced cyclin E accumulation (<xref ref-type="fig" rid="fig6">Figure 6C</xref>), similar to what was observed in <italic>MMTV-neu;MMTV-cre;Tasp1</italic><sup><italic>F</italic>/−</sup> mice (<xref ref-type="fig" rid="fig4">Figure 4D</xref>). Overall, these genetic and biochemical results demonstrate that Taspase1 and thus mature MLL<sup>N320/C180</sup>, generated upon Taspase1-mediated cleavage of the precursor MLL<sup>500</sup>, are required for <italic>MMTV-neu</italic>-driven breast carcinogenesis, which involves transcriptional activation of <italic>Cyclin E</italic> in the mammary glands.</p></sec></sec><sec sec-type="discussion"><title>Discussion</title><p>The cloning of Taspase1 (<underline>t</underline>hreonine <underline>asp</underline>art<underline>ase</underline>) founded a novel class of endopeptidases that employ the NH2-terminal threonine of the mature β subunit to cleave protein substrates after P1 aspartate<sup><xref ref-type="bibr" rid="bib20">20</xref></sup>. In this study, using genetically well-defined mouse breast cancer models we show that Taspase1 is required for <italic>MMTV-neu</italic>-driven mammary tumorigenesis, which represents the first <italic>in vivo</italic> study demonstrating that Taspase1 ablation suppresses tumor initiation.</p><p>The <italic>mixed-lineage leukemia</italic>(<italic>MLL</italic>) gene encodes an epigenetic transcriptional regulator belonging to the <italic>trithorax</italic> group family<sup><xref ref-type="bibr" rid="bib35">35</xref></sup>. MLL is a confirmed substrate of Taspase1<sup><xref ref-type="bibr" rid="bib22">22</xref></sup>. In its best-known developmental role, MLL maintains proper expression of <italic>Hox</italic> genes and thus coordinates the segmental body plan of vertebrates<sup><xref ref-type="bibr" rid="bib36">36</xref>,<xref ref-type="bibr" rid="bib37">37</xref></sup>. Mice deficient for <italic>MLL</italic> (<italic>MLL</italic><sup>+/−</sup> and <italic>MLL</italic><sup>−/−</sup>) accordingly display homeotic defects in their axial skeleton<sup><xref ref-type="bibr" rid="bib37">37</xref></sup>. In addition to patterning body axis, MLL also regulates hematopoiesis, cell cycle, and cancer cell invasion<sup><xref ref-type="bibr" rid="bib24">24</xref>,<xref ref-type="bibr" rid="bib37">37</xref>,<xref ref-type="bibr" rid="bib38">38</xref>,<xref ref-type="bibr" rid="bib39">39</xref>,<xref ref-type="bibr" rid="bib40">40</xref>,<xref ref-type="bibr" rid="bib41">41</xref>,<xref ref-type="bibr" rid="bib42">42</xref></sup>. MLL possesses histone H3 lysine 4 (H3K4) methyl transferase (HMT) activity<sup><xref ref-type="bibr" rid="bib43">43</xref>,<xref ref-type="bibr" rid="bib44">44</xref>,<xref ref-type="bibr" rid="bib45">45</xref>,<xref ref-type="bibr" rid="bib46">46</xref></sup>. MLL-catalyzed H3K4 trimethylation (H3K4me3) activates transcription, leading to orchestrated upregulation of key developmental, cell cycle, and cancer cell invasion genes such as <italic>Hox</italic> genes, <italic>Cyclins</italic>
<italic>E</italic> and <italic>A</italic>, and <italic>MMP1</italic> and <italic>MMP3</italic>, respectively<sup><xref ref-type="bibr" rid="bib22">22</xref>,<xref ref-type="bibr" rid="bib24">24</xref>,<xref ref-type="bibr" rid="bib28">28</xref>,<xref ref-type="bibr" rid="bib45">45</xref>,<xref ref-type="bibr" rid="bib46">46</xref></sup>. Notably, the activity of MLL can be modulated by post-translational modifications, such as phosphorylation, ubiquitination, and proteolysis<sup><xref ref-type="bibr" rid="bib20">20</xref>,<xref ref-type="bibr" rid="bib41">41</xref>,<xref ref-type="bibr" rid="bib42">42</xref>,<xref ref-type="bibr" rid="bib47">47</xref></sup>. The 500-kDa precursor MLL (MLL<sup>500</sup>) undergoes Taspase1-mediated proteolytic cleavage, which gives rise to the mature MLL<sup>N320/C180</sup> heterodimer<sup><xref ref-type="bibr" rid="bib20">20</xref>,<xref ref-type="bibr" rid="bib25">25</xref></sup> that binds to <italic>Cyclins E</italic> and <italic>A</italic> promoters<sup><xref ref-type="bibr" rid="bib22">22</xref>,<xref ref-type="bibr" rid="bib28">28</xref></sup>. MLL forms complexes with E2Fs to methylate H3K4 at promoters, and thereby transactivates <italic>Cyclins E</italic> and <italic>A</italic><sup><xref ref-type="bibr" rid="bib22">22</xref>,<xref ref-type="bibr" rid="bib28">28</xref></sup>. In the absence of Taspase1, MLL exists as MLL<sup>500</sup>, a noncleaved precursor with reduced HMT activity<sup><xref ref-type="bibr" rid="bib20">20</xref>,<xref ref-type="bibr" rid="bib22">22</xref></sup>, and is unable to fully activate the expression of <italic>Cyclins E, A and B</italic><sup><xref ref-type="bibr" rid="bib22">22</xref></sup>. The <italic>in vivo</italic> significance of MLL cleavage has nonetheless been questioned on the grounds that whereas <italic>Taspase1</italic><sup>−/−</sup> mice exhibit diverse developmental defects, <italic>MLL<sup>nc/nc</sup></italic> mice are born at Mendelian ratio, fertile, and grossly normal<sup><xref ref-type="bibr" rid="bib24">24</xref>,<xref ref-type="bibr" rid="bib33">33</xref></sup>. Our genetic study here unequivocally establishes the <italic>in vivo</italic> significance of the Taspase1-MLL-cyclin E pathway in carcinogenesis by demonstrating that <italic>MMTV-neu;MLL<sup>nc/nc</sup></italic> mice are protected from <italic>MMTV-neu</italic>-driven breast cancer formation. Taspase1-mediated cleavage of MLL is therefore required for HER2/neu-induced tumorigenesis. However, whether cleavages of other Taspase1 substrates, such as MLL2, have roles in tumorigenesis remains to be determined.</p><p>HER2-amplified/overexpressed breast cancer is characterized by an aggressive phenotype and poor overall survival. Although the application of anti-HER2 therapy has improved the clinical outcome of HER2-positive breast cancers, primary and secondary resistance constitute major obstacles to the further success of such treatment strategy. As cyclin E amplification/overexpression in HER2-positive human breast cancers results in decreased sensitivity to the anti-HER2 agent trastuzumab, high levels of cyclin E in human HER2-positive breast cancer may be predictive of resistance to anti-HER2 therapy<sup><xref ref-type="bibr" rid="bib7">7</xref>,<xref ref-type="bibr" rid="bib16">16</xref>,<xref ref-type="bibr" rid="bib17">17</xref></sup>. Pharmacological inhibition of cyclin E expression may therefore benefit HER2-positive breast cancer patients by delaying disease progression and/or preventing recurrence while they are receiving anti-HER2 therapy. A series of studies including this current report clearly demonstrate that Taspase1 cleaves MLL to promote cyclin E expression and this Taspase1-MLL-cyclin E axis is required for HER2/neu induced breast tumorigenesis<sup><xref ref-type="bibr" rid="bib22">22</xref>,<xref ref-type="bibr" rid="bib26">26</xref></sup>. Therefore, administering Taspase1 inhibitors in conjunction with anti-HER2 agents could produce therapeutic benefits for HER2-positive breast cancer patients. As proteases are drug targets, small-molecule inhibitors of Taspase1 may be developed for cancer therapy.</p><p>Lines of evidence indicate the active participation of Taspase1 in tumorigenesis, and thus support the development of small molecule inhibitors of Taspase1 for potential cancer therapy. However, caution should be exercised while exploiting Taspase1 inhibition as a therapeutic means in treating human subjects. First and foremost, the severe perinatal lethality resulting from the embryonic loss of Taspase1 suggests that inactivation of Taspase1 by genetic or pharmacological means is inadvisable in pregnant females and children in order to avoid potential developmental sequelae<sup><xref ref-type="bibr" rid="bib22">22</xref></sup>. Nevertheless, inactivation of Taspase1 in fully developed adult mammals appears to be well-tolerated<sup><xref ref-type="bibr" rid="bib29">29</xref></sup>. Cancer commonly hijacks key developmental pathways during tumorigenesis and thus frequently exhibits unique properties such as stem cell-like and dedifferentiated states<sup><xref ref-type="bibr" rid="bib48">48</xref></sup>, which may underlie the preferential therapeutic benefit conferred by targeting Taspase1 to treat cancers.</p><p>Primitive <underline><underline>Tasp</underline></underline>ase1 <underline><underline>in</underline></underline>hibitors (TASPINs) were designed and discovered, lending support to developing highly effective, specific Taspase1 inhibitors for cancer therapy<sup><xref ref-type="bibr" rid="bib29">29</xref></sup>. Interestingly, although mammary gland–specific knockout of <italic>Taspase1</italic> disrupts <italic>MMTV-neu</italic>–driven breast tumorigenesis, the same genetic deletion of Taspase1 in <italic>MMTV-wnt;MMTV-cre;Tasp1</italic><sup><italic>F</italic>/−</sup> mice did not deter <italic>MMTV-wnt</italic>–driven breast carcinogenesis (<xref ref-type="fig" rid="fig2">Figure 2C</xref>). These data highlight the various mechanisms underlying individual tumorigenesis and the importance of selecting responsive cancers that might benefit from the treatment with TASPINs. Although it is beyond the scope of this study, it is of our interests to discover biomarkers that are predictive of Taspase1 addiction. Based on our unpublished data, Taspase1 has a very long protein half-life and is likely under multilayers of regulations in addition to transcription. Nevertheless, we analyzed the data of Taspase1 expression in HER2-positive and -negative tumors from breast TCGA dataset, and did not observe significant differences in Taspase1 expression (<xref ref-type="supplementary-material" rid="sup1">Supplementary information, Figure S5</xref>). Since Taspase1 is important in many aspects of cancer biology, Taspase1 inhibitors likely will benefit patients with different cancer types. Further studies with regard to the involvement of Taspase1 in various oncogenic pathways and the pathogenesis of subtypes of cancer could guide selection of cancer patients who would benefit from the inhibition of Taspase1.</p></sec><sec sec-type="materials|methods"><title>Materials and Methods</title><sec><title>Animal studies</title><p>All animal work was performed in accordance with MSKCC guidelines and IACUC approval. Mice were monitored for tumors by palpation twice a week. Tumor free Kaplan-Meier survival was calculated using MedCalc analysis software.</p></sec><sec><title>Cell culture, knockdown, proliferation, cell cycle, cell death, and western blot assays</title><p>BT-474 and HCC1419 cell lines were obtained from American Type Culture Collection and cultivated for no more than 2 months after each frozen aliquot was thawed. Amphotropic retrovirus carrying Taspase1 specific knockdown hairpin was generated as described<sup><xref ref-type="bibr" rid="bib26">26</xref>,<xref ref-type="bibr" rid="bib29">29</xref></sup>. To assay cell proliferation, 1 × 10<sup>5</sup> cells were seeded onto each well of a 6-well plate and counted 4 days later. Cell cycle and cell death analyses were performed as described<sup><xref ref-type="bibr" rid="bib26">26</xref></sup>. For western blot, cells and tissues were lysed in standard RIPA buffer. The anti-Taspase1 rabbit polyclonal antibody is as described<sup><xref ref-type="bibr" rid="bib22">22</xref>,<xref ref-type="bibr" rid="bib26">26</xref></sup>. Antibodies for cyclin E2 (4132, Cell Signaling), cyclin A (C4710, Sigma), p21 (sc-397, Santa Cruz Biotechnology), p27(sc-528, Santa Cruz Biotechnology), cyclin D1(sc-450, Santa Cruz Biotechnology), p16(554079, BD Pharmingen), and ErbB2(OP-15, Calbiochem) were purchased from indicated companies. Antibodies were detected using the enhanced chemiluminescence method (Western Lightning, PerkinElmer). Immunoblot signals were acquired with the LAS-3000 Imaging system (FujiFilm) and were analyzed with ImageJ software.</p></sec><sec><title>Knockdown resistant Taspase1</title><p>Knockdown resistant Taspase1 (RT1) was generated by site-directed mutagenesis to create synonymous mutations at amino acids 387 to 392 (GGA AAG GCG AAA ACG CAT) of Taspase1. The cDNA was inserted into an MSCV-neo expression construct.</p></sec><sec><title>Soft agar assay</title><p>1 × 10<sup>5</sup> cells were seeded onto a 6 cm dish containing a top layer of 0.3% noble agar and a bottom layer of 0.6% noble agar base. Cells were fed with media every 3 days. After 3 weeks, colonies with diameter larger than 200 μm were scored. Three independent triplicate experiments were performed.</p></sec><sec><title>Quantitative RT-PCR</title><p>mRNA was isolated using TRIzol (Invitrogen) and further purified using the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. Reverse transcription reactions were carried out as described with Superscript II (Invitrogen) and random decamer primers (Ambion)<sup><xref ref-type="bibr" rid="bib20">20</xref>,<xref ref-type="bibr" rid="bib22">22</xref>,<xref ref-type="bibr" rid="bib34">34</xref></sup>. Quantitative RT-PCR was performed using TaqMan 2× buffer and an ABI Prism 7300 sequence detection system (Applied Biosystems). For cyclin E1 and E2 reactions, the TaqMan Hs01026536 and Hs00180319 probes were used, and for cyclin A, SYBR Green master mix and the following primers were used: CAA AGC ACC ACA GCA TGC ACA AC and GAT TTA GTG TCT CTG GTG GGT TGA GG. All reactions were normalized against 18s rRNA using an 18s rRNA TaqMan probe (Applied Biosystems).</p></sec><sec><title>Mammary gland whole-mount assays</title><p>Mouse mammary glands were surgically dissected, spread onto a glass slide, and fixed in a 1:3:6 mixture of glacial acetic acid/chloroform/100% ethanol. Following hydration, they were stained overnight in 0.2% carmine and 0.5% ALK(SO<sub>4</sub>)<sub>2</sub>; dehydrated in graded solutions of ethanol, cleared in xylenes; and mounted with Permount.</p></sec><sec><title>Immunohistochemistry</title><p>Tissue samples were fixed in 4% paraformaldehyde and embedded in paraffin. Sections of 5-μm thickness were prepared. pH3S10 was detected by immunohistochemistry using the antibody from Millipore (06-570).</p></sec><sec><title>Statistical analysis</title><p>Student's <italic>t</italic>-test was performed to compare means between two groups. Data were expressed as the mean ± SD or ± sem as indicated.</p></sec><sec><title>Taspase1 gene expression in human breast cancer</title><p>To determine whether Taspase1 (Tasp1) expression differs in HER2 positive and HER2 negative human breast cancer, normalized RNA sequencing (RNA-Seq) data produced by The Cancer Genome Atlas (TCGA)<sup><xref ref-type="bibr" rid="bib49">49</xref></sup> were downloaded from Broad GDAC Firehose. PAM50 subtype classifications were available for 500 of the 526 primary breast invasive carcinoma tumor samples TCGA subjected to mRNA expression profiling using the Illumina HiSeq 2000 RNA Sequencing Version 2 platform. The subtype classifications were obtained through cBioPortal for Cancer Genomics, and the 26 samples lacking classifications were discarded. The Tasp1 mRNA expression values of the remaining samples formed a dataset that was partitioned by HER2 status to form HER2 positive and HER2 negative datasets. Two-tailed Mann-Whitney tests were performed to compare the means of the HER2 positive and HER2 negative datasets.</p></sec></sec> |
Postzygotic single-nucleotide mosaicisms in whole-genome sequences of clinically unremarkable individuals | <p>Postzygotic single-nucleotide mutations (pSNMs) have been studied in cancer and a few other overgrowth human disorders at whole-genome scale and found to play critical roles. However, in clinically unremarkable individuals, pSNMs have never been identified at whole-genome scale largely due to technical difficulties and lack of matched control tissue samples, and thus the genome-wide characteristics of pSNMs remain unknown. We developed a new Bayesian-based mosaic genotyper and a series of effective error filters, using which we were able to identify 17 SNM sites from ∼80× whole-genome sequencing of peripheral blood DNAs from three clinically unremarkable adults. The pSNMs were thoroughly validated using pyrosequencing, Sanger sequencing of individual cloned fragments, and multiplex ligation-dependent probe amplification. The mutant allele fraction ranged from 5%-31%. We found that C→T and C→A were the predominant types of postzygotic mutations, similar to the somatic mutation profile in tumor tissues. Simulation data showed that the overall mutation rate was an order of magnitude lower than that in cancer. We detected varied allele fractions of the pSNMs among multiple samples obtained from the same individuals, including blood, saliva, hair follicle, buccal mucosa, urine, and semen samples, indicating that pSNMs could affect multiple sources of somatic cells as well as germ cells. Two of the adults have children who were diagnosed with Dravet syndrome. We identified two non-synonymous pSNMs in <italic>SCN1A</italic>, a causal gene for Dravet syndrome, from these two unrelated adults and found that the mutant alleles were transmitted to their children, highlighting the clinical importance of detecting pSNMs in genetic counseling.</p> | <contrib contrib-type="author"><name><surname>Huang</surname><given-names>August Y</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="aff" rid="aff2">2</xref><xref ref-type="author-notes" rid="note1"><sup>*</sup></xref></contrib><contrib contrib-type="author"><name><surname>Xu</surname><given-names>Xiaojing</given-names></name><xref ref-type="aff" rid="aff3">3</xref><xref ref-type="author-notes" rid="note1"><sup>*</sup></xref></contrib><contrib contrib-type="author"><name><surname>Ye</surname><given-names>Adam Y</given-names></name><xref ref-type="aff" rid="aff2">2</xref><xref ref-type="aff" rid="aff4">4</xref><xref ref-type="aff" rid="aff5">5</xref><xref ref-type="author-notes" rid="note1"><sup>*</sup></xref></contrib><contrib contrib-type="author"><name><surname>Wu</surname><given-names>Qixi</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="author-notes" rid="note1"><sup>*</sup></xref></contrib><contrib contrib-type="author"><name><surname>Yan</surname><given-names>Linlin</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Zhao</surname><given-names>Boxun</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="aff" rid="aff6">6</xref></contrib><contrib contrib-type="author"><name><surname>Yang</surname><given-names>Xiaoxu</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>He</surname><given-names>Yao</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="aff" rid="aff2">2</xref><xref ref-type="aff" rid="aff4">4</xref><xref ref-type="aff" rid="aff5">5</xref></contrib><contrib contrib-type="author"><name><surname>Wang</surname><given-names>Sheng</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Zhang</surname><given-names>Zheng</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="aff" rid="aff2">2</xref><xref ref-type="aff" rid="aff4">4</xref><xref ref-type="aff" rid="aff5">5</xref></contrib><contrib contrib-type="author"><name><surname>Gu</surname><given-names>Bowen</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Zhao</surname><given-names>Han-Qing</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Wang</surname><given-names>Meng</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Gao</surname><given-names>Hua</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Gao</surname><given-names>Ge</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Zhang</surname><given-names>Zhichao</given-names></name><xref ref-type="aff" rid="aff3">3</xref></contrib><contrib contrib-type="author"><name><surname>Yang</surname><given-names>Xiaoling</given-names></name><xref ref-type="aff" rid="aff3">3</xref></contrib><contrib contrib-type="author"><name><surname>Wu</surname><given-names>Xiru</given-names></name><xref ref-type="aff" rid="aff3">3</xref></contrib><contrib contrib-type="author"><name><surname>Zhang</surname><given-names>Yuehua</given-names></name><xref ref-type="aff" rid="aff3">3</xref><xref ref-type="corresp" rid="caf2">*</xref></contrib><contrib contrib-type="author"><name><surname>Wei</surname><given-names>Liping</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="aff" rid="aff2">2</xref><xref ref-type="corresp" rid="caf1">*</xref></contrib><aff id="aff1"><label>1</label><institution>National Institute of Biological Sciences</institution>, Beijing 102206, <country>China</country></aff><aff id="aff2"><label>2</label><institution>Center for Bioinformatics, State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University</institution>, Beijing 100871, <country>China</country></aff><aff id="aff3"><label>3</label><institution>Peking University First Hospital, Peking University</institution>, Beijing 100034, <country>China</country></aff><aff id="aff4"><label>4</label><institution>Peking-Tsinghua Center for Life Sciences</institution>, Beijing 100871, <country>China</country></aff><aff id="aff5"><label>5</label><institution>Academy for Advanced Interdisciplinary Studies, Peking University</institution>, Beijing 100871, <country>China</country></aff><aff id="aff6"><label>6</label><institution>Graduate School of Peking Union Medical College</institution>, Beijing 100730, <country>China</country></aff> | Cell Research | <sec sec-type="intro"><title>Introduction</title><p>Genomic mosaicism is a biological phenomenon in which genetic alterations occurring during development or aging give rise to two or more cell populations with distinct genome sequences within one individual<sup><xref ref-type="bibr" rid="bib1">1</xref>,<xref ref-type="bibr" rid="bib2">2</xref></sup>. The DNA alterations in a fraction of somatic and/or germ cells can occur at different genomic scales, varying from chromosomal abnormalities and copy number variations (CNVs) to small indels and single-nucleotide substitutions<sup><xref ref-type="bibr" rid="bib1">1</xref>,<xref ref-type="bibr" rid="bib3">3</xref></sup>. Comparisons of the whole-genome or whole-exome sequencing data from affected vs normal control tissues in the same person have discovered the role of mosaicism in multiple types of cancer<sup><xref ref-type="bibr" rid="bib4">4</xref>,<xref ref-type="bibr" rid="bib5">5</xref>,<xref ref-type="bibr" rid="bib6">6</xref></sup> as well as several overgrowth disorders including Proteus syndrome<sup><xref ref-type="bibr" rid="bib7">7</xref></sup>, Ollier disease and Maffucci syndrome<sup><xref ref-type="bibr" rid="bib8">8</xref></sup>, CLOVES syndrome<sup><xref ref-type="bibr" rid="bib9">9</xref></sup>, Schimmelpenning syndrome<sup><xref ref-type="bibr" rid="bib10">10</xref></sup>, Sturge-Weber syndrome<sup><xref ref-type="bibr" rid="bib11">11</xref></sup>, and several types of brain malformations<sup><xref ref-type="bibr" rid="bib12">12</xref>,<xref ref-type="bibr" rid="bib13">13</xref>,<xref ref-type="bibr" rid="bib14">14</xref></sup>.</p><p>In theory every person is a mosaic. Indeed, many sporadic cases of mosaicism have been reported in clinically unremarkable persons<sup><xref ref-type="bibr" rid="bib15">15</xref></sup>, sometimes parents of children with a genetic disease, highlighting the clinical importance of mosaicism in genetic counseling. Unfortunately, at the whole-genome scale, only relatively large mosaicisms have been identified in clinically unremarkable individuals<sup><xref ref-type="bibr" rid="bib16">16</xref></sup>. These include structural variations and CNVs by analyses of array comparative genomic hybridization or SNP microarray<sup><xref ref-type="bibr" rid="bib17">17</xref>,<xref ref-type="bibr" rid="bib18">18</xref>,<xref ref-type="bibr" rid="bib19">19</xref>,<xref ref-type="bibr" rid="bib20">20</xref></sup> and neuronal somatic retrotransposition events using transposon-specific targeted sequencing<sup><xref ref-type="bibr" rid="bib21">21</xref>,<xref ref-type="bibr" rid="bib22">22</xref></sup>.</p><p>The mosaicism caused by postzygotic single-nucleotide mutations (pSNMs), on the other hand, have not been identified in clinically unremarkable persons at genome scale. Existing algorithms for identifying pSNMs from whole-genome or targeted resequencing data require a matched control sample, such as JointSNVMix<sup><xref ref-type="bibr" rid="bib23">23</xref></sup>, Varscan 2<sup><xref ref-type="bibr" rid="bib24">24</xref></sup>, Strelka<sup><xref ref-type="bibr" rid="bib25">25</xref></sup>, EBCall<sup><xref ref-type="bibr" rid="bib26">26</xref></sup>, muTect<sup><xref ref-type="bibr" rid="bib27">27</xref></sup>, Mutascope<sup><xref ref-type="bibr" rid="bib28">28</xref></sup>, and LoFreq<sup><xref ref-type="bibr" rid="bib29">29</xref></sup>. As a result, fundamental patterns of the pSNMs in whole genomes of clinically unremarkable individuals remain largely unknown, such as the prevalence, allele fractions, mutation characteristics, tissue variations, and transmissions to offspring. The study of these patterns is the goal of our research, starting with the development of a new detection method based on next-generation sequencing, a Bayesian genotyper, and stringent error filters.</p></sec><sec sec-type="results"><title>Results</title><sec><title>A Bayesian model and error filters for the detection of pSNM</title><p>We detected the mosaic sites led by pSNMs and quantified their allele fractions in the peripheral blood of three clinically unremarkable adults using whole-genome sequences produced by Illumina Hiseq platform. Pre-processing of reads was done with standard protocols (see Materials and Methods). As we focused only on pSNMs here, we used CNVnator<sup><xref ref-type="bibr" rid="bib30">30</xref></sup> and GATK<sup><xref ref-type="bibr" rid="bib31">31</xref></sup> to mask CNVs and indels, respectively. The challenges of detecting pSNMs in non-overgrowth individuals without matched control samples involve distinguishing true mosaic sites from germline heterozygous and homozygous sites, and base-calling and alignment errors<sup><xref ref-type="bibr" rid="bib32">32</xref></sup>. To address these challenges, we developed a new Bayesian-based genotyper and a series of stringent error filters, summarized in <xref ref-type="fig" rid="fig1">Figure 1A</xref> and below and detailed in Materials and Methods.</p><p>Bayesian probabilistic models are commonly used to distinguish three germline genotypes: homozygous for the reference allele (ref-hom), heterozygous, and homozygous for the alternative allele (alt-hom)<sup><xref ref-type="bibr" rid="bib33">33</xref></sup>. To distinguish the mosaic sites from germline sites, we introduced a new genotype state, named “mosaic”, into the Bayesian model. Our new model aimed at identifying and measuring the departure of observed allele fractions from germline expectations (0, 0.5 and 1), which we formulated as:</p><p><disp-formula id="equ1"><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="cr2014131eq1.jpg"/></disp-formula></p><p>This model was able to incorporate known population genetics information and sequencing data characteristics to aid mosaic detection (<xref ref-type="fig" rid="fig1">Figure 1B</xref>). Specifically, <italic>Data</italic> was the bases, base qualities, and total sequencing depth from the aligned sequencing reads at a position. <italic>P</italic>(<italic>G<sub>i</sub></italic>) was the prior probability of each genotype, estimated using population genetic data from dbSNP and theoretically estimated somatic mutation rate<sup><xref ref-type="bibr" rid="bib34">34</xref></sup>. Non-pseudoautosomal regions of sex chromosomes in males were modeled as a haploid. <italic>P</italic>(<italic>Data</italic>|<italic>G<sub>i</sub></italic>) captures sequencing data characteristics such as base-calling errors and read depth biases with a likelihood estimation based on Bernoulli sampling and binomial distribution. Sites where the posterior probability of mosaic genotype was greater than 0.05 were considered candidate sites for the next step. As shown in <xref ref-type="fig" rid="fig1">Figure 1C</xref>, our genotyper was able to detect a mosaic site whose alternative allele fraction was around 0.05-0.35 and 0.65-0.95 when the sequencing depth reached 80. Increasing sequencing depth could improve the power to distinguish between mosaic and heterozygous sites, whereas increasing base quality could be helpful in distinguishing between mosaic and homozygous sites.</p><p>However, the Bayesian probabilistic model could not remove the large number of false positives caused by systematic errors in read alignment and base calling. We implemented a series of empirical error filters (<xref rid="tbl1" ref-type="table">Table 1</xref>). Genomic regions known to cause frequent errors were removed, including repetitive regions and homopolymers. Abnormal patterns of alignment were filtered out, such as extremely high or low read depth, high percentage of misaligned reads, high strand bias of the alternative allele, and skewed alignment position of the alternative allele. Abnormal local patterns were filtered out including clustered sites which were most likely to be within heterochromatin or missed CNV regions (see Materials and Methods), and sites with complete linkage with an adjacent polymorphic site which were most likely due to misalignment of paralogous regions. Finally, we filtered out sites whose allele fractions showed large deviations from germline expectations in two or more individuals because the likelihood of hotspot pSNMs in healthy individuals was presumed to be orders of magnitude smaller than the likelihood of recurrent systematic sequencing bias.</p><p>We demonstrated that applying the filters could dramatically decrease the discrepancies between the observed and expected distributions of allele fractions, suggesting that the filters successfully removed the majority of technical artifacts (<xref ref-type="supplementary-material" rid="sup1">Supplementary information, Figure S1</xref>). To further evaluate the efficacy of the error filters, we generated a benchmark dataset of simulated homozygous and heterozygous sites by <italic>in silico</italic> mixing of actual Hiseq sequencing reads from two well-genotyped individuals (see Materials and Methods). <xref ref-type="fig" rid="fig1">Figure 1D</xref> showed the effectiveness of the filters in removing false positives. Only 1 of 15 842 simulated heterozygous sites and none of 19 624 simulated homozygous sites were misclassified as mosaic.</p><p>In cases where matched control tissues are available, utilizing data from the matched controls may increase detection accuracy for pSNMs. We implemented a paired-sample mode of our pipeline to utilize sequencing information in control sample (see Materials and Methods). We used two sets of simulation data to evaluate the specificity and precision, and compared the performance of the single-sample mode and paired-sample mode of our pipeline against Varscan 2<sup><xref ref-type="bibr" rid="bib24">24</xref></sup> and muTect<sup><xref ref-type="bibr" rid="bib27">27</xref></sup>. Both the single-sample mode and the paired-sample mode of our pipeline achieved higher specificity than Varscan 2 and muTect in true reference sites (<xref ref-type="fig" rid="fig2">Figure 2A</xref>) and true non-reference sites (<xref ref-type="fig" rid="fig2">Figure 2B</xref>), suggesting that our pipeline can effectively remove false positives. As shown in <xref ref-type="fig" rid="fig2">Figure 2C</xref>, without the need of matched control sample, the precision of the single-sample mode of our pipeline was above 50% to identify pSNMs in healthy individuals when the allele fractions were 0.1-0.3. With matched control sample, the paired-sample mode of our pipeline achieved over 90% precision for all the pSNMs whose allele fraction were greater than 0.1, and outperformed both Varscan 2 and muTect (<xref ref-type="fig" rid="fig2">Figure 2C</xref>).</p></sec><sec><title>pSNMs in the peripheral blood of three clinically unremarkable individuals</title><p>We sequenced the whole genomes of the unamplified peripheral blood DNA samples from three unrelated adults, ACC1-II-1, DS1-II-2 and DS2-I-1, with an average sequencing depth of 76-90× (<xref ref-type="supplementary-material" rid="sup1">Supplementary information, Table S1</xref>). ACC1-II-1 and DS2-I-1 had no diagnosable symptoms at present and no clinical history. DS1-II-2 had two episodes of mild seizures at age 4-5 but had been subsequently seizure-free with normal cognitive function and no diagnosable symptoms at present. DS1-II-2 and DS2-I-1 were the mother and the father of two unrelated children diagnosed with Dravet syndrome (<xref ref-type="fig" rid="fig3">Figure 3A-3C</xref>). Applying our Bayesian model and error filters, we identified 38 candidate pSNMs in the three individuals (<xref ref-type="supplementary-material" rid="sup1">Supplementary information, Table S2</xref>). These sites showed a distinguishable pattern of allele fractions from the backgrounds of germline polymorphic sites (<xref ref-type="fig" rid="fig3">Figure 3D-3F</xref>).</p><p>Validation of the candidate pSNMs were performed by pyrosequencing, Sanger sequencing of individual cloned fragments, and multiplex ligation-dependent probe amplification (MLPA). The validated pSNMs were listed in <xref rid="tbl2" ref-type="table">Table 2</xref>.</p><p>First, eighteen of the 38 sites were confirmed by pyrosequencing above the allele fraction threshold of 5% which was the detection threshold of the pyrosequencing technology<sup><xref ref-type="bibr" rid="bib35">35</xref>,<xref ref-type="bibr" rid="bib36">36</xref></sup>. The alternative alleles were present in the corresponding sample, and absent in the control sample obtained from an unrelated individual (<xref ref-type="fig" rid="fig3">Figure 3G</xref>). In addition, two pSNMs whose alternative allele fractions did not exceed 5% by pyrosequencing showed a statistically significant difference between the case and three negative control samples (one-tailed <italic>Z</italic>-test, <italic>P</italic>-value < 2.2 × 10<sup>−16</sup>), and were thus also included in subsequent validation.</p><p>Second, for these 20 sites, we further confirmed the presence (not the allele fraction) of the alternative allele by Sanger sequencing of individual clones after TA-cloning the amplicons. The presence of reference and alternative alleles was confirmed for 19 sites with at least two, and in most cases three or more, independent clones (<xref rid="tbl2" ref-type="table">Table 2</xref> and <xref ref-type="supplementary-material" rid="sup1">Supplementary information, Table S3</xref>).</p><p>Third, as CNVnator may have missed some DNA copy number alterations which could cause abnormal allele fraction<sup><xref ref-type="bibr" rid="bib37">37</xref></sup>, we further performed MLPA on the 19 pSNMs which were validated by both pyrosequencing and clonal Sanger sequencing. DNA copy number gain was detected for two sites in DS2-I-1. These two sites had alternative allele fractions of ∼1/3 in both whole-genome sequencing and pyrosequencing data (<xref ref-type="fig" rid="fig3">Figure 3G</xref>), consistent with the expected allele fraction when three copies of DNA were present. The other 17 candidate pSNMs showed normal copy numbers (<xref ref-type="fig" rid="fig3">Figure 3H</xref>). These 17 pSNMs were validated by all three technologies and considered <italic>bona fide</italic> pSNMs.</p><p>In summary, we identified 17 pSNMs from the peripheral blood samples of three clinically unremarkable individuals and validated them using pyrosequencing (to validate the presence and fraction of the mosaic mutant allele), clonal Sanger sequencing (to validate the presence of the mosaic mutant allele), and MLPA (to rule out copy number alterations). Because the vast majority of genomic positions were not mosaic, some false positives were inevitable despite our stringent pipeline. The current validation rate was 45% (17/38). It could be increased to 70% (14/20) if we increased the Bayesian posterior probability threshold from 0.05 to 0.5.</p><p>By counting the allele numbers in the whole-genome sequencing data, we calculated the alternative allele fractions of the validated pSNMs, which range from 5% to 31% (<xref rid="tbl2" ref-type="table">Table 2</xref>). The quantification accuracy was justified by the significant correlation between the allele fractions estimated by whole-genome sequencing and pyrosequencing (Pearson's <italic>r</italic> = 0.79 and <italic>P</italic>-value = 0.0001, <xref ref-type="fig" rid="fig3">Figure 3I</xref>).</p><p>For ACC1-II-1 and DS1-II-2 whose parents' blood samples were available, pyrosequencing confirmed the absence of the alternative alleles in their parents (<xref ref-type="fig" rid="fig3">Figure 3G</xref>), which suggested postzygotic but not inherited origin of the mutant pSNM alleles. We also tried to assess the candidate pSNMs by direct Sanger sequencing of the PCR products, but found that mosaic could be unequivocally detected in only eight sites (<xref ref-type="supplementary-material" rid="sup1">Supplementary information, Table S3</xref>), which gave a warning that direct Sanger sequencing had low sensitivity in mosaic detection.</p></sec><sec><title>Characteristics of pSNMs in healthy individuals</title><p>The validated pSNMs enabled us to take a first look at the mutational spectrum of pSNMs in healthy individuals. Because it was unlikely that postzygotic mutations affected both alleles at a single genomic position, the allele generated by postzygotic mutation was expected to be the minor allele in pSNM and, therefore, distinguished from the ancestral allele. Among the validated sites, C→T and C→A were the two most common mutation types at 24% each (<xref ref-type="fig" rid="fig4">Figure 4A</xref>), followed by T→G and T→C mutations. These two predominant mutation types were also reported in previous cancer studies<sup><xref ref-type="bibr" rid="bib4">4</xref></sup>, suggesting possible shared mechanisms of somatic mutation between cancer and non-cancer samples.</p><p>A question of interest was, “what would be the allele fraction of the pSNMs in other samples collected non-invasively from the same healthy individuals?” In addition to the peripheral blood samples, we were able to collect saliva, hair follicle, buccal mucosa, and urine samples from ACC1-II-1 and DS1-II-2, as well as semen sample from ACC1-II-1. We performed pyrosequencing of the validated pSNMs in these samples. As shown in <xref ref-type="fig" rid="fig4">Figure 4B</xref>, for a few pSNMs, the mutant alleles were not detected in some samples, indicating the presence of lineage-specific pSNMs where the postzygotic mutation might have occurred after the differentiation of specific cell lineages. Most of the pSNMs, however, could be detected in multiple samples. Of particular interest, in three of the five pSNMs of ACC1-II-1, the mutant alleles were observed in both the blood and semen samples, suggesting that postzygotic mutations could affect both the somatic and germ cells (<xref ref-type="fig" rid="fig4">Figure 4B</xref>).</p><p>The inter-sample variations in allele fractions differed widely among the pSNM sites, with the coefficients of variation ranging from 25%-137%. We further demonstrated that the inter-sample variation was not caused by technical variation of DNA extraction and pyrosequencing: we extracted the genomic DNA from the blood sample of ACC1-II-1 three times, and each DNA sample was pyrosequenced three times. Among five mosaic sites, the inter-sample coefficients of variation (25%-137%) was an order of magnitude higher than the average coefficients of variation between different pyrosequencing runs (4.6%) and between different DNA extractions (5.2%) (<xref ref-type="supplementary-material" rid="sup1">Supplementary information, Figure S2</xref>). Hierarchical clustering of the minor allele fractions in the six types of samples showed that the peripheral blood and saliva samples were the most similar, followed by the buccal mucosa and semen samples (<xref ref-type="fig" rid="fig4">Figure 4C</xref>). This finding was consistent with previous reports that 74% of the DNA extracted from saliva samples and 21% from the buccal swab samples were from leukocytes<sup><xref ref-type="bibr" rid="bib38">38</xref></sup>. In addition, our results showed that the hair follicle and urine samples were the least similar to the other samples, indicating that these samples consisted of cell populations with more distant lineages compared to blood samples.</p><p>We showed that our detection pipeline had good specificity and was able to detect the small number of pSNMs among millions of germline polymorphic sites and sequencing errors. To estimate the sensitivity of the detection pipeline and infer the total number of pSNMs in clinically unremarkable individuals, we performed a computer simulation to generate ∼20 000 simulated mosaic sites each at varied minor allele fractions by mixing the real sequencing data (see Materials and Methods). The sensitivity of pSNM detection in non-repetitive genomic regions depended on the alternative allele fraction. The estimated sensitivity ranged from as high as 30% for pSNMs with alternative allele fraction 0.2 to as low as 1% for pSNMs with alternative allele fraction 0.4 (<xref ref-type="supplementary-material" rid="sup1">Supplementary information, Figure S3</xref>). Because we detected an average of 5.7 validated pSNMs in each individual, we propose that, based on the estimated sensitivity, a clinically unremarkable person might harbor ∼19-570 pSNMs in non-repetitive regions with minor allele fractions from 5%-40%, which corresponded to ∼1.5 × 10<sup>−8</sup>-4.4 × 10<sup>−7</sup> per nucleotide per individual. This was significantly lower than the somatic mutation rate in tumors, where non-silent somatic mutation rate had been estimated to be varied between 1 × 10<sup>−7</sup> and 1 × 10<sup>−4</sup> across different cancer types, with an average of 4 × 10<sup>−6</sup><sup><xref ref-type="bibr" rid="bib4">4</xref></sup>.</p></sec><sec><title>Clinical implications of detecting pSNMs</title><p>We found a non-synonymous c.5003C→G pSNM with 27% allele fraction and a non-synonymous c.4351C→A pSNM with 22% allele fraction in the <italic>SCN1A</italic> gene of DS1-II-2 and DS2-I-1, respectively. DS1-II-2 and DS2-I-1 each had a son with Dravet syndrome (DS1-III-1 and DS2-II-2). DS1-III-1 was heterozygous with c.5003C→G, and DS2-II-2 was heterozygous with c.4351C→A, in the <italic>SCN1A</italic> gene. Dravet syndrome is a rare and catastrophic form of intractable epilepsy that begins in infancy, and <italic>SCN1A</italic> is well established as the major causal gene for this disease<sup><xref ref-type="bibr" rid="bib39">39</xref>,<xref ref-type="bibr" rid="bib40">40</xref></sup>. We ruled out other possible causal mutations by sequencing all the exons of <italic>SCN1A</italic> and five other rare causal genes including <italic>PCDH19</italic><sup><xref ref-type="bibr" rid="bib41">41</xref></sup>, <italic>GABRG2</italic><sup><xref ref-type="bibr" rid="bib42">42</xref></sup>, <italic>SCN1B</italic><sup><xref ref-type="bibr" rid="bib43">43</xref></sup>, <italic>GABRA1</italic><sup><xref ref-type="bibr" rid="bib44">44</xref></sup>, and <italic>STXBP1</italic><sup><xref ref-type="bibr" rid="bib44">44</xref></sup> in these two boys. After filtering out the silent and common variants present in dbSNP, only one <italic>SCN1A</italic> non-synonymous mutation remained in each boy, which was exactly the same alternative allele at exactly the same positions as the two pSNMs identified in their respective parents. Among all the identified pSNMs sites in their parents, these two sites in <italic>SCN1A</italic> were the only sites where the mutant alleles were inherited. The two non-synonymous sites were located in the third and fourth domains of <italic>SCN1A</italic>, respectively, adjacent to previously identified pathogenic mutations of Dravet syndrome (<xref ref-type="fig" rid="fig5">Figure 5A</xref>). They were predicted to be deleterious by PolyPhen2 (score = 0.793 and 0.679)<sup><xref ref-type="bibr" rid="bib45">45</xref></sup>, SIFT (score = 0.001 and 0.000)<sup><xref ref-type="bibr" rid="bib46">46</xref></sup>, and SAPRED (likelihood = 0.878 and 0.867)<sup><xref ref-type="bibr" rid="bib47">47</xref></sup>. Using both pyrosequencing and Sanger sequencing, we confirmed that the two boys' other parents, DS1-II-1 and DS2-I-2, did not carry the mutant allele, and thus the causal <italic>SCN1A</italic> variants were inherited from the mosaic parents (<xref ref-type="supplementary-material" rid="sup1">Supplementary information, Table S4</xref>).</p><p>Our results highlighted the importance of accurate detection of pSNMs in genetic counseling. Consistent with previous studies<sup><xref ref-type="bibr" rid="bib48">48</xref>,<xref ref-type="bibr" rid="bib49">49</xref></sup>, our results showed that a clinically unremarkable carrier of a deleterious postzygotic mutation might transmit it to offspring and generate a heterozygous genotype that may cause serious genetic diseases (<xref ref-type="fig" rid="fig5">Figure 5B</xref>). Considering cell proliferation in embryogenesis, an pSNM with relatively higher mutant allele fraction might be more likely generated by postzygotic mutations at the early developmental stage and, therefore, be more likely shared between somatic and germ cells. As our results showed, simple Sanger sequencing was unable to detect more than half of the pSNMs. Furthermore, applying conventional genotyper GATK<sup><xref ref-type="bibr" rid="bib31">31</xref></sup> to our dataset resulted in 35% of the validated pSNMs being mistakenly genotyped as homozygous for the reference allele. Thus, some of the pathogenic genetic mutations currently believed to emerge <italic>de novo</italic> in affected children might be caused by transmitted parental mosaicism that were missed by Sanger sequencing or conventional genotypers<sup><xref ref-type="bibr" rid="bib16">16</xref>,<xref ref-type="bibr" rid="bib50">50</xref></sup>.</p></sec></sec><sec sec-type="discussion"><title>Discussion</title><p>Postzygotic single-nucleotide mutations had not been previously studied at genome scale in clinically unremarkable individuals, largely due to technical challenges caused by sequencing errors and the lack of matched control tissue. Our Bayesian model and error filters allowed us to detect pSNMs in all three clinically unremarkable individuals and enabled us to take a first look at the characteristics of pSNMs. Many factors may be involved in the generation of mutations, including external mutagens and spontaneous cellular processes<sup><xref ref-type="bibr" rid="bib51">51</xref></sup>. Interestingly, we observed similar mutational spectra as in cancer samples, but at lower mutation rate. The higher mutation rate in cancer may result from the occurrence of accelerated mutagenesis after the dysfunction of the DNA replication and repair systems that is common in many types of cancer<sup><xref ref-type="bibr" rid="bib51">51</xref></sup>.</p><p>The parent-to-offspring transmission of mutant <italic>SCN1A</italic> alleles highlighted the clinical implications of genome-wide identification of pSNMs in genetic counseling. In addition, mosaicisms have also previously been reported to cause diseases, often with milder symptoms than homozygous or heterozygous mutations<sup><xref ref-type="bibr" rid="bib3">3</xref>,<xref ref-type="bibr" rid="bib52">52</xref></sup>. DS1-II-2, who had a pSNM at <italic>SCN1A</italic> at allele fraction of 27% in her peripheral blood, had normal cognitive function and no diagnosable symptoms at present, but she had two episodes of mild seizure at four years old. In her whole genome sequence we identified non-synonymous mutations in three other genes that were in the epilepsy gene database CarpeDB (<ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://www.carpedb.ua.edu/">http://www.carpedb.ua.edu/</ext-link>), <italic>CASQ2</italic>, <italic>ALDH7A1</italic>, and <italic>CACNA1H</italic>, which were not present in the individuals of 1000 Genomes Project or dbSNP. Among them the mutation in <italic>ALDH7A1</italic> was predicted to be damaging by PolyPhen2<sup><xref ref-type="bibr" rid="bib45">45</xref></sup>, SIFT<sup><xref ref-type="bibr" rid="bib46">46</xref></sup>, and SAPRED<sup><xref ref-type="bibr" rid="bib47">47</xref></sup>. It would be interesting to investigate whether this mutation or her pSNM at <italic>SCN1A</italic> was the cause of her childhood seizure, but that was beyond the scope of this study.</p><p>Fetal cells had been found to remain in the circulatory system of some mothers long after birth and vice versa, a phenomenon called feto-maternal mosaicism<sup><xref ref-type="bibr" rid="bib53">53</xref></sup>. This is unlikely to be the case with the pSNMs that we identified. First, the proportion of extrinsic cells in feto-maternal mosaicism is < 0.5%<sup><xref ref-type="bibr" rid="bib54">54</xref></sup>, which is at least one order of magnitude lower than what we observed for the validated pSNMs. Second, pyrosequencing of the mothers of ACC1-II-1 and DS1-II-2 found no mutant alleles at the pSNM sites (<xref ref-type="fig" rid="fig3">Figure 3G</xref>). Finally, except for one site in <italic>SCN1A</italic>, the mutant alleles of all the other pSNMs identified in DS1-II-2 were found absent in her offspring. Therefore, the observed pSNMs were not likely to have resulted from feto-maternal mosaicism.</p><p>Clonal dominance led by proliferative or selection advantages was observed in peripheral blood cell population<sup><xref ref-type="bibr" rid="bib55">55</xref></sup>. Although we cannot completely exclude the possibility that the identified pSNMs were subjected to proliferative or selection advantages in blood cells, several lines of evidence did not support this hypothesis. First, except two non-synonymous pSNMs in <italic>SCN1A</italic>, 15 of the 17 pSNMs were located outside of the exonic regions and not likely to alter gene function. Second, <italic>SCN1A</italic> gene encodes a subunit of sodium channel which is critical for neuron functionality; however, there is no evidence about its roles in the proliferation of blood cell. Third, all the pSNMs present in blood samples were confirmed in the other non-blood samples from the same individuals, which suggested that the pSNMs are not limited to blood cells.</p><p>Single-cell sequencing is another possible approach to study pSNMs. However, because the mutant alleles of pSNMs are often present in only a small subpopulation of cells, a large number of cells would need to be sequenced to identify them. Furthermore, even larger number of cells need to be sequenced to quantify the allele fraction. In addition, the current whole-genome amplification step of single-cell sequencing might introduce unexpected locus or allele dropouts and thus cause false positives<sup><xref ref-type="bibr" rid="bib56">56</xref></sup>. Thus, bulk sequencing of a population of cells is more effective and less expensive for identifying and quantifying pSNMs.</p><p>The short read length and high error rate of next-generation sequencing make it difficult to remove false positive artifacts due to genomic variations or technical errors<sup><xref ref-type="bibr" rid="bib57">57</xref></sup>. Our pipeline implements a series of filters to reduce the false positives led by such artifacts, which might have potential values in other next-generation sequencing applications. Our current method could identify pSNMs with minor allele fraction of 5%-40% at sequencing depth of ∼80×. In next-generation sequencing, the observed minor allele fractions in two-allele sites are influenced by the random variation of binomial sampling. Thus, increasing sequencing depth could improve the sensitivity to distinguish pSNMs from inherited homozygous and heterozygous sites especially when their allele fractions are close to 0 and 0.5 (<xref ref-type="fig" rid="fig1">Figure 1C</xref>). Our simulation demonstrated that increasing the depth to 200× or increasing the base quality to 60 enabled the detection of pSNMs with minor allele fractions as low as 1%-2% (<xref ref-type="supplementary-material" rid="sup1">Supplementary information, Figure S4</xref>). The Bayesian model of our mosaic genotyper provides the opportunity to integrate more prior knowledge for better detection of mosaic sites, such as the genotyping information of the parents and the site-specific mutation rate which might be correlated to mutational spectrum, mRNA expression and DNA replication<sup><xref ref-type="bibr" rid="bib4">4</xref>,<xref ref-type="bibr" rid="bib58">58</xref></sup>. With next-generation sequencing technologies generating longer reads with higher sequencing depth and quality, the sensitivity and specificity of our pipeline will continue to be improved.</p><p>Previous cancer studies focused on postzygotic mutations that were restricted to be observed in only one tissue or even one clonal cell population, which might originate during later development and aging<sup><xref ref-type="bibr" rid="bib51">51</xref>,<xref ref-type="bibr" rid="bib59">59</xref></sup>. Our findings that most of the pSNMs that we identified were shared in multiple samples suggested the widespread nature of postzygotic mutations during embryogenesis and early development. This highlighted the importance of a control-free method to identify pSNMs. Indeed, when we applied conventional somatic mutation callers, Varscan 2 and muTect, to compare the whole-genome sequencing data of the blood (as case) and saliva (as control) samples of ACC1-II-1, none of the validated pSNMs in blood could be identified.</p><p>A recent paper reported that the number of substitution mutations per cell division in mouse small-bowel stem cells was estimated as ∼1.1 using organoid technology<sup><xref ref-type="bibr" rid="bib60">60</xref></sup>. The pSNMs might contribute to disease risks by either interrupting biological functions of the carriers or transmitting the mutant allele to the offspring<sup><xref ref-type="bibr" rid="bib1">1</xref>,<xref ref-type="bibr" rid="bib16">16</xref>,<xref ref-type="bibr" rid="bib50">50</xref></sup>. The accurate identification of pSNMs will reveal new avenues for studies on the mechanisms and functional consequences of postzygotic mutations and provide new insights into this previously overlooked genetic factor in applications such as finding the “missing heritability” and genetic counseling.</p></sec><sec sec-type="materials|methods"><title>Materials and Methods</title><sec><title>Sample collection and DNA processing</title><p>This study was officially approved by the Institutional Review Boards of Peking University, and informed consent was obtained from all participants or legal guardians. Blood and other samples were obtained from ACC1-II-1, DS1-II-2, and DS2-I-1 and their families whose pedigree structures were illustrated in <xref ref-type="fig" rid="fig3">Figure 3A-3C</xref>. ACC1-II-1 was a healthy adult with no clinical symptoms at present and no clinical history. DS1-II-2 had two episodes of mild seizures between the ages of 4 and 5 years but was subsequently seizure-free with normal cognitive function and no other symptoms. DS2-I-1 was a healthy adult with no clinical symptoms at present and no clinical history. In particular he had no seizures or epilepsy. DS1-II-2 and DS2-I-1 each had a child diagnosed with Dravet syndrome (DS1-III-1 and DS2-II-2). DS2-II-2 suffered sudden unexpected death at five years old. DS2-I-2 had several episodes of FS and DS2-II-1 had FS at an early stage and died of purulent meningitis eight months after birth. The clinical histories of all three families showed no symptoms of cancer or other known overgrowth disorders.</p><p>The genomic DNA from peripheral blood lymphocytes was extracted by the QIAamp DNA Blood Maxi Kit (Qiagen, Hilden, Germany) for family ACC1 and by a salting-out procedure<sup><xref ref-type="bibr" rid="bib61">61</xref></sup> for family DS1 and DS2. The TIANamp Micro DNA Kit (Tiangen Biotech, Beijing, China) was used to isolate genomic DNA from the hair follicle, buccal mucosa, urine, and semen samples, whereas the genomic DNA of the saliva samples was isolated using the Oragene DNA Kit (OG-500; DNA Genotek, Kanata, Canada), according to the manufacturer's instructions. Each sample of genomic DNA was divided into two parts with one part for whole-genome sequencing and the other part for low-throughput validations.</p><p>To screen for pathogenic variations in <italic>SCN1A</italic> in DS1-III-1 and DS2-II-2, 26 exons were PCR amplified and Sanger sequenced using primers as previously described<sup><xref ref-type="bibr" rid="bib62">62</xref></sup>. In addition, the canonical exons of five other rare causal genes of Dravet syndrome including <italic>PCDH19</italic><sup><xref ref-type="bibr" rid="bib41">41</xref></sup>, <italic>GABRG2</italic><sup><xref ref-type="bibr" rid="bib42">42</xref></sup>, <italic>SCN1B</italic><sup><xref ref-type="bibr" rid="bib43">43</xref></sup>, <italic>GABRA1</italic><sup><xref ref-type="bibr" rid="bib44">44</xref></sup>, and <italic>STXBP1</italic><sup><xref ref-type="bibr" rid="bib44">44</xref></sup> were also screened by Sanger sequencing. The exonic variations that were synonymous or present in dbSNP with minor allele fraction ≥ 5% were filtered out. Information about known <italic>SCN1A</italic> variants associated with Dravet syndrome was extracted from the <italic>SCN1A</italic> Variant Database<sup><xref ref-type="bibr" rid="bib63">63</xref></sup>.</p></sec><sec><title>Whole-genome sequencing and data analysis</title><p>Genomic DNA extracted from the peripheral blood samples of ACC1-II-1, DS1-II-2 and DS2-I-1 was selected for whole-genome sequencing. Sequencing libraries were constructed according to the manufacturer's protocol (Illumina, San Diego, CA, USA), with an average insert size of 400-500 bp. The libraries were sequenced by the Illumina HiSeq2000 platform using 100-bp paired-end reads. The reads were aligned against the GRCh37 human reference genome by BWA (version 0.6.1)<sup><xref ref-type="bibr" rid="bib64">64</xref></sup> in a paired-end mode, allowing for a maximum edit distance of four. The duplicate reads were then removed using Picard (<ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://picard.sourceforge.net/">http://picard.sourceforge.net/</ext-link>). To exclude ambiguous alignments, the reads flagged as improperly paired or those mapping to multiple positions were filtered out. In addition, we removed the reads with more than three mismatches, which were potentially error-prone in base calling or mapping. The remaining reads were processed by GATK (version 1.6-9)<sup><xref ref-type="bibr" rid="bib31">31</xref></sup> for indel realignment and base quality score recalibration, and piled-up by SAMtools<sup><xref ref-type="bibr" rid="bib65">65</xref></sup>. The average depth of the clean reads was ∼80× for the three peripheral blood samples (<xref ref-type="supplementary-material" rid="sup1">Supplementary information, Table S1</xref>). To further reduce errors in base calling and alignment, all the bases with base quality or mapping quality less than 20 were excluded from subsequent analyses. CNVs and indels were identified by CNVnator<sup><xref ref-type="bibr" rid="bib30">30</xref></sup> and GATK<sup><xref ref-type="bibr" rid="bib31">31</xref></sup>, respectively. The bin size of CNVnator was set to 100 bp, and the candidate lists of CNVs and indels were further filtered according to the developers' guidelines.</p></sec><sec><title>A new Bayesian genotyper for identifying pSNMs</title><p>We developed a new Bayesian-based genotyper, illustrated as a probabilistic graphical model in <xref ref-type="fig" rid="fig1">Figure 1B</xref>.</p><p>Four genotype states were considered in the probabilistic model: ref-hom, heterozygous, alt-hom, and mosaic. For each genomic position i, the genotype G<sub>i</sub> was inferred under Bayes' rule as described below:</p><p><disp-formula id="equ2"><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="cr2014131eq2.jpg"/></disp-formula></p><p>where the priors of each genotype, <italic>P</italic>(<italic>G<sub>i</sub></italic>), were estimated based on population genetics information, and the sequencing profiles incorporating read depth, allele counts and base qualities were modeled using the likelihood <italic>P</italic>(<italic>Data</italic>|<italic>G<sub>i</sub></italic>).</p><p>To generate the genotype priors, G<sub>i</sub> was considered as a random variable taken from a multinomial distribution with parameter π<sub>i</sub>, where π<sub>i</sub> was determined by the probability of observing a germline alternative allele at a given site, p<sub>i</sub>, and the probability of a site becoming mosaic by postzygotic mutation, p<sub>m</sub>. We assumed the Hardy-Weinberg equilibrium in calculating the genotypic prior probabilities of ref-hom, heterozygous and alt-hom according to p<sub>i</sub>. For the haploid regions, only the ref-hom and alt-hom genotypes were considered, and their prior probabilities were set to p<sub>i</sub> and 1−p<sub>i</sub>, respectively. To obtain the prior estimation of p<sub>i</sub>, the annotations from dbSNP v137 were extracted, and p<sub>i</sub> was set to be the allele frequency of the corresponding substitution. For the substitutions present in dbSNP that lacked allele frequency, p<sub>i</sub> was set to be 0.002, because the allele frequencies were estimated from 692 individuals (<ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/projects/SNP/">http://www.ncbi.nlm.nih.gov/projects/SNP/</ext-link>). If a substitution was completely unannotated in dbSNP, we set p<sub>i</sub> = 1/10 000, a relatively small probability of missing such a polymorphism in the existing data. We further set p<sub>m</sub> = 10<sup>−7</sup>, according to the estimated somatic mutation rate<sup><xref ref-type="bibr" rid="bib34">34</xref></sup>.</p><p>Conditional probability distributions in this model were shown as follows:</p><p><disp-formula id="equ3"><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="cr2014131eq3.jpg"/></disp-formula></p><p>At each position, we observed a pile of bases o<sub>i</sub> with base qualities q<sub>i</sub> and a total sequencing depth d<sub>i</sub> from the alignments of sequencing data. Because sequencing errors may have occurred, we set r<sub>i</sub> to be a vector of the real base states, showing whether a base is the reference or alternative, which could not be directly observed. r<sub>i</sub> was regarded as a series of independent random variables sampled from identical Bernoulli distributions with parameter θ<sub>i</sub>, where θ<sub>i</sub> is a determined variable depending only on the genotype state G<sub>i</sub>. Therefore, the calculation of the likelihood <italic>P</italic>(<italic>Data</italic>|<italic>G<sub>i</sub></italic>) could be separated into two parts as follows:</p><p><disp-formula id="equ4"><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="cr2014131eq4.jpg"/></disp-formula></p><p>Because the relationship between G<sub>i</sub> and θ<sub>i</sub> was determined by definition, we expected θ<sub>i</sub> = 0, 0.5, and 1 when G<sub>i</sub> is ref-hom, heterozygous and alt-hom, respectively. <italic>P</italic>(r<sub>i</sub>|G<sub>i</sub>, d<sub>i</sub>) was calculated based on the Bernoulli trial series r<sub>i</sub> with the success (alternative base state) count r<sub>i</sub> and probability θ<sub>i</sub>, which can be shown as follows:</p><p><disp-formula id="equ5"><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="cr2014131eq5.jpg"/></disp-formula></p><p>where,</p><p><disp-formula id="equ6"><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="cr2014131eq6.jpg"/></disp-formula></p><p>Specifically, the parameter θ<sub>i</sub> was considered to be a uniform random variable between 0 and 1 when G<sub>i</sub> is mosaic, because we assumed no special distribution of allele fractions in mosaic sites. The corresponding likelihood was computed by a beta function according to equation (<xref ref-type="disp-formula" rid="equ7">4</xref>):</p><p><disp-formula id="equ7"><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="cr2014131eq7.jpg"/></disp-formula></p><p>where <inline-formula id="equ12"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="cr2014131eq12.jpg"/></inline-formula> when <inline-formula id="equ13"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="cr2014131eq13.jpg"/></inline-formula></p><p>The second part <italic>P</italic>(o<sub>i</sub>|r<sub>i</sub>, q<sub>i</sub>) would be very easy to calculate if the real states r<sub>i</sub> were known. However, because r<sub>i</sub> was unknown, we traversed every possible Boolean vector r<sub>i</sub>, multiplied it by the corresponding first part <italic>P</italic>(r<sub>i</sub>|G<sub>i</sub>, d<sub>i</sub>), and added the values to obtain the final likelihood <italic>P</italic>(<italic>Data</italic>|G<sub>i</sub>). Because <italic>P</italic>(r<sub>i</sub>|G<sub>i</sub>, d<sub>i</sub>) is constant when the success count r<sub>i</sub> is fixed, the calculation of <italic>P</italic>(<italic>Data</italic>|G<sub>i</sub>) in equation (<xref ref-type="disp-formula" rid="equ4">2</xref>) can be further simplified as follows:</p><p><disp-formula id="equ8"><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="cr2014131eq8.jpg"/></disp-formula></p><p>where, <italic>P</italic>(o<sub>i</sub>|r<sub>i</sub>, q<sub>i</sub>) denotes the sum of the probabilities <italic>P</italic>(o<sub>i</sub>|r<sub>i</sub>, q<sub>i</sub>) for all Boolean vectors r<sub>i</sub> with the same r<sub>i</sub>, as shown in equation (<xref ref-type="disp-formula" rid="equ9">6</xref>):</p><p><disp-formula id="equ9"><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="cr2014131eq9.jpg"/></disp-formula></p><p>To compute <italic>P</italic>(o<sub>i</sub>|r<sub>i</sub>, q<sub>i</sub>), an iterative algorithm traveling over every base state was used. Each base state r<sub>ij</sub> in the Boolean vector r<sub>i</sub> was assumed to be independent of the others, with a sequencing error probability p<sub>error</sub> derived from its Phred-scaled quality score q<sub>ij</sub>. Therefore,</p><p><disp-formula id="equ10"><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="cr2014131eq10.jpg"/></disp-formula></p><p>First, the initial <italic>P</italic>(o<sub>i1</sub>|r<sub>i1</sub>, q<sub>i1</sub>) for r<sub>i1</sub> = 0 or 1 was set using the corresponding p<sub>error</sub> or 1-p<sub>error</sub>, according to the match between the observed and read base states, o<sub>i1</sub> and r<sub>i1</sub>, respectively. Then, the iterative formula was employed according to equation (<xref ref-type="disp-formula" rid="equ11">7</xref>) to calculate <italic>P</italic>(o<sub>i</sub>|r<sub>i</sub>, q<sub>i</sub>) by traversing all d<sub>i</sub> bases:</p><p><disp-formula id="equ11"><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="cr2014131eq11.jpg"/></disp-formula></p><p>where <italic>P</italic>(o<sub>i,1..k</sub>|r<sub>i,1..k</sub>, q<sub>i,1..k</sub>) is the summed probability for the first k bases, as summarized in the count r<sub>i,1..k</sub> = x, which can be taken from 0 to k.</p><p>As a result, the <italic>P</italic>(<italic>Data</italic>|<italic>G<sub>i</sub></italic>) could be easily calculated according to equation (<xref ref-type="disp-formula" rid="equ8">5</xref>), and then <italic>P</italic>(<italic>G<sub>i</sub></italic>|<italic>Data</italic>) for each genotype state was calculated by further multiplying <italic>P</italic>(<italic>G<sub>i</sub></italic>) as shown in equation (<xref ref-type="disp-formula" rid="equ2">1</xref>) and normalized to set the summed probability equal to 1. The sites with < 3 reads, or < 5% reads supporting the minor allele were skipped for quality control. To achieve a high sensitivity of pSNM detection at this genotyping step, a relatively low threshold (<italic>P</italic><sub>mosaic</sub> > 0.05) was applied for the posterior probability of the mosaic genotype. As expected, the specificity could be improved when the threshold was increased (<italic>P</italic><sub>mosaic</sub> > 0.5) (<xref ref-type="supplementary-material" rid="sup1">Supplementary information, Table S2</xref>). To avoid potential computational underflow, our calculations were generally performed in the log-probability space.</p></sec><sec><title>Filtering of candidate pSNMs</title><p>There were artifacts caused by systematic errors in sequencing, base calling, and read alignment that the probabilistic model could not remove. We developed a series of error filters and integrated them into the identification pipeline. The descriptions of the filters that we implemented were summarized in <xref rid="tbl1" ref-type="table">Table 1</xref>.</p><p>First, we excluded the sites that were located near repetitive DNA elements and homopolymers which were known to be prone to errors from existing experimental methods<sup><xref ref-type="bibr" rid="bib57">57</xref></sup>. The annotations of repetitive regions were downloaded from the UCSC genome browser<sup><xref ref-type="bibr" rid="bib66">66</xref></sup>, including transposons, microsatellites, simple tandem repeats, interrupted repeats, segmental duplications, self-alignment regions with similarity score > 80, and other repeats masked by RepeatMasker (<ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://www.repeatmasker.org">http://www.repeatmasker.org</ext-link>). Sites within 2 bp from homopolymers of 4-6 nt or within 3 bp from longer homopolymers were also filtered out. Theoretically pSNMs should be scattered, rather than clustered, along the chromosomes. We found that clustered sites were enriched in heterochromatic regions, including the centromeres and telomeres, and regions with copy number alterations (<xref ref-type="supplementary-material" rid="sup1">Supplementary information, Figure S5</xref>). Thus we filtered out clustered sites with abnormal allele fractions.</p><p>Next, we implemented several filters to remove artifacts caused by alignment errors. Reads with discordant alignment between BWA<sup><xref ref-type="bibr" rid="bib64">64</xref></sup> and BLAT<sup><xref ref-type="bibr" rid="bib67">67</xref></sup> were removed. All contigs in the hg19 assembly were added to the GRCh37 human genome sequence and applied as the reference genome in BLAT to minimize potential misalignment due to the incompleteness of the human reference genome. The sites meeting either of the following criteria were also excluded: (1) predominantly supported by alignment near the ends of reads or near gaps which were known to be error-prone; (2) one allele showing complete co-occurrence with an adjacent polymorphic site within the same sequencing read-pair. To further exclude the reads that were misaligned due to unexpected structural variations, we rejected the sites with significant bias in strand distribution of the reads or sites with skewed within-read position between the reference and alternative alleles. These criteria are known to be efficient for removing misalignment artifacts<sup><xref ref-type="bibr" rid="bib27">27</xref>,<xref ref-type="bibr" rid="bib31">31</xref></sup>.</p><p>To exclude the artifacts caused by base-calling errors, a statistical test was performed for each site following the algorithm developed in LoFreq<sup><xref ref-type="bibr" rid="bib29">29</xref></sup> to distinguish the true alternative allele from sequencing errors, and the sites with <italic>P</italic>-value > 0.05 after Bonferroni correction were filtered out. The sites with extreme depth (< 25 or > 150, the 10th and 90th percentile among all genomic positions) were also excluded because they were often caused by sequencing gaps, CNVs or alignment errors.</p></sec><sec><title>Simulating benchmark datasets to estimate sensitivity and specificity</title><p>To evaluate the performance of our pipeline, we generated a benchmark data set of simulated mosaic and polymorphic sites <italic>in silico</italic> by mixing the whole-genome sequencing data from two individuals (the “<italic>in silico</italic> mixture dataset”), according to Cibulskis <italic>et al</italic>.<sup><xref ref-type="bibr" rid="bib27">27</xref></sup>. We selected two individuals, NA12878 and NA12891, for whom the sequencing depth was similar to our samples. The source of the sequence data and high-quality genotyping files were shown in <xref ref-type="supplementary-material" rid="sup1">Supplementary information, Table S5</xref>. By comparing the genotypes of the two individuals, we identified positions that were heterozygous in NA12878 and homozygous for the reference allele in NA12891. Because the genders of NA12878 and NA12891 were different, the sites located in X and Y chromosomes were excluded. For each position with enough depth to be sampled, the paired-end reads overlapping with the candidate site were extracted for both individuals. Then some of the NA12891 reads were randomly replaced with the corresponding reads of NA12891 following a binomial sampling with given alternative allele fraction and read depth. We generated ∼20 000 simulated sites for each of seven expected alternative allele fractions, or more specifically, 19 989, 19 986, 19 985, 19 968, 19 883, 19 365, and 16 224 sites with expected allele fractions of 0, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5, respectively. <xref ref-type="supplementary-material" rid="sup1">Supplementary information, Figure S6</xref> showed that the distributions of the simulated polymorphic sites mimicked the distributions of stochastic sampling of real sequenced reads at the polymorphic sites.</p><p>Sites with alternative allele fractions 0 and 0.5 were considered homozygous for the reference allele and heterozygous, respectively. Specificity of our pipeline was calculated as the proportion of reference-homozygous and heterozygous sites, respectively, that were correctly rejected as “not mosaic”. Sensitivity was calculated as the proportion of simulated pSNMs correctly identified as “mosaic” for each of the alternative allele fractions ranging from 0.05, 0.1, 0.2, 0.3, to 0.4 in non-repetitive regions.</p><p>The performance of our pSNM identification pipeline was compared against two conventional somatic mutation callers, Varscan 2<sup><xref ref-type="bibr" rid="bib24">24</xref></sup> and muTect<sup><xref ref-type="bibr" rid="bib27">27</xref></sup>. The latest versions of Varscan 2 (version 2.2.11) and muTect (version 1.1.4) were run under their default parameters, and the candidate lists were filtered following their instructions. Since the sequencing data from the matched control samples were required for both of the tools, we implemented a paired-sample mode of our pipeline: we extracted the candidate sites which were predicted as not homozygous for the reference allele in the case sample (<italic>P</italic><sub>ref-hom</sub> < 0.05) and homozygous for the reference allele in the corresponding control sample (<italic>P</italic><sub>ref-hom</sub> > 0.5) by our Bayesian genotyper, and our error filters were then applied for the candidates in the case sample. The reads of libraries Solexa-18483 and Solexa-18484 of the same individual, NA12878, were treated as the case and control datasets, respectively, following the strategy described in<sup><xref ref-type="bibr" rid="bib27">27</xref></sup>. All the identified postzygotic mutations were considered as false positives and the false positive rates were reported. The depth-dependent specificities were calculated and subsequently used.</p><p>We next estimated the identification precision from paired samples. We used the original NA12891 sequencing data as the control to compare against the <italic>in silico</italic> mixture data set described above, and calculated sensitivity as the fraction of identified simulated sites in non-repetitive regions. Precision was calculated from this sensitivity and the depth-dependent specificities. The proportions of reference sites and non-reference sites were set based on estimates from previous population-based study<sup><xref ref-type="bibr" rid="bib67">67</xref></sup>, which were several orders of magnitude larger than the mosaic mutation rate estimated in this work.</p><p>In addition, we compared the performance between our pipeline and the pooled-sample model of GATK when the matched control tissue was unavailable. We set the haplotype number to be 20 for practical reasons. Because GATK pooled-sample model only estimated the proportion of haplotypes carrying the alternative allele without reporting the genotype of each position, all the sites with alternative allele proportions differing from 0 and 0.5 were reported as mosaic. Only about 1 out of 10 000 candidate mosaic sites reported by the GATK pooled-sample model were expected to be real, whereas our pipeline achieved order of magnitude higher specificity (<xref ref-type="supplementary-material" rid="sup1">Supplementary information, Figure S7</xref>). We further showed here that all the false positives in homozygous sites identified by GATK pooled-sample model could be filtered by our stringent filters, which suggested the power of our filters to remove technical artifacts, but a large number of false positives in heterozygous sites still remained even after we combined GATK pooled-sample model with our filters (<xref ref-type="supplementary-material" rid="sup1">Supplementary information, Figure S7</xref>).</p></sec><sec><title>Validation of pSNMs by pyrosequencing</title><p>To validate the presence and allele fraction of the candidate pSNMs detected by our pipeline, pyrosequencing was performed on the genomic DNA obtained from all available samples and family members. The PCR and sequencing primers were designed using PyroMark Assay Design (2.0; Qiagen, Venlo, the Netherlands) and listed in <xref ref-type="supplementary-material" rid="sup1">Supplementary information, Table S6</xref>. The PCR amplification, product processing and pyrosequencing were performed using the PyroMark Q96 ID System (Qiagen) with the corresponding reagents. The raw data were analyzed using the PyroMark Q96 ID Software (Qiagen) for allele quantification. Pyrosequencing has a detection limit of 5% allele fraction<sup><xref ref-type="bibr" rid="bib35">35</xref>,<xref ref-type="bibr" rid="bib36">36</xref></sup>, and any sites with an alternative signal < 5% were usually considered technical noise. The differences in allele fraction between different samples within the same individuals were assessed using the Euclidean distance of the minor allele fractions for all the validated sites, and the six samples were further clustered using Ward's method.</p></sec><sec><title>Validation of pSNMs by Sanger sequencing of TA clones</title><p>To further confirm the presence of the alternative alleles in pSNMs by another independent validation platform, all the pyrosequencing-validated sites were Sanger sequenced in individual clones selected from TA-cloned PCR amplicons. The genomic DNA was amplified using primers flanking these sites (<xref ref-type="supplementary-material" rid="sup1">Supplementary information, Table S7</xref>) and the PCR products were purified. The amplicons were cloned into the Trans1-T1 phage resistant chemically competent cells using the pEASY-T1 Simple Cloning Kit (Transgen Biotech, Beijing, China). The DNA from the positive colonies was PCR amplified using the M13 universal primers, and then the purified products were sequenced using the Applied Biosystems 3730 DNA Analyzer (Life Technologies, Carlsbad, CA, USA). All the pSNMs were confirmed by the independent validation of at least two reference and mutation calls each by Sanger sequencing. We also sequenced the original PCR amplicons in both directions in the Applied Biosystems 3730 DNA Analyzer (Life Technologies).</p></sec><sec><title>MLPA</title><p>To rule out potential copy number abnormalities at the candidate pSNM sites, MLPA was performed on the case sample and a reference control sample obtained from an unrelated individual in whom no mosaicism was observed at the corresponding site. A pair of custom synthetic probes was designed for each validated pSNM to target its flanking regions; the distance to the pSNMs varied from 5 to 1 267 bp (<xref ref-type="supplementary-material" rid="sup1">Supplementary information, Table S8</xref>). The steps of probe preparation, ligation, and PCR amplification were performed using the EK1-FAM Probe Kit and the P300-100R Reference Probemix (MRC-Holland, Amsterdam, the Netherlands), following the manufacturer's protocol. The PCR products were analyzed on the Applied Biosystems 3730 DNA Analyzer (Life Technologies), and the signal processing, normalization and comparison were performed using Coffalyser.NET software (MRC-Holland). Each MLPA experiment on the reference sample was repeated three times. The genomic copy number analysis was reported as normal when the ratio of the normalized peak areas between the case and reference samples was 0.7-1.3, which were the default parameters on Coffalyser.NET.</p></sec><sec><title>Data availability</title><p>The raw whole-genome sequencing data from this study have been deposited in the Short Reads Archive of NCBI (<ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/sra/">http://www.ncbi.nlm.nih.gov/sra/</ext-link>) under accession number SRP028833.</p><p>We made the scripts which implemented the Bayesian-based mosaic genotyper and error filters publicly available at <ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="https://github.com/AugustHuang/MosaicHunter">https://github.com/AugustHuang/MosaicHunter</ext-link>. The users can change the running order and the parameters of running the genotyper and the filters.</p></sec></sec> |
<italic>De novo</italic> mutation in <italic>ATP6V1B2</italic> impairs lysosome acidification and causes dominant deafness-onychodystrophy syndrome | Could not extract abstract | <contrib contrib-type="author"><name><surname>Yuan</surname><given-names>Yongyi</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="aff" rid="aff2">2</xref><xref ref-type="author-notes" rid="note1"><sup>*</sup></xref></contrib><contrib contrib-type="author"><name><surname>Zhang</surname><given-names>Jianguo</given-names></name><xref ref-type="aff" rid="aff3">3</xref><xref ref-type="aff" rid="aff4">4</xref><xref ref-type="author-notes" rid="note1"><sup>*</sup></xref></contrib><contrib contrib-type="author"><name><surname>Chang</surname><given-names>Qing</given-names></name><xref ref-type="aff" rid="aff2">2</xref><xref ref-type="author-notes" rid="note1"><sup>*</sup></xref></contrib><contrib contrib-type="author"><name><surname>Zeng</surname><given-names>Jin</given-names></name><xref ref-type="aff" rid="aff5">5</xref></contrib><contrib contrib-type="author"><name><surname>Xin</surname><given-names>Feng</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Wang</surname><given-names>Jianjun</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Zhu</surname><given-names>Qingyan</given-names></name><xref ref-type="aff" rid="aff3">3</xref></contrib><contrib contrib-type="author"><name><surname>Wu</surname><given-names>Jing</given-names></name><xref ref-type="aff" rid="aff6">6</xref></contrib><contrib contrib-type="author"><name><surname>Lu</surname><given-names>Jingqiao</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Guo</surname><given-names>Weiwei</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Yan</surname><given-names>Xukun</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Jiang</surname><given-names>Hui</given-names></name><xref ref-type="aff" rid="aff3">3</xref></contrib><contrib contrib-type="author"><name><surname>Zhou</surname><given-names>Binfei</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Li</surname><given-names>Qi</given-names></name><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Gao</surname><given-names>Xue</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Yuan</surname><given-names>Huijun</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Yang</surname><given-names>Shiming</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Han</surname><given-names>Dongyi</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Mao</surname><given-names>Zixu</given-names></name><xref ref-type="aff" rid="aff5">5</xref><xref ref-type="aff" rid="aff7">7</xref></contrib><contrib contrib-type="author"><name><surname>Chen</surname><given-names>Ping</given-names></name><xref ref-type="aff" rid="aff8">8</xref></contrib><contrib contrib-type="author"><name><surname>Lin</surname><given-names>Xi</given-names></name><xref ref-type="aff" rid="aff2">2</xref><xref ref-type="corresp" rid="caf1">*</xref></contrib><contrib contrib-type="author"><name><surname>Dai</surname><given-names>Pu</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="aff" rid="aff9">9</xref><xref ref-type="corresp" rid="caf2">*</xref></contrib><aff id="aff1"><label>1</label><institution>Department of Otolaryngology, Chinese PLA General Hospital</institution>, Beijing 100853, <country>China</country></aff><aff id="aff2"><label>2</label><institution>Department of Otolaryngology, Emory University School of Medicine</institution>, Atlanta, GA 30322-3030, <country>USA</country></aff><aff id="aff3"><label>3</label><institution>BGI-Shenzhen</institution>, Shenzhen, Guangdong 518083, <country>China</country></aff><aff id="aff4"><label>4</label><institution>T-Life Research Center, Fudan University</institution>, Shanghai 200433, <country>China</country></aff><aff id="aff5"><label>5</label><institution>Department of Pharmacology, Emory University School of Medicine</institution>, Atlanta, GA 30322-3030, <country>USA</country></aff><aff id="aff6"><label>6</label><institution>BGI-Tianjin</institution>, Tianjin 200000, <country>China</country></aff><aff id="aff7"><label>7</label><institution>Department of Neurology, Emory University School of Medicine</institution>, Atlanta, GA 30322-3030, <country>USA</country></aff><aff id="aff8"><label>8</label><institution>Department of Cell biology, Emory University School of Medicine</institution>, Atlanta, GA 30322-3030, <country>USA</country></aff><aff id="aff9"><label>9</label><institution>Department of Otolaryngology, Hainan Branch of PLA General Hospital</institution>, Sanya, Hainan 572000, <country>China</country></aff> | Cell Research | <sec><title>Dear Editor,</title><p>Dominant deafness-onychodystrophy syndrome (DDOD syndrome; MIM 124480) is characterized mainly by congenital sensorineural hearing loss accompanied by dystrophic or absent nails. Prominent differences between DDOD syndrome and DOORS syndrome (deafness, onychodystrophy, osteodystrophy, mental retardation and seizures; MIM 220500) are the intellectual disability and seizure aspects of DOORS<sup><xref ref-type="bibr" rid="bib1">1</xref></sup>. <italic>TBC1D24</italic> mutations were recently identified as a cause of DOORS syndrome<sup><xref ref-type="bibr" rid="bib2">2</xref></sup>. To date, six families with DDOD syndrome in various ethnic populations have been reported<sup><xref ref-type="bibr" rid="bib3">3</xref></sup>. However, the molecular etiology of DDOD remains unknown.</p><p>We collected three Chinese DDOD pedigrees during the past two years. The probands displayed identical phenotypes including severe congenital sensorineural hearing loss, absence of nails and aplasia of the middle phalanx in the fifth fingers (<xref ref-type="fig" rid="fig1">Figure 1A</xref> and <xref ref-type="fig" rid="fig1">1B</xref>). None showed inner ear malformation and intellectual disability. All three individuals had unilateral cochlea implantation at the ages of 2.5, 2 and 18 years, respectively. The successful language rehabilitation in the DDOD probands further confirmed their normal mental development.</p><p>Whole-exome sequencing was performed in pedigrees 1 and 2, including the probands and their parents (BGI-Shenzhen, China). In each sample, we obtained approximately 5.9-6.9 Gb of data. The data mapped to the targeted region have a mean depth of 145.74 folds, and 99.41% of the targeted bases were covered. For bioinformatics analysis, we focused on variants in coding regions. Variants were filtered by four databases, the 1000 Genomes Project, the HapMap database, the EVS database, and an in-house database from BGI, with the Minor Allele Frequency lower than 0.005. Based on 1) the dominant inheritance of DDOD, 2) the identical phenotype of the two probands, and 3) the pedigree traits (only one individual and neither parent had symptoms), we assumed that there may be a <italic>de novo</italic> mutation following the dominant inheritance characteristics. After completing such a filtering process, 6 genes with variants shared by the two probands were identified (<xref ref-type="supplementary-material" rid="sup1">Supplementary information, Table S1</xref>). The 14 variants in the 6 shared genes (<italic>CIB1</italic>, <italic>MUC4</italic>, <italic>OR5H6</italic>, <italic>PRAMEF1</italic>, <italic>AMBN</italic> and <italic>ATP6V1B2</italic>) were then tested by Sanger sequencing. Combining the sequencing results, the prediction results by SIFT, Polyphen, Mutationtaster and the genes' pathway, and their expression in human fetal cochlear EST database, <italic>ATP6V1B2</italic> was identified as one potential gene that associates with DDOD. An identical heterozygous <italic>de novo</italic> c.1516 C>T (p.Arg506X) mutation in <italic>ATP6V1B2</italic> was verified in two probands (<xref ref-type="fig" rid="fig1">Figure 1C</xref>). The result was further confirmed by Sanger sequencing in another DDOD family (pedigree 3) (data not shown). We then used a restriction enzyme assay to perform a molecular epidemiology analysis of the mutation in 1 053 ethnically matched normal controls. The mutation was not detected in the normal-hearing population (<xref ref-type="supplementary-material" rid="sup1">Supplementary information, Figure S1A</xref>). Although <italic>de novo</italic> mutations have recently been shown to play a major role in human diseases with intellectual disability such as Dravet's syndrome, Kabuki syndrome and Schinzel-Giedion syndrome<sup><xref ref-type="bibr" rid="bib4">4</xref>,<xref ref-type="bibr" rid="bib5">5</xref>,<xref ref-type="bibr" rid="bib6">6</xref>,<xref ref-type="bibr" rid="bib7">7</xref>,<xref ref-type="bibr" rid="bib8">8</xref></sup>, the identication of a same <italic>de novo</italic> mutation in 3 unrelated DDOD individuals is extremely rare.</p><p>The p.Arg506X mutation in <italic>ATP6V1B2</italic> inserts a premature stop codon and results in a truncated protein. Conservation analysis of amino acids in 8 ATP6V1B2 orthologs indicates that the last six amino acids, from residues 506 to 511, are highly conserved (<xref ref-type="fig" rid="fig1">Figure 1D</xref>). Three-dimensional protein structure modeling suggests that the p.Arg506X mutation results in failure of hydrogen bond formation between Tyr504 and Asp507 in ATP6V1B2 (<xref ref-type="supplementary-material" rid="sup1">Supplementary information, Figure S1B</xref>). Expression analysis performed by quantitative real-time PCR on total RNA isolated from leukocytes in pedigree 3 showed that the average expression level of <italic>ATP6V1B2</italic> in case 3 was comparable to that in her parent controls, indicating that the mutant <italic>ATP6V1B2</italic> mRNA is stable. The identification of <italic>ATP6V1B2</italic> c.1516 C>T mutation in three independently identified DDOD patients provides evidence that defect in <italic>ATP6V1B2</italic> is the genetic etiology for DDOD syndrome.</p><p><italic>ATP6V1B2</italic> encodes a component of the vacuolar ATPase (V-ATPase), which is a multisubunit enzyme mediating acidification of eukaryotic intracellular organelles. V-ATPase is composed of a cytosolic V1 domain responsible for ATP hydrolysis and a transmembrane V0 domain responsible for protein translocation. ATP6V1B2 is one of the two V1 domain B subunit isoforms, and as it is highly expressed in the organ of cerebrum and in the organelle of lysosome, it is usually called a brain isoform or lysosomal V1 subunit B2. Deficiencies of <italic>ATP6V1B1</italic> and <italic>ATPV0A4</italic> are related to distal renal tubular acidosis and hearing loss<sup><xref ref-type="bibr" rid="bib9">9</xref></sup>. To the best of our knowledge, no report has linked the function of ATP6V1B2 to hearing. The gene related to DOORS syndrome, <italic>TBC1D24</italic><sup><xref ref-type="bibr" rid="bib2">2</xref></sup>, encodes a member of the Tre2-Bub2-Cdc16 (TBC) domain-containing Rab (Ras-related proteins in brain)-specific GTPase-activating proteins, which coordinate Rab proteins and other GTPases for the regulation of membrane trafficking. TBC1D24 and ATP6V1B2 are all known to be widely expressed, most highly in the brain and kidneys. Immunostaining of mouse cochlear sections and cultured cochlear tissues showed <italic>Atp6v1b2</italic> expression mainly in the organ of Corti and spiral ganglion neurons (<xref ref-type="supplementary-material" rid="sup1">Supplementary information, Figure S1C</xref>). This expression pattern was found in both the early postnatal (postnatal day 2, P2) and the adult (P30) cochlea. Tbc1d24 expresses in the stereocilia of hair cells as well as in spiral ganglion neurons<sup><xref ref-type="bibr" rid="bib10">10</xref></sup>. The identical distribution regions of TBC1D24 and ATP6V1B2 and their function with GTPases or ATPases activity indicate that they may have some physiological link.</p><p>To investigate the function of <italic>ATP6V1B2</italic> in the cochlea, we generated a cochlea-specific <italic>Atp6v1b2</italic>-knockdown mouse model using morpholino oligomer (MO) (<xref ref-type="supplementary-material" rid="sup1">Supplementary information, Data S1</xref>). The oligomer was designed to anneal at the junction of intron 12 and exon 13, which resulted in a partial inclusion of intron 12 followed by a stop codon that excluded the expression of exons 13 and 14 in the mRNA (<xref ref-type="fig" rid="fig1">Figure 1E</xref>). The <italic>Atp6v1b2</italic> MO was microinjected (0.05-5.0 μg/μl) into the scala media of the basal turn of the mouse cochlea before postnatal day 3. Hearing sensitivity has been shown to be unaffected by such an injection procedure<sup><xref ref-type="bibr" rid="bib11">11</xref></sup>. RT-PCR was used to verify the abnormal transcript product containing part of intron 12 in the mouse cochlea 3 days after injection (<xref ref-type="fig" rid="fig1">Figure 1E</xref>). Four weeks post-injection, auditory brainstem response (ABR) tests showed that hearing thresholds in the mice injected with the MO at the concentration of 0.5, 1.25, 2.5 and 5.0 μg/μl were elevated by 30-50 dB compared to wild-type (WT) mice (<xref ref-type="fig" rid="fig1">Figure 1F</xref>). However, hearing thresholds in mice injected with 0.05 μg/μl of MO was within the normal range, indicating the dosage-dependence of the hearing loss phenotype. All the mice receiving scrambled MO injection (0.5 μg/μl) displayed normal hearing (<xref ref-type="fig" rid="fig1">Figure 1F</xref>). Immunological staining revealed that <italic>Atp6v1b2</italic> expression was knocked down significantly in the whole cochlea, especially in hair cells and spiral ganglion neurons (<xref ref-type="fig" rid="fig1">Figure 1G</xref>). Western blot analysis showed that 7 days after injection, the level of Atp6v1b2 was decreased distinctly in spiral ganglia neurons and slightly in the organ of Corti, whereas 21 days after injection, the Atp6v1b2 level was significantly decreased in the organ of Corti (<xref ref-type="supplementary-material" rid="sup1">Supplementary information, Figure S1D</xref>).</p><p>To evaluate the pathogenicity of the <italic>ATP6V1B2</italic> c.1516 C>T mutation, we transfected the pIRES2-EGFP-<italic>ATP6V1B2</italic> WT and pIRES2-EGFP-<italic>ATP6V1B2</italic> c.1516 C>T mutant plasmids into HEK293 cells. We found that the ATPase hydrolysis activity significantly decreased in the transfected cells when the ratio of the mutant/WT increased. This trend indicates that the c.1516 C>T mutant reduced ATPase hydrolysis activity compared with the WT. The proton transport activity of V-ATPase in lysosomes was measured using a lysosome-specific dye. A statistically significant difference in lysosomal pH was detected between <italic>ATP6V1B2</italic> WT- and c.1516 C>T mutant-transfected cells (LSD test, <italic>p</italic> = 0.02, <xref ref-type="supplementary-material" rid="sup1">Supplementary information, Figure S1E</xref>), indicating the reduced acidification caused by c.1516 C>T mutation. Taken together, these results suggest that <italic>ATP6V1B2</italic> c.1516 C>T mutation is a haploinsufficient mutation.</p><p>In summary, we identified a <italic>de novo</italic> mutation (c.1516 C>T (p.Arg506X)) in <italic>ATP6V1B2</italic> as the cause of DDOD syndrome in three independently identified individuals using whole-exome sequencing. Molecular epidemiology analysis showed that the mutation was not present in 1 053 ethnically matched normal hearing controls. We generated a cochlea-specific <italic>Atp6v1b2</italic>-knockdown mouse model and found that <italic>Atp6v1b2</italic> deficiency leads to severe sensorineural hearing loss. <italic>In vitro</italic> pathogenic evaluation showed that the <italic>ATP6V1B2</italic> p.Arg506X is a haploinsufficient mutation and resulted in abnormal acidification in lysosomes. These findings provide the molecular basis for DDOD genetic diagnosis as well as future therapeutic interventions.</p></sec> |
Disease-specific mortality burdens in a rural Gambian population using verbal autopsy, 1998–2007 | <sec id="st1"><title>Objective</title><p>To estimate and evaluate the cause-of-death structure and disease-specific mortality rates in a rural area of The Gambia as determined using the InterVA-4 model.</p></sec><sec id="st2"><title>Design</title><p>Deaths and person-years of observation were determined by age group for the population of the Farafenni Health and Demographic Surveillance area from January 1998 to December 2007. Causes of death were determined by verbal autopsy (VA) using the InterVA-4 model and ICD-10 disease classification. Assigned causes of death were classified into six broad groups: infectious and parasitic diseases; cancers; other non-communicable diseases; neonatal; maternal; and external causes. Poisson regression was used to estimate age and disease-specific mortality rates, and likelihood ratio tests were used to determine statistical significance.</p></sec><sec id="st3"><title>Results</title><p>A total of 3,203 deaths were recorded and VA administered for 2,275 (71%). All-age mortality declined from 15 per 1,000 person-years in 1998–2001 to 8 per 1,000 person-years in 2005–2007. Children aged 1–4 years registered the most marked (74%) decline from 27 to 7 per 1,000 person-years. Communicable diseases accounted for half (49.9%) of the deaths in all age groups, dominated by acute respiratory infections (ARI) (13.7%), malaria (12.9%) and pulmonary tuberculosis (10.2%). The leading causes of death among infants were ARI (5.59 per 1,000 person-years [95% CI: 4.38–7.15]) and malaria (4.11 per 1,000 person-years [95% CI: 3.09–5.47]). Mortality rates in children aged 1–4 years were 3.06 per 1,000 person-years (95% CI: 2.58–3.63) for malaria, and 1.05 per 1,000 person-years (95% CI: 0.79–1.41) for ARI. The HIV-related mortality rate in this age group was 1.17 per 1,000 person-years (95% CI: 0.89–1.54). Pulmonary tuberculosis and communicable diseases other than malaria, HIV/AIDS and ARI were the main killers of adults aged 15 years and over. Stroke-related mortality increased to become the leading cause of death among the elderly aged 60 years or more in 2005–2007.</p></sec><sec id="st4"><title>Conclusions</title><p>Mortality in the Farafenni HDSS area was dominated by communicable diseases. Malaria and ARI were the leading causes of death in the general population. In addition to these, diarrhoeal disease was a particularly important cause of death among children under 5 years of age, as was pulmonary tuberculosis among adults aged 15 years and above.</p></sec> | <contrib contrib-type="author"><name><surname>Jasseh</surname><given-names>Momodou</given-names></name><xref ref-type="aff" rid="AF0001">1</xref><xref ref-type="aff" rid="AF0002">2</xref><xref ref-type="corresp" rid="cor1">*</xref></contrib><contrib contrib-type="author"><name><surname>Howie</surname><given-names>Stephen R. C.</given-names></name><xref ref-type="aff" rid="AF0001">1</xref></contrib><contrib contrib-type="author"><name><surname>Gomez</surname><given-names>Pierre</given-names></name><xref ref-type="aff" rid="AF0001">1</xref></contrib><contrib contrib-type="author"><name><surname>Scott</surname><given-names>Susana</given-names></name><xref ref-type="aff" rid="AF0001">1</xref><xref ref-type="aff" rid="AF0003">3</xref></contrib><contrib contrib-type="author"><name><surname>Roca</surname><given-names>Anna</given-names></name><xref ref-type="aff" rid="AF0001">1</xref></contrib><contrib contrib-type="author"><name><surname>Cham</surname><given-names>Mamady</given-names></name><xref ref-type="aff" rid="AF0004">4</xref></contrib><contrib contrib-type="author"><name><surname>Greenwood</surname><given-names>Brian</given-names></name><xref ref-type="aff" rid="AF0003">3</xref></contrib><contrib contrib-type="author"><name><surname>Corrah</surname><given-names>Tumani</given-names></name><xref ref-type="aff" rid="AF0001">1</xref></contrib><contrib contrib-type="author"><name><surname>D'Alessandro</surname><given-names>Umberto</given-names></name><xref ref-type="aff" rid="AF0001">1</xref></contrib> | Global Health Action | <p>The development of a successful health care system should be supported by information on the frequency of specific diseases and their contribution to morbidity and mortality in the population. Therefore, measuring mortality, including cause-of-death determination, enhances every level of health care planning, resource allocation and service delivery systems (<xref rid="CIT0001" ref-type="bibr">1</xref>, <xref rid="CIT0002" ref-type="bibr">2</xref>). Unfortunately, many countries with the greatest demand for this information have weak health systems that do not generate the requisite information to establish cause-of-death distributions (<xref rid="CIT0003" ref-type="bibr">3</xref>, <xref rid="CIT0004" ref-type="bibr">4</xref>) as they also lack functional vital registration systems to capture all the deaths in their populations at specific periods (<xref rid="CIT0005" ref-type="bibr">5</xref>). This is particularly true for sub-Saharan Africa where the majority of deaths occur at home with little or no chance of the cause of death being certified by qualified medical personnel. In this context, verbal autopsy (VA), despite its known limitations, is the most viable option for generating representative cause-of-death information.</p><p>The VA approach to determination of the cause of death is described in detail elsewhere (<xref rid="CIT0005" ref-type="bibr">5</xref>). Used predominantly in research settings, the range of methods adopted for cause-of-death assignment includes physician-certification, data-derived algorithms, application of Bayes’ theorem, and direct statistical estimation of cause fractions (<xref rid="CIT0002" ref-type="bibr">2</xref>). Physician-certified VA (PCVA) is an established method for assigning cause of death, but it is time consuming. Different approaches more suitable for resource-challenged settings have been developed (<xref rid="CIT0006" ref-type="bibr">6</xref>). These include the InterVA, a computer-based automated VA coding model, and the adoption by WHO of a set of standardised questionnaires that facilitate comparison across different geographical and environmental settings (<xref rid="CIT0007" ref-type="bibr">7</xref>). InterVA has been validated in different settings throughout its evolution (<xref rid="CIT0008" ref-type="bibr">8</xref>–<xref rid="CIT0011" ref-type="bibr">11</xref>).</p><p>The Gambia, like many other low-income countries, does not have a functional civil and vital registration system. Health facilities are also unable to provide such information. The only available information on causes of death derives from studies focused on specific target groups (mainly children aged under 5 years) (<xref rid="CIT0012" ref-type="bibr">12</xref>–<xref rid="CIT0014" ref-type="bibr">14</xref>) and from a hospital-based study in Banjul, representing a largely urban area (<xref rid="CIT0015" ref-type="bibr">15</xref>). There are no data on the cause-of-death structure and its evolution over time at the national or sub-national level. However, relevant data have been collected routinely in a population of about 45,000 people in the town of Farafenni and its environs in the North Bank Region of The Gambia as part of the Farafenni Health and Demographic Surveillance System (HDSS). VAs conducted for deaths that occurred between 1998 and 2007 were used to evaluate the cause-of-death patterns and to establish the disease-specific mortality burdens for this part of The Gambia.</p><sec sec-type="methods" id="S0002"><title>Methods</title><sec id="S0002-S20001"><title>Study area</title><p>The Farafenni HDSS was established in the North Bank Region of The Gambia in 1981 in two geographically contiguous clusters of 42 rural villages to the east and west of the town of Farafenni (population 25,000). In October 2002, the surveillance area was expanded to include Farafenni Town and 23 settlements within a 5-km radius of the town (see <xref ref-type="fig" rid="F0001">Fig. 1</xref>). The majority of the residents are subsistence farmers with very few earning salaries from employment. Detailed socio-demographic characteristics of the population are described elsewhere (<xref rid="CIT0013" ref-type="bibr">13</xref>).</p><fig id="F0001" position="float"><label>Fig. 1</label><caption><p>Location of the urban and contiguous rural areas east and west of Farafenni Town, which collectively constitute the Farafenni Health and Demographic Surveillance area.</p></caption><graphic xlink:href="GHA-7-25598-g001"/></fig><p>Malaria transmission is highly seasonal with most infections occurring between September and November. It was highly endemic in the past but has now declined substantially (<xref rid="CIT0016" ref-type="bibr">16</xref>). The health care system consists of an under-resourced village-based primary health care system that has been operational since 1983 made up of 16 PHC Posts and five Dispensaries, a Health Centre for reproductive and child health services, and a 250-bed referral district hospital in the town of Farafenni, which was commissioned in 1999. Immunisation coverage levels for individual vaccines against childhood diseases are high (<xref rid="CIT0017" ref-type="bibr">17</xref>), and use of ITNs is widespread (<xref rid="CIT0018" ref-type="bibr">18</xref>). For the period 2000–2001, HIV prevalence among pregnant women in The Gambia was 1.0% for HIV-1 and 0.8% for HIV-2, and the corresponding levels for Farafenni were 0.4 and 0.3%, respectively (<xref rid="CIT0019" ref-type="bibr">19</xref>). From a similar sentinel surveillance in 2008, the national prevalence levels were 1.6% for HIV1 and 0.4% for HIV2 (<xref rid="CIT0020" ref-type="bibr">20</xref>). The majority of deaths occur at home and burials are held almost immediately or within a few hours after death, in line with religious beliefs. This constitutes a barrier to having deaths medically certified or being captured by vital registration.</p></sec><sec id="S0002-S20002"><title>Data and statistical analysis</title><p>The surveillance procedures of the Farafenni HDSS are described in detail elsewhere (<xref rid="CIT0013" ref-type="bibr">13</xref>, <xref rid="CIT0021" ref-type="bibr">21</xref>). The administration of VAs was introduced in 1998 for a sample of deaths and for specific study purposes. Routine administration of VA for every death in the area using the 2002 standard WHO VA questionnaires for neonates, children and adults commenced in 2005. VAs were administered retrospectively for deaths that occurred before 2005. VA interviews are conducted at least 40 days after death by trained fieldworkers, but the time lapse between death and interview has spanned up to 3 years in some cases. The main carers of the deceased persons prior to their deaths are targeted for the interviews, or if they are not available, other close relatives who were present during the period of illness and death. Interviews were conducted in one of the three main local languages spoken in the area.</p><p>The data collected using the three different age-specific questionnaires have been systematically transformed into the InterVA-4 input format and merged together. Responses for questions in the InterVA-4 format that could not be derived from the original data were coded as missing. InterVA-4 was applied with malaria prevalence set as ‘high’ and HIV prevalence as ‘low’, reflecting the situation in the study area for the period covered by this study and as recommended for West African regions. The data forms part of the INDEPTH Network pooled dataset for cause-of-death analyses covering Farafenni HDSS and 21 other INDEPTH HDSS sites (<xref rid="CIT0022" ref-type="bibr">22</xref>).</p><p>The assigned causes of death were classified into six broad groups: infectious and parasitic diseases, cancers, other non-communicable diseases, neonatal, maternal, and external causes. In the analysis presented here, cause-specific mortality fractions (CSMFs) are derived from the three most likely causes of death and the residual indeterminate fraction assigned by InterVA-4 using disease codes in version 10 of the International Statistical Classification of Diseases and Related Health Problems, ICD-10. The output was merged with the detailed individual level data and analysed using STATA version 12 (Stata Corporation, College Station, TX, USA). Cause-specific mortality rates were calculated as the number of cause-specific deaths per 1,000 person-years of follow-up. They were derived using survival analysis techniques and Poisson regression, and individual deaths were associated with the most likely cause of death. Statistical significance at the 95% confidence level was determined using likelihood ratio tests. Since annual cause-specific mortality rates would not be meaningful due to small numbers of cause-specific deaths, the study period was divided up into three short periods with roughly similar numbers of deaths for the assessment of cause-specific mortality trends. These were defined <italic>a priori</italic> as 1998–2001, 2002–2004, and 2005–2007.</p><p>The Joint MRC/Gambia Government Ethics committee approved the establishment of the Farafenni HDSS and all instruments used to collect household and individual level information, including the VA questionnaires. Verbal consent was obtained for the administration of all VA questionnaires.</p></sec></sec><sec sec-type="results" id="S0003"><title>Results</title><p>The mid-year population of the Farafenni HDSS in 1998 was 15,960 with a mean age of 23.0 years; and 44,644 in 2007 with a mean age 22.4 years. The age structure remained the same with 18% of the population aged less than 5 years in both periods; whilst the proportion aged 15 years and over was 53% in 1998 and 55% in 2007. Between 1 January 1998 and 31 December 2007, 3,203 deaths were recorded and VAs were administered for 2,275 (71%) of them. Annual VA coverage improved progressively from about 50% in the first 3 years of the study period and peaked at 87% in 2003. Coverage was generally high among adults aged 15 years or more throughout the study period. There was no difference in VA coverage by gender or area of residence.</p><p>Overall mortality declined by a third in the rural part of the demographic surveillance area over the 10-year period, a decline from 15 per 1,000 person-years in 1998–2001 to 10 per 1,000 person-years in 2005–2007 and by 12.5% in the urban part between 2002–2004 and 2005–2007, a decline from 8 to 7 per 1,000 person-years (<xref ref-type="table" rid="T0001">Table 1</xref>). Mortality dropped by 55% among infants (from 78 to 35 per 1,000 person-years) and by 74% among children 1–4 years old (from 27 to 7 per 1,000 person-years). Overall mortality rates did not vary appreciably between ethnic groups in any of the three periods; and females had a lower mortality rate than males throughout.</p><table-wrap id="T0001" position="float"><label>Table 1</label><caption><p>Number of deaths, proportion of deaths with verbal autopsy and death rates by period and population</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1"/><th align="center" colspan="3" rowspan="1">All</th><th align="center" colspan="3" rowspan="1">1998–2001</th><th align="center" colspan="3" rowspan="1">2002–2004</th><th align="center" colspan="3" rowspan="1">2005–2007</th></tr><tr><th align="left" rowspan="1" colspan="1"/><th align="center" colspan="3" rowspan="1">
<hr/>
</th><th align="center" colspan="3" rowspan="1">
<hr/>
</th><th align="center" colspan="3" rowspan="1">
<hr/>
</th><th align="center" colspan="3" rowspan="1">
<hr/>
</th></tr><tr><th align="left" rowspan="1" colspan="1"/><th align="center" rowspan="1" colspan="1">No. of deaths</th><th align="center" rowspan="1" colspan="1">Number of VAs conducted (% of deaths)</th><th align="center" rowspan="1" colspan="1">Death rate (per 1,000 PY)</th><th align="center" rowspan="1" colspan="1">No. of deaths</th><th align="center" rowspan="1" colspan="1">Number of VAs conducted (% of deaths)</th><th align="center" rowspan="1" colspan="1">Death rate (per 1,000 PY)</th><th align="center" rowspan="1" colspan="1">No. of deaths</th><th align="center" rowspan="1" colspan="1">Number of VAs conducted (% of deaths)</th><th align="center" rowspan="1" colspan="1">Death rate (per 1,000 PY)</th><th align="center" rowspan="1" colspan="1">No. of deaths</th><th align="center" rowspan="1" colspan="1">Number of VAs conducted (% of deaths)</th><th align="center" rowspan="1" colspan="1">Death rate (per 1,000 PY)</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">Age group</td><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1"> <1 year</td><td align="center" rowspan="1" colspan="1">536</td><td align="center" rowspan="1" colspan="1">224 (42)</td><td align="center" rowspan="1" colspan="1">47</td><td align="center" rowspan="1" colspan="1">201</td><td align="center" rowspan="1" colspan="1">20 (10)</td><td align="center" rowspan="1" colspan="1">78</td><td align="center" rowspan="1" colspan="1">162</td><td align="center" rowspan="1" colspan="1">122 (75)</td><td align="center" rowspan="1" colspan="1">42</td><td align="center" rowspan="1" colspan="1">173</td><td align="center" rowspan="1" colspan="1">82 (47)</td><td align="center" rowspan="1" colspan="1">35</td></tr><tr><td align="left" rowspan="1" colspan="1"> 1–4 years</td><td align="center" rowspan="1" colspan="1">559</td><td align="center" rowspan="1" colspan="1">295 (53)</td><td align="center" rowspan="1" colspan="1">13</td><td align="center" rowspan="1" colspan="1">246</td><td align="center" rowspan="1" colspan="1">24 (10)</td><td align="center" rowspan="1" colspan="1">27</td><td align="center" rowspan="1" colspan="1">188</td><td align="center" rowspan="1" colspan="1">163 (87)</td><td align="center" rowspan="1" colspan="1">12</td><td align="center" rowspan="1" colspan="1">125</td><td align="center" rowspan="1" colspan="1">108 (86)</td><td align="center" rowspan="1" colspan="1">7</td></tr><tr><td align="left" rowspan="1" colspan="1"> 5–14 years</td><td align="center" rowspan="1" colspan="1">204</td><td align="center" rowspan="1" colspan="1">137 (67)</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">81</td><td align="center" rowspan="1" colspan="1">38 (47)</td><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">67</td><td align="center" rowspan="1" colspan="1">59 (88)</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">56</td><td align="center" rowspan="1" colspan="1">40 (71)</td><td align="center" rowspan="1" colspan="1">2</td></tr><tr><td align="left" rowspan="1" colspan="1"> 15–59 years</td><td align="center" rowspan="1" colspan="1">891</td><td align="center" rowspan="1" colspan="1">715 (80)</td><td align="center" rowspan="1" colspan="1">6</td><td align="center" rowspan="1" colspan="1">187</td><td align="center" rowspan="1" colspan="1">180 (96)</td><td align="center" rowspan="1" colspan="1">6</td><td align="center" rowspan="1" colspan="1">368</td><td align="center" rowspan="1" colspan="1">303 (82)</td><td align="center" rowspan="1" colspan="1">6</td><td align="center" rowspan="1" colspan="1">336</td><td align="center" rowspan="1" colspan="1">232 (69)</td><td align="center" rowspan="1" colspan="1">5</td></tr><tr><td align="left" rowspan="1" colspan="1"> 60+ years</td><td align="center" rowspan="1" colspan="1">1,013</td><td align="center" rowspan="1" colspan="1">904 (89)</td><td align="center" rowspan="1" colspan="1">58</td><td align="center" rowspan="1" colspan="1">287</td><td align="center" rowspan="1" colspan="1">280 (98)</td><td align="center" rowspan="1" colspan="1">63</td><td align="center" rowspan="1" colspan="1">359</td><td align="center" rowspan="1" colspan="1">331 (92)</td><td align="center" rowspan="1" colspan="1">59</td><td align="center" rowspan="1" colspan="1">367</td><td align="center" rowspan="1" colspan="1">293 (80)</td><td align="center" rowspan="1" colspan="1">54</td></tr><tr><td align="left" rowspan="1" colspan="1">Sex</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1"> Male</td><td align="center" rowspan="1" colspan="1">1,727</td><td align="center" rowspan="1" colspan="1">1,217 (70)</td><td align="center" rowspan="1" colspan="1">12</td><td align="center" rowspan="1" colspan="1">531</td><td align="center" rowspan="1" colspan="1">287 (54)</td><td align="center" rowspan="1" colspan="1">17</td><td align="center" rowspan="1" colspan="1">609</td><td align="center" rowspan="1" colspan="1">522 (86)</td><td align="center" rowspan="1" colspan="1">11</td><td align="center" rowspan="1" colspan="1">587</td><td align="center" rowspan="1" colspan="1">408 (70)</td><td align="center" rowspan="1" colspan="1">9</td></tr><tr><td align="left" rowspan="1" colspan="1"> Female</td><td align="center" rowspan="1" colspan="1">1,476</td><td align="center" rowspan="1" colspan="1">1,058 (72)</td><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1">471</td><td align="center" rowspan="1" colspan="1">255 (54)</td><td align="center" rowspan="1" colspan="1">14</td><td align="center" rowspan="1" colspan="1">535</td><td align="center" rowspan="1" colspan="1">456 (85)</td><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1">470</td><td align="center" rowspan="1" colspan="1">347 (74)</td><td align="center" rowspan="1" colspan="1">7</td></tr><tr><td align="left" rowspan="1" colspan="1">Ethnic group</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1"> Wollof</td><td align="center" rowspan="1" colspan="1">1,192</td><td align="center" rowspan="1" colspan="1">825 (69)</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">349</td><td align="center" rowspan="1" colspan="1">172 (49)</td><td align="center" rowspan="1" colspan="1">15</td><td align="center" rowspan="1" colspan="1">418</td><td align="center" rowspan="1" colspan="1">355 (85)</td><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1">425</td><td align="center" rowspan="1" colspan="1">298 (70)</td><td align="center" rowspan="1" colspan="1">8</td></tr><tr><td align="left" rowspan="1" colspan="1"> Mandinka</td><td align="center" rowspan="1" colspan="1">1,262</td><td align="center" rowspan="1" colspan="1">950 (75)</td><td align="center" rowspan="1" colspan="1">11</td><td align="center" rowspan="1" colspan="1">453</td><td align="center" rowspan="1" colspan="1">272 (60)</td><td align="center" rowspan="1" colspan="1">16</td><td align="center" rowspan="1" colspan="1">428</td><td align="center" rowspan="1" colspan="1">386 (90)</td><td align="center" rowspan="1" colspan="1">11</td><td align="center" rowspan="1" colspan="1">381</td><td align="center" rowspan="1" colspan="1">292 (77)</td><td align="center" rowspan="1" colspan="1">9</td></tr><tr><td align="left" rowspan="1" colspan="1"> Fula</td><td align="center" rowspan="1" colspan="1">629</td><td align="center" rowspan="1" colspan="1">416 (66)</td><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1">194</td><td align="center" rowspan="1" colspan="1">95 (49)</td><td align="center" rowspan="1" colspan="1">14</td><td align="center" rowspan="1" colspan="1">239</td><td align="center" rowspan="1" colspan="1">195 (82)</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">196</td><td align="center" rowspan="1" colspan="1">126 (64)</td><td align="center" rowspan="1" colspan="1">7</td></tr><tr><td align="left" rowspan="1" colspan="1"> Other</td><td align="center" rowspan="1" colspan="1">120</td><td align="center" rowspan="1" colspan="1">84 (70)</td><td align="center" rowspan="1" colspan="1">7</td><td align="center" rowspan="1" colspan="1">6</td><td align="center" rowspan="1" colspan="1">3 (50)</td><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1">59</td><td align="center" rowspan="1" colspan="1">42 (71)</td><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1">55</td><td align="center" rowspan="1" colspan="1">39 (71)</td><td align="center" rowspan="1" colspan="1">6</td></tr><tr><td align="left" rowspan="1" colspan="1">Area of residence</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1"> Rural</td><td align="center" rowspan="1" colspan="1">2,145</td><td align="center" rowspan="1" colspan="1">1,491 (70)</td><td align="center" rowspan="1" colspan="1">13</td><td align="center" rowspan="1" colspan="1">1,002</td><td align="center" rowspan="1" colspan="1">542 (54)</td><td align="center" rowspan="1" colspan="1">15</td><td align="center" rowspan="1" colspan="1">635</td><td align="center" rowspan="1" colspan="1">572 (90)</td><td align="center" rowspan="1" colspan="1">12</td><td align="center" rowspan="1" colspan="1">508</td><td align="center" rowspan="1" colspan="1">377 (74)</td><td align="center" rowspan="1" colspan="1">10</td></tr><tr><td align="left" rowspan="1" colspan="1"> Urban<xref ref-type="table-fn" rid="TF0001">a</xref>
</td><td align="center" rowspan="1" colspan="1">1,058</td><td align="center" rowspan="1" colspan="1">784 (74)</td><td align="center" rowspan="1" colspan="1">7</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">509</td><td align="center" rowspan="1" colspan="1">406 (80)</td><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1">549</td><td align="center" rowspan="1" colspan="1">378 (69)</td><td align="center" rowspan="1" colspan="1">7</td></tr><tr><td align="left" rowspan="1" colspan="1">All</td><td align="center" rowspan="1" colspan="1">3,203</td><td align="center" rowspan="1" colspan="1">2,275 (71)</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">1,002</td><td align="center" rowspan="1" colspan="1">542 (54)</td><td align="center" rowspan="1" colspan="1">15</td><td align="center" rowspan="1" colspan="1">1,144</td><td align="center" rowspan="1" colspan="1">978 (85)</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">1,057</td><td align="center" rowspan="1" colspan="1">755 (71)</td><td align="center" rowspan="1" colspan="1">8</td></tr></tbody></table><table-wrap-foot><fn id="TF0001"><label>a</label><p>Surveillance in this area started in October 2002.</p></fn></table-wrap-foot></table-wrap><p>CSMFs generated by InterVA-4 are presented in <xref ref-type="table" rid="T0002">Table 2</xref> by age group, sex, area of residence and ethnicity. Almost half (49.9%) of deaths over the 10-year period were attributed to communicable diseases. These were dominated by acute respiratory infections (ARI) (13.7%), malaria (12.9%) and pulmonary tuberculosis (10.2%). A similar pattern of cause-specific mortality proportions was maintained in all three periods. However, the proportion of HIV-related deaths showed an increasing trend over the three periods.</p><table-wrap id="T0002" position="float"><label>Table 2</label><caption><p>Cause-specific mortality fractions (CSMFs) derived using recommended InterVA-4 settings by age group, period, sex, residence and ethnicity</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1">Classification and causes of death</th><th align="center" rowspan="1" colspan="1">Total</th><th align="center" rowspan="1" colspan="1"><1 year</th><th align="center" rowspan="1" colspan="1">1–4 years</th><th align="center" rowspan="1" colspan="1">5–14 years</th><th align="center" rowspan="1" colspan="1">15–59 years</th><th align="center" rowspan="1" colspan="1">60+ years</th><th align="center" rowspan="1" colspan="1">1998–2001</th><th align="center" rowspan="1" colspan="1">2002–2004</th><th align="center" rowspan="1" colspan="1">2005–2007</th><th align="center" rowspan="1" colspan="1">Male</th><th align="center" rowspan="1" colspan="1">Female</th><th align="center" rowspan="1" colspan="1">Rural</th><th align="center" rowspan="1" colspan="1">Urban</th><th align="center" rowspan="1" colspan="1">Wollof</th><th align="center" rowspan="1" colspan="1">Mandinka</th><th align="center" rowspan="1" colspan="1">Fula</th><th align="center" rowspan="1" colspan="1">Other</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">Number of deaths</td><td align="center" rowspan="1" colspan="1">2,275</td><td align="center" rowspan="1" colspan="1">224</td><td align="center" rowspan="1" colspan="1">295</td><td align="center" rowspan="1" colspan="1">137</td><td align="center" rowspan="1" colspan="1">715</td><td align="center" rowspan="1" colspan="1">904</td><td align="center" rowspan="1" colspan="1">542</td><td align="center" rowspan="1" colspan="1">978</td><td align="center" rowspan="1" colspan="1">755</td><td align="center" rowspan="1" colspan="1">1,217</td><td align="center" rowspan="1" colspan="1">1,058</td><td align="center" rowspan="1" colspan="1">1,491</td><td align="center" rowspan="1" colspan="1">784</td><td align="center" rowspan="1" colspan="1">825</td><td align="center" rowspan="1" colspan="1">950</td><td align="center" rowspan="1" colspan="1">416</td><td align="center" rowspan="1" colspan="1">84</td></tr><tr><td align="left" rowspan="1" colspan="1">Infectious diseases</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1"> Malaria</td><td align="center" rowspan="1" colspan="1">12.9</td><td align="center" rowspan="1" colspan="1">15.8</td><td align="center" rowspan="1" colspan="1">38.3</td><td align="center" rowspan="1" colspan="1">28.3</td><td align="center" rowspan="1" colspan="1">7.5</td><td align="center" rowspan="1" colspan="1">5.9</td><td align="center" rowspan="1" colspan="1">10.7</td><td align="center" rowspan="1" colspan="1">14</td><td align="center" rowspan="1" colspan="1">12.9</td><td align="center" rowspan="1" colspan="1">12.3</td><td align="center" rowspan="1" colspan="1">13.5</td><td align="center" rowspan="1" colspan="1">13</td><td align="center" rowspan="1" colspan="1">12.6</td><td align="center" rowspan="1" colspan="1">15.4</td><td align="center" rowspan="1" colspan="1">10.3</td><td align="center" rowspan="1" colspan="1">13.5</td><td align="center" rowspan="1" colspan="1">13.9</td></tr><tr><td align="left" rowspan="1" colspan="1"> ARI, including pneumonia</td><td align="center" rowspan="1" colspan="1">13.7</td><td align="center" rowspan="1" colspan="1">28.1</td><td align="center" rowspan="1" colspan="1">15.2</td><td align="center" rowspan="1" colspan="1">11.7</td><td align="center" rowspan="1" colspan="1">9.9</td><td align="center" rowspan="1" colspan="1">12.9</td><td align="center" rowspan="1" colspan="1">13.5</td><td align="center" rowspan="1" colspan="1">13.8</td><td align="center" rowspan="1" colspan="1">13.6</td><td align="center" rowspan="1" colspan="1">15</td><td align="center" rowspan="1" colspan="1">12.2</td><td align="center" rowspan="1" colspan="1">14</td><td align="center" rowspan="1" colspan="1">13</td><td align="center" rowspan="1" colspan="1">14.5</td><td align="center" rowspan="1" colspan="1">13.1</td><td align="center" rowspan="1" colspan="1">13.8</td><td align="center" rowspan="1" colspan="1">11.3</td></tr><tr><td align="left" rowspan="1" colspan="1"> Pulmonary tuberculosis</td><td align="center" rowspan="1" colspan="1">10.2</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.4</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">14</td><td align="center" rowspan="1" colspan="1">14.3</td><td align="center" rowspan="1" colspan="1">10.2</td><td align="center" rowspan="1" colspan="1">9.7</td><td align="center" rowspan="1" colspan="1">10.8</td><td align="center" rowspan="1" colspan="1">12.6</td><td align="center" rowspan="1" colspan="1">7.4</td><td align="center" rowspan="1" colspan="1">9.6</td><td align="center" rowspan="1" colspan="1">11.3</td><td align="center" rowspan="1" colspan="1">7.4</td><td align="center" rowspan="1" colspan="1">12.5</td><td align="center" rowspan="1" colspan="1">8.8</td><td align="center" rowspan="1" colspan="1">17.5</td></tr><tr><td align="left" rowspan="1" colspan="1"> HIV/AIDS-related</td><td align="center" rowspan="1" colspan="1">5.8</td><td align="center" rowspan="1" colspan="1">5.9</td><td align="center" rowspan="1" colspan="1">15</td><td align="center" rowspan="1" colspan="1">8.7</td><td align="center" rowspan="1" colspan="1">6.6</td><td align="center" rowspan="1" colspan="1">1.9</td><td align="center" rowspan="1" colspan="1">3.3</td><td align="center" rowspan="1" colspan="1">6.2</td><td align="center" rowspan="1" colspan="1">7.2</td><td align="center" rowspan="1" colspan="1">4.3</td><td align="center" rowspan="1" colspan="1">7.6</td><td align="center" rowspan="1" colspan="1">4.8</td><td align="center" rowspan="1" colspan="1">7.8</td><td align="center" rowspan="1" colspan="1">6.5</td><td align="center" rowspan="1" colspan="1">4.5</td><td align="center" rowspan="1" colspan="1">7.3</td><td align="center" rowspan="1" colspan="1">8</td></tr><tr><td align="left" rowspan="1" colspan="1"> Diarrhoeal diseases</td><td align="center" rowspan="1" colspan="1">3.9</td><td align="center" rowspan="1" colspan="1">10.2</td><td align="center" rowspan="1" colspan="1">8.3</td><td align="center" rowspan="1" colspan="1">0.9</td><td align="center" rowspan="1" colspan="1">3.7</td><td align="center" rowspan="1" colspan="1">1.7</td><td align="center" rowspan="1" colspan="1">4.2</td><td align="center" rowspan="1" colspan="1">3.8</td><td align="center" rowspan="1" colspan="1">3.9</td><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">3.8</td><td align="center" rowspan="1" colspan="1">4.3</td><td align="center" rowspan="1" colspan="1">3.3</td><td align="center" rowspan="1" colspan="1">3.4</td><td align="center" rowspan="1" colspan="1">4.2</td><td align="center" rowspan="1" colspan="1">4.7</td><td align="center" rowspan="1" colspan="1">2.1</td></tr><tr><td align="left" rowspan="1" colspan="1"> Meningitis and encephalitis</td><td align="center" rowspan="1" colspan="1">2.2</td><td align="center" rowspan="1" colspan="1">1.5</td><td align="center" rowspan="1" colspan="1">1.3</td><td align="center" rowspan="1" colspan="1">6.5</td><td align="center" rowspan="1" colspan="1">2.7</td><td align="center" rowspan="1" colspan="1">1.5</td><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">1.8</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">2.1</td><td align="center" rowspan="1" colspan="1">2.2</td><td align="center" rowspan="1" colspan="1">2.1</td><td align="center" rowspan="1" colspan="1">2.2</td><td align="center" rowspan="1" colspan="1">2.5</td><td align="center" rowspan="1" colspan="1">1.4</td><td align="center" rowspan="1" colspan="1">2.8</td><td align="center" rowspan="1" colspan="1">4.2</td></tr><tr><td align="left" rowspan="1" colspan="1"> Other infectious diseases</td><td align="center" rowspan="1" colspan="1">1.2</td><td align="center" rowspan="1" colspan="1">0.1</td><td align="center" rowspan="1" colspan="1">0.1</td><td align="center" rowspan="1" colspan="1">2.8</td><td align="center" rowspan="1" colspan="1">1.6</td><td align="center" rowspan="1" colspan="1">1.2</td><td align="center" rowspan="1" colspan="1">1.1</td><td align="center" rowspan="1" colspan="1">1.4</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1.7</td><td align="center" rowspan="1" colspan="1">0.6</td><td align="center" rowspan="1" colspan="1">1.2</td><td align="center" rowspan="1" colspan="1">1.2</td><td align="center" rowspan="1" colspan="1">0.9</td><td align="center" rowspan="1" colspan="1">1.2</td><td align="center" rowspan="1" colspan="1">1.6</td><td align="center" rowspan="1" colspan="1">0.7</td></tr><tr><td align="left" rowspan="1" colspan="1">Cancers</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1"> Digestive neoplasms</td><td align="center" rowspan="1" colspan="1">3.1</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">5.3</td><td align="center" rowspan="1" colspan="1">3.5</td><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">3.3</td><td align="center" rowspan="1" colspan="1">3.6</td><td align="center" rowspan="1" colspan="1">2.5</td><td align="center" rowspan="1" colspan="1">2.8</td><td align="center" rowspan="1" colspan="1">3.6</td><td align="center" rowspan="1" colspan="1">3.1</td><td align="center" rowspan="1" colspan="1">3.1</td><td align="center" rowspan="1" colspan="1">3.4</td><td align="center" rowspan="1" colspan="1">1.1</td></tr><tr><td align="left" rowspan="1" colspan="1"> Respiratory neoplasms</td><td align="center" rowspan="1" colspan="1">1.6</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">1.6</td><td align="center" rowspan="1" colspan="1">2.7</td><td align="center" rowspan="1" colspan="1">2.3</td><td align="center" rowspan="1" colspan="1">1.5</td><td align="center" rowspan="1" colspan="1">1.3</td><td align="center" rowspan="1" colspan="1">2.4</td><td align="center" rowspan="1" colspan="1">0.7</td><td align="center" rowspan="1" colspan="1">1.8</td><td align="center" rowspan="1" colspan="1">1.3</td><td align="center" rowspan="1" colspan="1">0.8</td><td align="center" rowspan="1" colspan="1">2.2</td><td align="center" rowspan="1" colspan="1">1.5</td><td align="center" rowspan="1" colspan="1">3.3</td></tr><tr><td align="left" rowspan="1" colspan="1"> Reproductive neoplasms</td><td align="center" rowspan="1" colspan="1">1.3</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">1.6</td><td align="center" rowspan="1" colspan="1">2.1</td><td align="center" rowspan="1" colspan="1">1.8</td><td align="center" rowspan="1" colspan="1">1.3</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1.7</td><td align="center" rowspan="1" colspan="1">1.4</td><td align="center" rowspan="1" colspan="1">1.1</td><td align="center" rowspan="1" colspan="1">1.3</td><td align="center" rowspan="1" colspan="1">1.4</td><td align="center" rowspan="1" colspan="1">1.2</td><td align="center" rowspan="1" colspan="1">0.9</td></tr><tr><td align="left" rowspan="1" colspan="1"> Other neoplasms</td><td align="center" rowspan="1" colspan="1">0.9</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">3.2</td><td align="center" rowspan="1" colspan="1">3.8</td><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">2.4</td><td align="center" rowspan="1" colspan="1">2.3</td><td align="center" rowspan="1" colspan="1">2.7</td><td align="center" rowspan="1" colspan="1">2.3</td><td align="center" rowspan="1" colspan="1">2.5</td><td align="center" rowspan="1" colspan="1">2.5</td><td align="center" rowspan="1" colspan="1">2.4</td><td align="center" rowspan="1" colspan="1">2.9</td><td align="center" rowspan="1" colspan="1">1.9</td><td align="center" rowspan="1" colspan="1">2.3</td></tr><tr><td align="left" rowspan="1" colspan="1">Other NCDs</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1"> Stroke</td><td align="center" rowspan="1" colspan="1">6.4</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">4.2</td><td align="center" rowspan="1" colspan="1">12.8</td><td align="center" rowspan="1" colspan="1">5.6</td><td align="center" rowspan="1" colspan="1">6.8</td><td align="center" rowspan="1" colspan="1">6.5</td><td align="center" rowspan="1" colspan="1">5.4</td><td align="center" rowspan="1" colspan="1">7.6</td><td align="center" rowspan="1" colspan="1">5.9</td><td align="center" rowspan="1" colspan="1">7.5</td><td align="center" rowspan="1" colspan="1">5.9</td><td align="center" rowspan="1" colspan="1">6.4</td><td align="center" rowspan="1" colspan="1">7.6</td><td align="center" rowspan="1" colspan="1">6.3</td></tr><tr><td align="left" rowspan="1" colspan="1"> Acute abdomen</td><td align="center" rowspan="1" colspan="1">4.8</td><td align="center" rowspan="1" colspan="1">0.4</td><td align="center" rowspan="1" colspan="1">0.2</td><td align="center" rowspan="1" colspan="1">9.7</td><td align="center" rowspan="1" colspan="1">5.6</td><td align="center" rowspan="1" colspan="1">6</td><td align="center" rowspan="1" colspan="1">6.5</td><td align="center" rowspan="1" colspan="1">3.9</td><td align="center" rowspan="1" colspan="1">4.8</td><td align="center" rowspan="1" colspan="1">4.5</td><td align="center" rowspan="1" colspan="1">5.1</td><td align="center" rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1">4.5</td><td align="center" rowspan="1" colspan="1">4.2</td><td align="center" rowspan="1" colspan="1">6.2</td><td align="center" rowspan="1" colspan="1">3.2</td><td align="center" rowspan="1" colspan="1">3.4</td></tr><tr><td align="left" rowspan="1" colspan="1"> Severe malnutrition</td><td align="center" rowspan="1" colspan="1">1.6</td><td align="center" rowspan="1" colspan="1">3.4</td><td align="center" rowspan="1" colspan="1">6</td><td align="center" rowspan="1" colspan="1">1.5</td><td align="center" rowspan="1" colspan="1">0.2</td><td align="center" rowspan="1" colspan="1">0.8</td><td align="center" rowspan="1" colspan="1">0.8</td><td align="center" rowspan="1" colspan="1">1.7</td><td align="center" rowspan="1" colspan="1">2.1</td><td align="center" rowspan="1" colspan="1">1.6</td><td align="center" rowspan="1" colspan="1">1.5</td><td align="center" rowspan="1" colspan="1">1.5</td><td align="center" rowspan="1" colspan="1">1.9</td><td align="center" rowspan="1" colspan="1">2.1</td><td align="center" rowspan="1" colspan="1">1.4</td><td align="center" rowspan="1" colspan="1">1.3</td><td align="center" rowspan="1" colspan="1">0.9</td></tr><tr><td align="left" rowspan="1" colspan="1"> Acute cardiac diseases</td><td align="center" rowspan="1" colspan="1">0.9</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.8</td><td align="center" rowspan="1" colspan="1">1.5</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0.6</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0.8</td><td align="center" rowspan="1" colspan="1">0.9</td><td align="center" rowspan="1" colspan="1">0.9</td><td align="center" rowspan="1" colspan="1">0.8</td><td align="center" rowspan="1" colspan="1">1.3</td><td align="center" rowspan="1" colspan="1">0.7</td><td align="center" rowspan="1" colspan="1">0.5</td><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1"> Other cardiac diseases</td><td align="center" rowspan="1" colspan="1">1.3</td><td align="center" rowspan="1" colspan="1">0.5</td><td align="center" rowspan="1" colspan="1">0.8</td><td align="center" rowspan="1" colspan="1">2.2</td><td align="center" rowspan="1" colspan="1">1.1</td><td align="center" rowspan="1" colspan="1">1.7</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">1.5</td><td align="center" rowspan="1" colspan="1">1.4</td><td align="center" rowspan="1" colspan="1">1.3</td><td align="center" rowspan="1" colspan="1">1.3</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">1.6</td><td align="center" rowspan="1" colspan="1">1.2</td><td align="center" rowspan="1" colspan="1">1.1</td><td align="center" rowspan="1" colspan="1">1</td></tr><tr><td align="left" rowspan="1" colspan="1"> Other unspecified NCDs</td><td align="center" rowspan="1" colspan="1">2.5</td><td align="center" rowspan="1" colspan="1">0.1</td><td align="center" rowspan="1" colspan="1">0.8</td><td align="center" rowspan="1" colspan="1">1.2</td><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">3.5</td><td align="center" rowspan="1" colspan="1">2.5</td><td align="center" rowspan="1" colspan="1">2.2</td><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">2.2</td><td align="center" rowspan="1" colspan="1">2.9</td><td align="center" rowspan="1" colspan="1">2.6</td><td align="center" rowspan="1" colspan="1">2.3</td><td align="center" rowspan="1" colspan="1">2.4</td><td align="center" rowspan="1" colspan="1">3.1</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1"> Liver cirrhosis</td><td align="center" rowspan="1" colspan="1">2.5</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">1.8</td><td align="center" rowspan="1" colspan="1">0.8</td><td align="center" rowspan="1" colspan="1">1.3</td><td align="center" rowspan="1" colspan="1">0.5</td><td align="center" rowspan="1" colspan="1">0.9</td><td align="center" rowspan="1" colspan="1">1.1</td><td align="center" rowspan="1" colspan="1">1.1</td><td align="center" rowspan="1" colspan="1">0.7</td><td align="center" rowspan="1" colspan="1">1.1</td><td align="center" rowspan="1" colspan="1">0.6</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0.5</td><td align="center" rowspan="1" colspan="1">1.6</td><td align="center" rowspan="1" colspan="1">1.2</td></tr><tr><td align="left" rowspan="1" colspan="1">Neonatal</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">20.6</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">1.4</td><td align="center" rowspan="1" colspan="1">3.2</td><td align="center" rowspan="1" colspan="1">0.9</td><td align="center" rowspan="1" colspan="1">1.5</td><td align="center" rowspan="1" colspan="1">2.6</td><td align="center" rowspan="1" colspan="1">2.9</td><td align="center" rowspan="1" colspan="1">0.4</td><td align="center" rowspan="1" colspan="1">2.1</td><td align="center" rowspan="1" colspan="1">2.1</td><td align="center" rowspan="1" colspan="1">1.6</td><td align="center" rowspan="1" colspan="1">2.4</td></tr><tr><td align="left" rowspan="1" colspan="1">External</td><td align="center" rowspan="1" colspan="1">2.5</td><td align="center" rowspan="1" colspan="1">0.7</td><td align="center" rowspan="1" colspan="1">1.2</td><td align="center" rowspan="1" colspan="1">5.9</td><td align="center" rowspan="1" colspan="1">3.8</td><td align="center" rowspan="1" colspan="1">1.7</td><td align="center" rowspan="1" colspan="1">2.9</td><td align="center" rowspan="1" colspan="1">2.1</td><td align="center" rowspan="1" colspan="1">2.6</td><td align="center" rowspan="1" colspan="1">2.7</td><td align="center" rowspan="1" colspan="1">2.2</td><td align="center" rowspan="1" colspan="1">2.4</td><td align="center" rowspan="1" colspan="1">2.5</td><td align="center" rowspan="1" colspan="1">2.2</td><td align="center" rowspan="1" colspan="1">2.9</td><td align="center" rowspan="1" colspan="1">2.2</td><td align="center" rowspan="1" colspan="1">1.8</td></tr><tr><td align="left" rowspan="1" colspan="1">Maternal</td><td align="center" rowspan="1" colspan="1">2.1</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">6.8</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">2.6</td><td align="center" rowspan="1" colspan="1">2.3</td><td align="center" rowspan="1" colspan="1">1.5</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">4.6</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">2.4</td><td align="center" rowspan="1" colspan="1">2.7</td><td align="center" rowspan="1" colspan="1">1.5</td><td align="center" rowspan="1" colspan="1">2.4</td><td align="center" rowspan="1" colspan="1">2.9</td></tr><tr><td align="left" rowspan="1" colspan="1">Indeterminate</td><td align="center" rowspan="1" colspan="1">16.6</td><td align="center" rowspan="1" colspan="1">12.8</td><td align="center" rowspan="1" colspan="1">12.4</td><td align="center" rowspan="1" colspan="1">17.8</td><td align="center" rowspan="1" colspan="1">16.2</td><td align="center" rowspan="1" colspan="1">19.1</td><td align="center" rowspan="1" colspan="1">19.1</td><td align="center" rowspan="1" colspan="1">15.9</td><td align="center" rowspan="1" colspan="1">15.8</td><td align="center" rowspan="1" colspan="1">17.1</td><td align="center" rowspan="1" colspan="1">16</td><td align="center" rowspan="1" colspan="1">17.3</td><td align="center" rowspan="1" colspan="1">15.3</td><td align="center" rowspan="1" colspan="1">16.5</td><td align="center" rowspan="1" colspan="1">17.2</td><td align="center" rowspan="1" colspan="1">16</td><td align="center" rowspan="1" colspan="1">14.7</td></tr><tr><td align="left" rowspan="1" colspan="1">Total</td><td align="center" rowspan="1" colspan="1">100</td><td align="center" rowspan="1" colspan="1">100</td><td align="center" rowspan="1" colspan="1">100</td><td align="center" rowspan="1" colspan="1">100</td><td align="center" rowspan="1" colspan="1">100</td><td align="center" rowspan="1" colspan="1">100</td><td align="center" rowspan="1" colspan="1">100</td><td align="center" rowspan="1" colspan="1">100</td><td align="center" rowspan="1" colspan="1">100</td><td align="center" rowspan="1" colspan="1">100</td><td align="center" rowspan="1" colspan="1">100</td><td align="center" rowspan="1" colspan="1">100</td><td align="center" rowspan="1" colspan="1">100</td><td align="center" rowspan="1" colspan="1">100</td><td align="center" rowspan="1" colspan="1">100</td><td align="center" rowspan="1" colspan="1">100</td><td align="center" rowspan="1" colspan="1">100</td></tr></tbody></table></table-wrap><p>Malaria, ARI and diarrhoeal diseases accounted for more than half (58.5%) of deaths among children aged under 5 years (<xref ref-type="fig" rid="F0002">Fig. 2</xref>); 11% of deaths were attributed to HIV-related causes in this age group. More than a third (38%) of deaths among those aged 15–59 years were due to four communicable diseases: pulmonary tuberculosis (14%), ARI (including pneumonia) (9.9%), malaria (7.5%) and HIV/AIDS (6.5%). Overall, 46 and 37.8% of adult deaths were attributed to communicable and non-communicable diseases respectively.</p><fig id="F0002" position="float"><label>Fig. 2</label><caption><p>Comparison of the distribution of causes of death among children under 5 years of age by InterVA-4 for Farafenni HDSS, 1998–2007 and CHERG estimates for The Gambia, 2008 (<xref rid="CIT0027" ref-type="bibr">27</xref>).</p></caption><graphic xlink:href="GHA-7-25598-g002"/></fig><p>
<xref ref-type="table" rid="T0003">Table 3</xref> shows the top five cause-specific mortality rates for each age group and period. Overall, ARI, malaria, diarrhoeal diseases, neonatal sepsis, HIV/AIDS-related illnesses and severe malnutrition had the highest cause-specific mortality rates among children under 5 years of age. ARI mortality was consistently high among infants throughout the study period with an average rate of 5.59 per 1,000 person-years (95% CI: 4.38–7.15), followed by malaria with a rate of 4.11 per 1,000 person-years (95% CI: 3.09–5.47). The reverse pattern was observed among children aged 1–4 years who had a malaria mortality rate of 3.06 per 1,000 person-years (95% CI: 2.58–3.63) and an ARI mortality rate of 1.05 per 1,000 person-years (95% CI: 0.79–1.41). HIV-related mortality had the second highest rate in this age group (1.17 per 1,000 person-years [95% CI: 0.89–1.54]). Disease-specific mortality rates among children aged under 5 years increased overall between 1998–2001 and 2002–2004 due to increased VA coverage, but only significantly for malaria in infants (<italic>p=</italic>0.001) and children 1–4 years (<italic>p<</italic>0.0001). Although at comparatively lower levels, malaria, ARI, acute abdomen and HIV/AIDS constituted the greatest mortality burden among older children aged 5–14 years.</p><table-wrap id="T0003" position="float"><label>Table 3</label><caption><p>Top five causes of death by age group and period</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1"/><th align="center" colspan="3" rowspan="1">1998–2007</th><th align="center" colspan="3" rowspan="1">1998–2001</th><th align="center" colspan="3" rowspan="1">2002–2004</th><th align="center" colspan="3" rowspan="1">2005–2007</th></tr><tr><th align="left" rowspan="1" colspan="1"/><th align="center" colspan="3" rowspan="1">
<hr/>
</th><th align="center" colspan="3" rowspan="1">
<hr/>
</th><th align="center" colspan="3" rowspan="1">
<hr/>
</th><th align="center" colspan="3" rowspan="1">
<hr/>
</th></tr><tr><th align="left" rowspan="1" colspan="1">Age group and rank</th><th align="center" rowspan="1" colspan="1">Cause</th><th align="center" rowspan="1" colspan="1">No. of deaths</th><th align="center" rowspan="1" colspan="1">Cause-specific mortality rate per 1,000 PY (95% CI)</th><th align="center" rowspan="1" colspan="1">Cause</th><th align="center" rowspan="1" colspan="1">No. of deaths</th><th align="center" rowspan="1" colspan="1">Cause-specific mortality rate per 1,000 PY (95% CI)</th><th align="center" rowspan="1" colspan="1">Cause</th><th align="center" rowspan="1" colspan="1">No. of deaths</th><th align="center" rowspan="1" colspan="1">Cause-specific mortality rate per 1,000 PY (95% CI)</th><th align="center" rowspan="1" colspan="1">Cause</th><th align="center" rowspan="1" colspan="1">No. of deaths</th><th align="center" rowspan="1" colspan="1">Cause-specific mortality rate per 1,000 PY (95% CI)</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1"><1 year</td><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">ARI, including pneumonia</td><td align="center" rowspan="1" colspan="1">64</td><td align="center" rowspan="1" colspan="1">5.59 (4.38–7.15)</td><td align="left" rowspan="1" colspan="1">Neonatal sepsis</td><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">1.54 (0.58–4.11)</td><td align="left" rowspan="1" colspan="1">ARI, including pneumonia</td><td align="center" rowspan="1" colspan="1">36</td><td align="center" rowspan="1" colspan="1">9.34 (6.74–13.00)</td><td align="left" rowspan="1" colspan="1">ARI, including pneumonia</td><td align="center" rowspan="1" colspan="1">25</td><td align="center" rowspan="1" colspan="1">5.01 (3.38–7.41)</td></tr><tr><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1">Malaria</td><td align="center" rowspan="1" colspan="1">47</td><td align="center" rowspan="1" colspan="1">4.11 (3.09–5.47)</td><td align="left" rowspan="1" colspan="1">Malaria</td><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">1.54 (0.58–4.11)</td><td align="left" rowspan="1" colspan="1">Malaria</td><td align="center" rowspan="1" colspan="1">21</td><td align="center" rowspan="1" colspan="1">5.45 (3.55–8.36)</td><td align="left" rowspan="1" colspan="1">Malaria</td><td align="center" rowspan="1" colspan="1">22</td><td align="center" rowspan="1" colspan="1">4.41 (2.90–6.69)</td></tr><tr><td align="left" rowspan="1" colspan="1">3</td><td align="left" rowspan="1" colspan="1">Neonatal sepsis</td><td align="center" rowspan="1" colspan="1">31</td><td align="center" rowspan="1" colspan="1">2.71 (1.91–3.85)</td><td align="left" rowspan="1" colspan="1">ARI, including pneumonia</td><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">1.16 (0.37–3.59)</td><td align="left" rowspan="1" colspan="1">Neonatal Sepsis</td><td align="center" rowspan="1" colspan="1">21</td><td align="center" rowspan="1" colspan="1">5.45 (3.55–8.36)</td><td align="left" rowspan="1" colspan="1">Diarrhoeal diseases</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">2.00 (1.08–3.72)</td></tr><tr><td align="left" rowspan="1" colspan="1">4</td><td align="left" rowspan="1" colspan="1">Diarrhoeal diseases</td><td align="center" rowspan="1" colspan="1">24</td><td align="center" rowspan="1" colspan="1">2.10 (1.41–3.13)</td><td align="left" rowspan="1" colspan="1">Neonatal pneumonia</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">0.77 (0.19–3.08)</td><td align="left" rowspan="1" colspan="1">Diarrhoeal diseases</td><td align="center" rowspan="1" colspan="1">12</td><td align="center" rowspan="1" colspan="1">3.11 (1.77–5.48)</td><td align="left" rowspan="1" colspan="1">Neonatal sepsis</td><td align="center" rowspan="1" colspan="1">6</td><td align="center" rowspan="1" colspan="1">1.20 (0.54–2.68)</td></tr><tr><td align="left" rowspan="1" colspan="1">5</td><td align="left" rowspan="1" colspan="1">HIV/AIDS-related</td><td align="center" rowspan="1" colspan="1">14</td><td align="center" rowspan="1" colspan="1">1.22 (0.72–2.07)</td><td align="left" rowspan="1" colspan="1">Other neonatal causes</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">0.77 (0.19–3.08)</td><td align="left" rowspan="1" colspan="1">Neonatal pneumonia</td><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1">2.33 (1.21–4.49)</td><td align="left" rowspan="1" colspan="1">HIV/AIDS-related</td><td align="center" rowspan="1" colspan="1">6</td><td align="center" rowspan="1" colspan="1">1.20 (0.54–2.68)</td></tr><tr><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1">Indeterminate</td><td align="center" rowspan="1" colspan="1">11</td><td align="center" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1">Indeterminate</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1">Indeterminate</td><td align="center" rowspan="1" colspan="1">7</td><td align="center" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1">Indeterminate</td><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">1–4 years</td><td align="left" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">Malaria</td><td align="center" rowspan="1" colspan="1">131</td><td align="center" rowspan="1" colspan="1">3.06 (2.58–3.63)</td><td align="left" rowspan="1" colspan="1">Malaria</td><td align="center" rowspan="1" colspan="1">16</td><td align="center" rowspan="1" colspan="1">1.78 (1.09–2.91)</td><td align="left" rowspan="1" colspan="1">Malaria</td><td align="center" rowspan="1" colspan="1">78</td><td align="center" rowspan="1" colspan="1">4.92 (3.94–6.14)</td><td align="left" rowspan="1" colspan="1">Malaria</td><td align="center" rowspan="1" colspan="1">37</td><td align="center" rowspan="1" colspan="1">2.06 (1.49–2.84)</td></tr><tr><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1">HIV/AIDS-related</td><td align="center" rowspan="1" colspan="1">50</td><td align="center" rowspan="1" colspan="1">1.17 (0.89–1.54)</td><td align="left" rowspan="1" colspan="1">Diarrhoeal diseases</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1">0.22 (0.06–0.89)</td><td align="left" rowspan="1" colspan="1">HIV/AIDS-related</td><td align="center" rowspan="1" colspan="1">27</td><td align="center" rowspan="1" colspan="1">1.70 (1.17–2.48)</td><td align="left" rowspan="1" colspan="1">HIV/AIDS-related</td><td align="center" rowspan="1" colspan="1">23</td><td align="center" rowspan="1" colspan="1">1.28 (0.85–1.93)</td></tr><tr><td align="left" rowspan="1" colspan="1">3</td><td align="left" rowspan="1" colspan="1">ARI, including pneumonia</td><td align="center" rowspan="1" colspan="1">45</td><td align="center" rowspan="1" colspan="1">1.05 (0.79–1.41)</td><td align="left" rowspan="1" colspan="1">ARI, including pneumonia</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0.11 (0.02–0.79)</td><td align="left" rowspan="1" colspan="1">ARI, including pneumonia</td><td align="center" rowspan="1" colspan="1">25</td><td align="center" rowspan="1" colspan="1">1.58 (1.06–2.33)</td><td align="left" rowspan="1" colspan="1">ARI, including pneumonia</td><td align="center" rowspan="1" colspan="1">19</td><td align="center" rowspan="1" colspan="1">1.06 (0.67–1.66)</td></tr><tr><td align="left" rowspan="1" colspan="1">4</td><td align="left" rowspan="1" colspan="1">Diarrhoeal diseases</td><td align="center" rowspan="1" colspan="1">26</td><td align="center" rowspan="1" colspan="1">0.61 (0.41–0.89)</td><td align="left" rowspan="1" colspan="1">Severe malnutrition</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0.11 (0.02–0.79)</td><td align="left" rowspan="1" colspan="1">Diarrhoeal diseases</td><td align="center" rowspan="1" colspan="1">13</td><td align="center" rowspan="1" colspan="1">0.82 (0.48–1.41)</td><td align="left" rowspan="1" colspan="1">Diarrhoeal diseases</td><td align="center" rowspan="1" colspan="1">11</td><td align="center" rowspan="1" colspan="1">0.61 (0.34–1.11)</td></tr><tr><td align="left" rowspan="1" colspan="1">5</td><td align="left" rowspan="1" colspan="1">Severe malnutrition</td><td align="center" rowspan="1" colspan="1">18</td><td align="center" rowspan="1" colspan="1">0.42 (0.26–0.67)</td><td align="left" rowspan="1" colspan="1">Meningitis and encephalitis</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0.11 (0.02–0.79)</td><td align="left" rowspan="1" colspan="1">Severe malnutrition</td><td align="center" rowspan="1" colspan="1">7</td><td align="center" rowspan="1" colspan="1">0.44 (0.21–0.93)</td><td align="left" rowspan="1" colspan="1">Severe malnutrition</td><td align="center" rowspan="1" colspan="1">10</td><td align="center" rowspan="1" colspan="1">0.56 (0.30–1.03)</td></tr><tr><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1">Indeterminate</td><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1">Indeterminate</td><td align="center" rowspan="1" colspan="1">2</td><td align="center" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1">Indeterminate</td><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1">Indeterminate</td><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1"/></tr><tr><td colspan="2" align="left" rowspan="1">5–14 years</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">Malaria</td><td align="center" rowspan="1" colspan="1">47</td><td align="center" rowspan="1" colspan="1">0.53 (0.40–0.70)</td><td align="left" rowspan="1" colspan="1">Malaria</td><td align="center" rowspan="1" colspan="1">11</td><td align="center" rowspan="1" colspan="1">0.58 (0.32–1.05)</td><td align="left" rowspan="1" colspan="1">Malaria</td><td align="center" rowspan="1" colspan="1">25</td><td align="center" rowspan="1" colspan="1">0.76 (0.51–1.12)</td><td align="left" rowspan="1" colspan="1">Malaria</td><td align="center" rowspan="1" colspan="1">11</td><td align="center" rowspan="1" colspan="1">0.30 (0.17–0.54)</td></tr><tr><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1">ARI, including pneumonia</td><td align="center" rowspan="1" colspan="1">16</td><td align="center" rowspan="1" colspan="1">0.18 (0.11–0.29)</td><td align="left" rowspan="1" colspan="1">Acute abdomen</td><td align="center" rowspan="1" colspan="1">6</td><td align="center" rowspan="1" colspan="1">0.32 (0.14–0.71)</td><td align="left" rowspan="1" colspan="1">HIV/AIDS-related</td><td align="center" rowspan="1" colspan="1">6</td><td align="center" rowspan="1" colspan="1">0.18 (0.08–0.41)</td><td align="left" rowspan="1" colspan="1">ARI, including pneumonia</td><td align="center" rowspan="1" colspan="1">6</td><td align="center" rowspan="1" colspan="1">0.16 (0.07–0.36)</td></tr><tr><td align="left" rowspan="1" colspan="1">3</td><td align="left" rowspan="1" colspan="1">Acute abdomen</td><td align="center" rowspan="1" colspan="1">15</td><td align="center" rowspan="1" colspan="1">0.17 (0.10–0.28)</td><td align="left" rowspan="1" colspan="1">ARI, including pneumonia</td><td align="center" rowspan="1" colspan="1">6</td><td align="center" rowspan="1" colspan="1">0.32 (0.14–0.71)</td><td align="left" rowspan="1" colspan="1">Acute abdomen</td><td align="center" rowspan="1" colspan="1">6</td><td align="center" rowspan="1" colspan="1">0.18 (0.08–0.41)</td><td align="left" rowspan="1" colspan="1">HIV/AIDS-related</td><td align="center" rowspan="1" colspan="1">6</td><td align="center" rowspan="1" colspan="1">0.16 (0.07–0.36)</td></tr><tr><td align="left" rowspan="1" colspan="1">4</td><td align="left" rowspan="1" colspan="1">HIV/AIDS-related</td><td align="center" rowspan="1" colspan="1">14</td><td align="center" rowspan="1" colspan="1">0.16 (0.09–0.27)</td><td align="left" rowspan="1" colspan="1">External causes</td><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">0.16 (0.05–0.49)</td><td align="left" rowspan="1" colspan="1">ARI, including pneumonia</td><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">0.12 (0.05–0.32)</td><td align="left" rowspan="1" colspan="1">Acute abdomen</td><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">0.08 (0.03–0.25)</td></tr><tr><td align="left" rowspan="1" colspan="1">5</td><td align="left" rowspan="1" colspan="1">External causes</td><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1">0.10 (0.05–0.19)</td><td align="left" rowspan="1" colspan="1">Meningitis and encephalitis</td><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">0.16 (0.05–0.49)</td><td align="left" rowspan="1" colspan="1">External causes</td><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">0.09 (0.03–0.28)</td><td align="left" rowspan="1" colspan="1">Meningitis and encephalitis</td><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1">0.08 (0.03–0.25)</td></tr><tr><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1">Indeterminate</td><td align="center" rowspan="1" colspan="1">8</td><td align="center" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1">Indeterminate</td><td align="center" rowspan="1" colspan="1">3</td><td align="center" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1">Indeterminate</td><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1">Indeterminate</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1"/></tr><tr><td colspan="2" align="left" rowspan="1">15–59 years</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">Pulmonary tuberculosis</td><td align="center" rowspan="1" colspan="1">107</td><td align="center" rowspan="1" colspan="1">0.68 (0.57–0.83)</td><td align="left" rowspan="1" colspan="1">ARI, including pneumonia</td><td align="center" rowspan="1" colspan="1">23</td><td align="center" rowspan="1" colspan="1">0.76 (0.50–1.14)</td><td align="left" rowspan="1" colspan="1">Pulmonary tuberculosis</td><td align="center" rowspan="1" colspan="1">44</td><td align="center" rowspan="1" colspan="1">0.75 (0.56–1.01)</td><td align="left" rowspan="1" colspan="1">Pulmonary tuberculosis</td><td align="center" rowspan="1" colspan="1">44</td><td align="center" rowspan="1" colspan="1">0.65 (0.49–0.88)</td></tr><tr><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1">Other infectious diseases</td><td align="center" rowspan="1" colspan="1">107</td><td align="center" rowspan="1" colspan="1">0.68 (0.57–0.83)</td><td align="left" rowspan="1" colspan="1">Pulmonary tuberculosis</td><td align="center" rowspan="1" colspan="1">19</td><td align="center" rowspan="1" colspan="1">0.62 (0.40–0.98)</td><td align="left" rowspan="1" colspan="1">Other infectious diseases</td><td align="center" rowspan="1" colspan="1">44</td><td align="center" rowspan="1" colspan="1">0.75 (0.56–1.01)</td><td align="left" rowspan="1" colspan="1">Other infectious diseases</td><td align="center" rowspan="1" colspan="1">44</td><td align="center" rowspan="1" colspan="1">0.65 (0.49–0.88)</td></tr><tr><td align="left" rowspan="1" colspan="1">3</td><td align="left" rowspan="1" colspan="1">ARI, including pneumonia</td><td align="center" rowspan="1" colspan="1">78</td><td align="center" rowspan="1" colspan="1">0.50 (0.40–0.62)</td><td align="left" rowspan="1" colspan="1">Other infectious diseases</td><td align="center" rowspan="1" colspan="1">19</td><td align="center" rowspan="1" colspan="1">0.62 (0.40–0.98)</td><td align="left" rowspan="1" colspan="1">ARI, including pneumonia</td><td align="center" rowspan="1" colspan="1">37</td><td align="center" rowspan="1" colspan="1">0.63 (0.46–0.87)</td><td align="left" rowspan="1" colspan="1">Malaria</td><td align="center" rowspan="1" colspan="1">27</td><td align="center" rowspan="1" colspan="1">0.40 (0.27–0.58)</td></tr><tr><td align="left" rowspan="1" colspan="1">4</td><td align="left" rowspan="1" colspan="1">Malaria</td><td align="center" rowspan="1" colspan="1">61</td><td align="center" rowspan="1" colspan="1">0.39 (0.30–0.50)</td><td align="left" rowspan="1" colspan="1">Maternal causes<xref ref-type="table-fn" rid="TF0002">a</xref>
</td><td align="center" rowspan="1" colspan="1">18</td><td align="center" rowspan="1" colspan="1">0.59 (0.37–0.94)</td><td align="left" rowspan="1" colspan="1">Maternal causes<xref ref-type="table-fn" rid="TF0002">a</xref>
</td><td align="center" rowspan="1" colspan="1">25</td><td align="center" rowspan="1" colspan="1">0.43 (0.29–0.63)</td><td align="left" rowspan="1" colspan="1">HIV/AIDS-related</td><td align="center" rowspan="1" colspan="1">19</td><td align="center" rowspan="1" colspan="1">0.28 (0.18–0.44)</td></tr><tr><td align="left" rowspan="1" colspan="1">5</td><td align="left" rowspan="1" colspan="1">Maternal causes<xref ref-type="table-fn" rid="TF0002">a</xref>
</td><td align="center" rowspan="1" colspan="1">56</td><td align="center" rowspan="1" colspan="1">0.36 (0.28–0.47)</td><td align="left" rowspan="1" colspan="1">Acute abdomen</td><td align="center" rowspan="1" colspan="1">15</td><td align="center" rowspan="1" colspan="1">0.49 (0.30–0.82)</td><td align="left" rowspan="1" colspan="1">HIV/AIDS-related</td><td align="center" rowspan="1" colspan="1">24</td><td align="center" rowspan="1" colspan="1">0.41 (0.28–0.61)</td><td align="left" rowspan="1" colspan="1">ARI, including pneumonia</td><td align="center" rowspan="1" colspan="1">18</td><td align="center" rowspan="1" colspan="1">0.27 (0.17–0.42)</td></tr><tr><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1">Indeterminate</td><td align="center" rowspan="1" colspan="1">47</td><td align="center" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1">Indeterminate</td><td align="center" rowspan="1" colspan="1">9</td><td align="center" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1">Indeterminate</td><td align="center" rowspan="1" colspan="1">21</td><td align="center" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1">Indeterminate</td><td align="center" rowspan="1" colspan="1">17</td><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">60+ years</td><td align="left" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">1</td><td align="left" rowspan="1" colspan="1">Pulmonary tuberculosis</td><td align="center" rowspan="1" colspan="1">137</td><td align="center" rowspan="1" colspan="1">7.84 (6.63–9.27)</td><td align="left" rowspan="1" colspan="1">ARI, including pneumonia</td><td align="center" rowspan="1" colspan="1">47</td><td align="center" rowspan="1" colspan="1">10.00 (7.79–14.00)</td><td align="left" rowspan="1" colspan="1">Stroke</td><td align="center" rowspan="1" colspan="1">59</td><td align="center" rowspan="1" colspan="1">9.62 (7.45–12.00)</td><td align="left" rowspan="1" colspan="1">Stroke</td><td align="center" rowspan="1" colspan="1">43</td><td align="center" rowspan="1" colspan="1">6.32 (4.69–8.52)</td></tr><tr><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1">Other infectious diseases</td><td align="center" rowspan="1" colspan="1">137</td><td align="center" rowspan="1" colspan="1">7.84 (6.63–9.27)</td><td align="left" rowspan="1" colspan="1">Pulmonary tuberculosis</td><td align="center" rowspan="1" colspan="1">43</td><td align="center" rowspan="1" colspan="1">9.49 (7.04–13.00)</td><td align="left" rowspan="1" colspan="1">Other infectious diseases</td><td align="center" rowspan="1" colspan="1">54</td><td align="center" rowspan="1" colspan="1">8.80 (6.74–11.00)</td><td align="left" rowspan="1" colspan="1">ARI, including pneumonia</td><td align="center" rowspan="1" colspan="1">42</td><td align="center" rowspan="1" colspan="1">6.17 (4.56–8.35)</td></tr><tr><td align="left" rowspan="1" colspan="1">3</td><td align="left" rowspan="1" colspan="1">Stroke</td><td align="center" rowspan="1" colspan="1">134</td><td align="center" rowspan="1" colspan="1">7.67 (6.48–9.09)</td><td align="left" rowspan="1" colspan="1">Other infectious diseases</td><td align="center" rowspan="1" colspan="1">43</td><td align="center" rowspan="1" colspan="1">9.49 (7.04–13.00)</td><td align="left" rowspan="1" colspan="1">Pulmonary tuberculosis</td><td align="center" rowspan="1" colspan="1">54</td><td align="center" rowspan="1" colspan="1">8.80 (6.74–11.00)</td><td align="left" rowspan="1" colspan="1">Pulmonary tuberculosis</td><td align="center" rowspan="1" colspan="1">40</td><td align="center" rowspan="1" colspan="1">5.88 (4.31–8.02)</td></tr><tr><td align="left" rowspan="1" colspan="1">4</td><td align="left" rowspan="1" colspan="1">ARI, including pneumonia</td><td align="center" rowspan="1" colspan="1">130</td><td align="center" rowspan="1" colspan="1">7.44 (6.27–8.84)</td><td align="left" rowspan="1" colspan="1">Stroke</td><td align="center" rowspan="1" colspan="1">32</td><td align="center" rowspan="1" colspan="1">7.06 (4.99–9.99)</td><td align="left" rowspan="1" colspan="1">ARI, including pneumonia</td><td align="center" rowspan="1" colspan="1">41</td><td align="center" rowspan="1" colspan="1">6.68 (4.92–9.08)</td><td align="left" rowspan="1" colspan="1">Other infectious diseases</td><td align="center" rowspan="1" colspan="1">40</td><td align="center" rowspan="1" colspan="1">5.88 (4.31–8.02)</td></tr><tr><td align="left" rowspan="1" colspan="1">5</td><td align="left" rowspan="1" colspan="1">Malaria</td><td align="center" rowspan="1" colspan="1">63</td><td align="center" rowspan="1" colspan="1">3.61 (2.82–4.62)</td><td align="left" rowspan="1" colspan="1">Malaria</td><td align="center" rowspan="1" colspan="1">24</td><td align="center" rowspan="1" colspan="1">5.30 (3.55–7.90)</td><td align="left" rowspan="1" colspan="1">Acute abdomen</td><td align="center" rowspan="1" colspan="1">20</td><td align="center" rowspan="1" colspan="1">3.26 (2.10–5.05)</td><td align="left" rowspan="1" colspan="1">Acute abdomen</td><td align="center" rowspan="1" colspan="1">24</td><td align="center" rowspan="1" colspan="1">3.53 (2.36–5.26)</td></tr><tr><td align="left" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1">Indeterminate</td><td align="center" rowspan="1" colspan="1">82</td><td align="center" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1">Indeterminate</td><td align="center" rowspan="1" colspan="1">28</td><td align="center" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1">Indeterminate</td><td align="center" rowspan="1" colspan="1">32</td><td align="center" rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1">Indeterminate</td><td align="center" rowspan="1" colspan="1">22</td><td align="center" rowspan="1" colspan="1"/></tr></tbody></table><table-wrap-foot><fn id="TF0002"><label>a</label><p>Rate based on person-time contributed by women aged 15–49 years who constitute the population at risk of maternal causes of death.</p></fn></table-wrap-foot></table-wrap><p>Pulmonary tuberculosis, ARI, malaria and other infectious diseases (i.e. communicable diseases other than those listed in <xref ref-type="table" rid="T0002">Table 2</xref>) were the main causes of death among adults aged 15–59 years. Maternity-related causes also had a relatively high mortality burden among women of reproductive age (15–49 years) (mortality rate of 0.36 per 1,000 person-years [95% CI: 0.28–0.47]). Overall, pulmonary tuberculosis and other infectious diseases were the main causes of death of adults in the Farafenni area during the 10-year period, each with a disease-specific mortality rate of 0.68 per 1,000 person-years (95% CI: 0.57–0.83). There was no significant change in their respective mortality rates in the latter two periods compared to the first (<italic>p=</italic>0.567 for 2002–2004 and 0.703 for 2005–2007 for TB; and <italic>p=</italic>0.276 and 0.885 for other infectious diseases for the same periods).</p><p>Pulmonary tuberculosis and other infectious diseases, stroke, ARI and malaria were the main causes of death among the elderly population aged 60 years and over. Mortality from stroke emerged in the last 6 years of the study period as the most important cause of death in this age group – 9.62 per 1,000 person-years in 2002–2004 (95% CI: 7.45–12.00) and 6.32 per 1,000 person-years in 2005–2008 (95% CI: 4.69–8.52).</p><p>The mortality burdens of both communicable and non-communicable diseases are summarised in <xref ref-type="table" rid="T0004">Table 4</xref> by period, age group, sex, ethnicity and area of residence. Mortality rates for both categories of disease dropped significantly in 2005–2007 compared with levels in 1998–2001. For all categories of disease, mortality burdens were greater in the rural than in the urban area. Males had a 42% (95% CI: 27–59%) and a 26% (95% CI: 9–47%) higher risk of dying from communicable and non-communicable diseases respectively compared to females. Mandinkas were more likely to die from a non-communicable disease than any other ethnic group (RR 1.45, 95% CI: 1.22–1.71). Cancer-related mortality was significantly higher among men than women (RR 1.66, 95% CI: 1.29–2.15) and among rural rather than urban residents (RR 1.73, 95% CI: 1.33–2.26). Only cancers arising from the reproductive system were more prevalent among females than males.</p><table-wrap id="T0004" position="float"><label>Table 4</label><caption><p>Comparison of incidence of communicable and non-communicable diseases in the Farafenni demographic surveillance area by period and socio-demographic characteristics</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1"/><th align="center" rowspan="1" colspan="1"/><th align="center" rowspan="1" colspan="1"/><th align="center" colspan="6" rowspan="1">Non-communicable diseases</th></tr><tr><th align="left" rowspan="1" colspan="1"/><th align="center" rowspan="1" colspan="1"/><th align="center" rowspan="1" colspan="1"/><th align="center" colspan="6" rowspan="1">
<hr/>
</th></tr><tr><th align="left" rowspan="1" colspan="1"/><th align="center" colspan="2" rowspan="1">Communicable diseases</th><th align="center" colspan="2" rowspan="1">All</th><th align="center" colspan="2" rowspan="1">Cancers</th><th align="center" colspan="2" rowspan="1">Others</th></tr><tr><th align="left" rowspan="1" colspan="1"/><th align="center" colspan="2" rowspan="1">
<hr/>
</th><th align="center" colspan="2" rowspan="1">
<hr/>
</th><th align="center" colspan="2" rowspan="1">
<hr/>
</th><th align="center" colspan="2" rowspan="1">
<hr/>
</th></tr><tr><th align="left" rowspan="1" colspan="1"/><th align="center" rowspan="1" colspan="1">Rate ratio (95% CI)</th><th align="center" rowspan="1" colspan="1">
<italic>p</italic>
</th><th align="center" rowspan="1" colspan="1">Rate ratio (95% CI)</th><th align="center" rowspan="1" colspan="1">
<italic>p</italic>
</th><th align="center" rowspan="1" colspan="1">Rate ratio (95% CI)</th><th align="center" rowspan="1" colspan="1">
<italic>p</italic>
</th><th align="center" rowspan="1" colspan="1">Rate ratio (95% CI)</th><th align="center" rowspan="1" colspan="1">
<italic>p</italic>
</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">Period</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1"> 1998–2001</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1"> 2002–2004</td><td align="center" rowspan="1" colspan="1">1.09 (0.94–1.25)</td><td align="center" rowspan="1" colspan="1">0.265</td><td align="center" rowspan="1" colspan="1">0.91 (0.75–1.10)</td><td align="center" rowspan="1" colspan="1">0.317</td><td align="center" rowspan="1" colspan="1">0.85 (0.62–1.16)</td><td align="center" rowspan="1" colspan="1">0.310</td><td align="center" rowspan="1" colspan="1">0.94 (0.74–1.19)</td><td align="center" rowspan="1" colspan="1">0.625</td></tr><tr><td align="left" rowspan="1" colspan="1"> 2005–2007</td><td align="center" rowspan="1" colspan="1">0.75 (0.64–0.87)</td><td align="center" rowspan="1" colspan="1"><0.0001</td><td align="center" rowspan="1" colspan="1">0.67 (0.55–0.81)</td><td align="center" rowspan="1" colspan="1"><0.0001</td><td align="center" rowspan="1" colspan="1">0.59 (0.43–0.82)</td><td align="center" rowspan="1" colspan="1">0.002</td><td align="center" rowspan="1" colspan="1">0.71 (0.55–0.90)</td><td align="center" rowspan="1" colspan="1">0.006</td></tr><tr><td align="left" rowspan="1" colspan="1">Age group</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">  < 1 year</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1"> 1–4 years</td><td align="center" rowspan="1" colspan="1">0.45 (0.37–0.55)</td><td align="center" rowspan="1" colspan="1"><0.0001</td><td align="center" rowspan="1" colspan="1">0.64 (0.31–1.34)</td><td align="center" rowspan="1" colspan="1">0.238</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.64 (0.31–1.34)</td><td align="center" rowspan="1" colspan="1">0.238</td></tr><tr><td align="left" rowspan="1" colspan="1"> 5–14 years</td><td align="center" rowspan="1" colspan="1">0.08 (0.06–0.10)</td><td align="center" rowspan="1" colspan="1"><0.0001</td><td align="center" rowspan="1" colspan="1">0.34 (0.16–0.69)</td><td align="center" rowspan="1" colspan="1">0.003</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.30 (0.14–0.62)</td><td align="center" rowspan="1" colspan="1">0.001</td></tr><tr><td align="left" rowspan="1" colspan="1"> 15–60 years</td><td align="center" rowspan="1" colspan="1">0.17 (0.14–0.21)</td><td align="center" rowspan="1" colspan="1"><0.0001</td><td align="center" rowspan="1" colspan="1">1.64 (0.87–3.09)</td><td align="center" rowspan="1" colspan="1">0.126</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">0.90 (0.47–1.72)</td><td align="center" rowspan="1" colspan="1">0.750</td></tr><tr><td align="left" rowspan="1" colspan="1"> 60+ years</td><td align="center" rowspan="1" colspan="1">1.68 (1.40–2.03)</td><td align="center" rowspan="1" colspan="1"><0.0001</td><td align="center" rowspan="1" colspan="1">26.85 (14.34–50.28)</td><td align="center" rowspan="1" colspan="1"><0.0001</td><td align="center" rowspan="1" colspan="1">12.31 (9.53–15.91)</td><td align="center" rowspan="1" colspan="1"><0.0001</td><td align="center" rowspan="1" colspan="1">17.75 (9.44–33.36)</td><td align="center" rowspan="1" colspan="1"><0.0001</td></tr><tr><td align="left" rowspan="1" colspan="1">Sex</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1"> Female</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1"> Male</td><td align="center" rowspan="1" colspan="1">1.42 (1.27–1.59)</td><td align="center" rowspan="1" colspan="1"><0.0001</td><td align="center" rowspan="1" colspan="1">1.26 (1.09–1.47)</td><td align="center" rowspan="1" colspan="1">0.002</td><td align="center" rowspan="1" colspan="1">1.66 (1.29–2.15)</td><td align="center" rowspan="1" colspan="1"><0.0001</td><td align="center" rowspan="1" colspan="1">1.09 (0.91–1.31)</td><td align="center" rowspan="1" colspan="1">0.359</td></tr><tr><td align="left" rowspan="1" colspan="1">Ethnic group</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1"> Wollof</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1"> Mandinka</td><td align="center" rowspan="1" colspan="1">1.18 (1.04–1.33)</td><td align="center" rowspan="1" colspan="1">0.012</td><td align="center" rowspan="1" colspan="1">1.45 (1.22–1.71)</td><td align="center" rowspan="1" colspan="1"><0.0001</td><td align="center" rowspan="1" colspan="1">1.53 (1.15–2.05)</td><td align="center" rowspan="1" colspan="1">0.004</td><td align="center" rowspan="1" colspan="1">1.40 (1.14–1.72)</td><td align="center" rowspan="1" colspan="1">0.001</td></tr><tr><td align="left" rowspan="1" colspan="1"> Fula</td><td align="center" rowspan="1" colspan="1">0.96 (0.82–1.12)</td><td align="center" rowspan="1" colspan="1">0.615</td><td align="center" rowspan="1" colspan="1">0.87 (0.70–1.09)</td><td align="center" rowspan="1" colspan="1">0.235</td><td align="center" rowspan="1" colspan="1">1.07 (0.74–1.53)</td><td align="center" rowspan="1" colspan="1">0.728</td><td align="center" rowspan="1" colspan="1">0.78 (0.59–1.03)</td><td align="center" rowspan="1" colspan="1">0.085</td></tr><tr><td align="left" rowspan="1" colspan="1"> Other</td><td align="center" rowspan="1" colspan="1">0.84 (0.64–1.11)</td><td align="center" rowspan="1" colspan="1">0.232</td><td align="center" rowspan="1" colspan="1">0.56 (0.35–0.89)</td><td align="center" rowspan="1" colspan="1">0.015</td><td align="center" rowspan="1" colspan="1">0.82 (0.41–1.63)</td><td align="center" rowspan="1" colspan="1">0.562</td><td align="center" rowspan="1" colspan="1">0.44 (0.23–0.83)</td><td align="center" rowspan="1" colspan="1">0.011</td></tr><tr><td align="left" rowspan="1" colspan="1">Area of residence</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1"> Urban</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1"/><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1"> Rural</td><td align="center" rowspan="1" colspan="1">1.59 (1.42–1.79)</td><td align="center" rowspan="1" colspan="1"><0.0001</td><td align="center" rowspan="1" colspan="1">1.56 (1.33–1.82)</td><td align="center" rowspan="1" colspan="1"><0.0001</td><td align="center" rowspan="1" colspan="1">1.73 (1.33–2.26)</td><td align="center" rowspan="1" colspan="1"><0.0001</td><td align="center" rowspan="1" colspan="1">1.47 (1.22–1.78)</td><td align="center" rowspan="1" colspan="1"><0.0001</td></tr></tbody></table></table-wrap></sec><sec sec-type="discussion" id="S0004"><title>Discussion</title><p>There has been an appreciable decline in all-cause mortality in the Farafenni HDSS area over the 10-year period considered in this study. However, the pace of decline varied markedly between different age groups. Reduction in childhood mortality, especially among children aged between 1 and 4 years, accounted for much of the overall decline, similar to other countries in sub-Saharan Africa (<xref rid="CIT0023" ref-type="bibr">23</xref>, <xref rid="CIT0024" ref-type="bibr">24</xref>). Improvements in the death rates for adults and the elderly were very modest.</p><p>With respect to causes of death, the results presented here give the first indication of the cause-specific pattern of mortality for all age groups over a period of time for any geographically defined population in The Gambia. Previous <italic>ad hoc</italic> investigations in the Farafenni HDSS
area and other parts of the country used the physician coding method of VAs and focused on populations of interest such as children (<xref rid="CIT0012" ref-type="bibr">12</xref>, <xref rid="CIT0013" ref-type="bibr">13</xref>, <xref rid="CIT0025" ref-type="bibr">25</xref>), women of reproductive age (<xref rid="CIT0026" ref-type="bibr">26</xref>), or hospital patients (<xref rid="CIT0015" ref-type="bibr">15</xref>). The outcomes of this study are therefore relevant from two perspectives. First, it provides an opportunity to evaluate the cause-of-death structure produced by the InterVA-4 model against the established disease epidemiology of the region and secondly it helps in defining the public health priorities of the area.</p><p>Using the recommended settings for malaria and HIV prevalence levels in West Africa, InterVA-4 has produced results that suggest three preventable and treatable diseases accounted for more than half (58%) of all deaths among children aged under 5 years between 1998 and 2007. These were malaria (28%), ARI (21%) and diarrhoeal diseases (9%) (<xref ref-type="fig" rid="F0002">Fig. 2</xref>). These proportions compare reasonably well with the distribution of under-5 deaths in 2008 estimated for The Gambia by the Child Health Epidemiology Reference Group (CHERG), with 53% of under-5 deaths being attributed to malaria, pneumonia and diarrhoeal diseases (<xref rid="CIT0027" ref-type="bibr">27</xref>). These three diseases also dominate the overall cause-of-death structure compiled for children aged under 5 years in sub-Saharan Africa (<xref rid="CIT0027" ref-type="bibr">27</xref>, <xref rid="CIT0028" ref-type="bibr">28</xref>). A similar study in Niakhar in central Senegal about 150 km north of the Farafenni HDSS, showed the same pattern of mortality burden among children under 5 years old for these three specific diseases (<xref rid="CIT0029" ref-type="bibr">29</xref>), although malaria cases were included in a broader classification of fevers of unknown origin.</p><p>The InterVA-4 results suggest that only about one-tenth (9%) of under-5 deaths were due to neonatal causes, substantially lower than the CHERG estimate of 27% for The Gambia (<xref ref-type="fig" rid="F0002">Fig. 2</xref>). This is likely to be due to the relatively low VA coverage for infants during the study period; only a quarter (51/199) of neonatal deaths had a VA administered. Assuming that all neonatal deaths were due to neonatal causes, this will increase the proportion of under-5 deaths due to neonatal causes to 30%, which is similar to the CHERG estimate.</p><p>In view of the relatively low HIV prevalence in Farafenni, InterVA appears to have overestimated the proportion of under-5 deaths from HIV/AIDS, ranking it as the second highest killer among children aged 1–4 years (<xref ref-type="table" rid="T0003">Table 3</xref>). Malnutrition is prevalent in rural Gambia and constitutes a major public health concern. About a quarter of children aged less than 5 years in the North Bank Region of The Gambia are underweight (<xref rid="CIT0018" ref-type="bibr">18</xref>). It is therefore possible that the InterVA-4 model misinterpreted symptoms of malnourishment in children and returned the cause of their deaths as HIV-related, thus overestimating deaths resulting from HIV/AIDS. A similar conclusion was reached in another study in Kenya that compared the outcomes of an earlier version of InterVA and physician coding of the same VAs (<xref rid="CIT0011" ref-type="bibr">11</xref>).</p><p>The plausibility of the cause-specific mortality proportions for adults aged 15–59 years was assessed by re-grouping all causes by sex into the categories indicated in <xref ref-type="fig" rid="F0003">Fig. 3</xref> and comparing the resulting patterns of cause and sex-specific mortality rates with those derived for the African region as a whole in 2008 (<xref rid="CIT0028" ref-type="bibr">28</xref>). Apart from differences in the magnitudes of the burdens of HIV/AIDS and external (including injury) mortality, the InterVA-derived cause and sex-specific patterns of mortality among adults in the Farafenni HDSS area are remarkably similar to those of the African region. Common features between the two sets of mortality patterns include higher mortality rates among males for external, communicable and non-communicable disease-related causes of death and a higher HIV/AIDS mortality rate among females than among males. Communicable diseases, especially pulmonary tuberculosis, constitute the greatest mortality burden among adults aged 15–59 years in the Farafenni HDSS area.</p><fig id="F0003" position="float"><label>Fig. 3</label><caption><p>Cause and sex-specific mortality rates among Farafenni adults aged 15–59 years, 1998–2007.</p></caption><graphic xlink:href="GHA-7-25598-g003"/></fig><p>As expected, there were more deaths among the elderly population aged 60 years and over, the group who had the highest VA coverage (89%). However, in the absence of an established cause-of-death structure for the elderly in an epidemiological setting such as that of the Farafenni HDSS, the plausibility of the disease-specific mortality burdens in this age group cannot be assessed with certainty. Judging from the reasonable cause-of-death distributions obtained for children and adults, and the fact that the InterVA-4 model has not returned any unexpected or unreasonable cause or causes of death in the elderly, the cause-of-death structure obtained can be accepted as a reasonable reflection of the pattern of mortality among the elderly in the area. The results show that stroke has emerged as the leading cause of death among this population in the latter part of the period considered. The rise in cardiovascular risk factors in this population to levels prevalent in the main urban centre of The Gambia was established between 1996 and 1997 (<xref rid="CIT0030" ref-type="bibr">30</xref>). Hence, the level of stroke mortality produced by the model is plausible. Decreases in cause-specific mortality rates for this age group were not statistically significant and imply a stable cause-of-death pattern over the study period.</p><p>Despite using VA data covering less than three-quarters of all deaths in the period, especially among children aged under 5 years, and transforming these data from their original structure to meet InterVA-4 requirements, the model has proven useful in providing a population-based cause-of-death structure and disease-specific mortality rates for a sub-national population of The Gambia. The derived cause-specific mortality rates should be interpreted as the minimal possible rates because verbal autopsies were not administered for 29% of the deaths considered in the study, especially among children aged less than 5 years. In its Health Policy document for the period 2012–2020, The Gambia government identified the main causes of death in the country, albeit without strong supporting evidence, as malaria, pneumonia, anaemia, diarrhoeal diseases, road traffic accidents, pregnancy complications and cardiovascular diseases (<xref rid="CIT0020" ref-type="bibr">20</xref>). Concern was also expressed over the increasing incidences of tuberculosis and HIV/AIDS. The results of this study reflect the same pattern of disease-specific mortality burden in the Farafenni HDSS area, and confirm the double burden of communicable and non-communicable diseases, which constitute a huge strain on a relatively weak local health care delivery system.</p><p>The InterVA-4 model should be subjected to further methodological tests and fine tuning to enhance its performance in an epidemiologically dynamic setting like that of the Farafenni HDSS area. Whilst an earlier study confirmed that the model is capable of detecting a significant epidemic of HIV-related mortality in South Africa (<xref rid="CIT0031" ref-type="bibr">31</xref>), and that the use of VA tracked effectively an epidemiological transition in rural India (<xref rid="CIT0032" ref-type="bibr">32</xref>), it has not been sufficiently validated in settings with changing malaria epidemiology and lower HIV prevalence than the regional level prescribed by the model. With the recent decline in malaria incidence in The Gambia (<xref rid="CIT0016" ref-type="bibr">16</xref>, <xref rid="CIT0033" ref-type="bibr">33</xref>), a comparison of the results obtained from this study with those generated by setting the HIV and malaria prevalence levels that reflect the current epidemiological situation will show the extent to which death from communicable diseases will be redistributed. Non-communicable disease-related deaths will not be affected. The outcome of such a comparison will identify and document potential methodological limitations of the model in an environment like the Farafenni HDSS and facilitate future enhancement of the model.</p></sec> |
Controlling for population structure and genotyping platform bias in the eMERGE multi-institutional biobank linked to electronic health records | <p>Combining samples across multiple cohorts in large-scale scientific research programs is often required to achieve the necessary power for genome-wide association studies. Controlling for genomic ancestry through principal component analysis (PCA) to address the effect of population stratification is a common practice. In addition to local genomic variation, such as copy number variation and inversions, other factors directly related to combining multiple studies, such as platform and site recruitment bias, can drive the correlation patterns in PCA. In this report, we describe the combination and analysis of multi-ethnic cohort with biobanks linked to electronic health records for large-scale genomic association discovery analyses. First, we outline the observed site and platform bias, in addition to ancestry differences. Second, we outline a general protocol for selecting variants for input into the subject variance-covariance matrix, the conventional PCA approach. Finally, we introduce an alternative approach to PCA by deriving components from subject loadings calculated from a reference sample. This alternative approach of generating principal components controlled for site and platform bias, in addition to ancestry differences, has the advantage of fewer covariates and degrees of freedom.</p> | <contrib contrib-type="author"><name><surname>Crosslin</surname><given-names>David R.</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref><xref ref-type="author-notes" rid="fn001"><sup>*</sup></xref><xref ref-type="author-notes" rid="fn003"><sup>†</sup></xref><uri xlink:type="simple" xlink:href="http://community.frontiersin.org/people/u/120229"/></contrib><contrib contrib-type="author"><name><surname>Tromp</surname><given-names>Gerard</given-names></name><xref ref-type="aff" rid="aff3"><sup>3</sup></xref><xref ref-type="author-notes" rid="fn003"><sup>†</sup></xref><uri xlink:type="simple" xlink:href="http://community.frontiersin.org/people/u/130461"/></contrib><contrib contrib-type="author"><name><surname>Burt</surname><given-names>Amber</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref><uri xlink:type="simple" xlink:href="http://community.frontiersin.org/people/u/173254"/></contrib><contrib contrib-type="author"><name><surname>Kim</surname><given-names>Daniel S.</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Verma</surname><given-names>Shefali S.</given-names></name><xref ref-type="aff" rid="aff4"><sup>4</sup></xref><uri xlink:type="simple" xlink:href="http://community.frontiersin.org/people/u/120194"/></contrib><contrib contrib-type="author"><name><surname>Lucas</surname><given-names>Anastasia M.</given-names></name><xref ref-type="aff" rid="aff4"><sup>4</sup></xref></contrib><contrib contrib-type="author"><name><surname>Bradford</surname><given-names>Yuki</given-names></name><xref ref-type="aff" rid="aff4"><sup>4</sup></xref></contrib><contrib contrib-type="author"><name><surname>Crawford</surname><given-names>Dana C.</given-names></name><xref ref-type="aff" rid="aff5"><sup>5</sup></xref><xref ref-type="aff" rid="aff6"><sup>6</sup></xref><uri xlink:type="simple" xlink:href="http://community.frontiersin.org/people/u/124512"/></contrib><contrib contrib-type="author"><name><surname>Armasu</surname><given-names>Sebastian M.</given-names></name><xref ref-type="aff" rid="aff7"><sup>7</sup></xref><uri xlink:type="simple" xlink:href="http://community.frontiersin.org/people/u/186096"/></contrib><contrib contrib-type="author"><name><surname>Heit</surname><given-names>John A.</given-names></name><xref ref-type="aff" rid="aff8"><sup>8</sup></xref></contrib><contrib contrib-type="author"><name><surname>Hayes</surname><given-names>M. Geoffrey</given-names></name><xref ref-type="aff" rid="aff9"><sup>9</sup></xref></contrib><contrib contrib-type="author"><name><surname>Kuivaniemi</surname><given-names>Helena</given-names></name><xref ref-type="aff" rid="aff3"><sup>3</sup></xref><uri xlink:type="simple" xlink:href="http://community.frontiersin.org/people/u/111492"/></contrib><contrib contrib-type="author"><name><surname>Ritchie</surname><given-names>Marylyn D.</given-names></name><xref ref-type="aff" rid="aff4"><sup>4</sup></xref><uri xlink:type="simple" xlink:href="http://community.frontiersin.org/people/u/41302"/></contrib><contrib contrib-type="author"><name><surname>Jarvik</surname><given-names>Gail P.</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref><uri xlink:type="simple" xlink:href="http://community.frontiersin.org/people/u/140814"/></contrib><contrib contrib-type="author"><name><surname>de Andrade</surname><given-names>Mariza</given-names></name><xref ref-type="aff" rid="aff7"><sup>7</sup></xref><uri xlink:type="simple" xlink:href="http://community.frontiersin.org/people/u/37559"/></contrib><contrib contrib-type="author"><collab>the electronic Medical Records and Genomics (eMERGE) Network</collab></contrib> | Frontiers in Genetics | <sec id="s1"><title>1. Introduction</title><p>To reach the statistical power needed for genome-wide association studies, large numbers of participants are needed. This can be achieved through large research networks such as the Electronic Medical Records and Genomics (eMERGE) Network, which comprises a multi-ethnic cohort of ~57,000 participants linked to electronic health records (EHRs) for phenotype mining from nine participating sites (seven adult; two pediatric) in the United States (U.S.) (Gottesman et al., <xref rid="B7" ref-type="bibr">2013</xref>). When combining genetic data from diverse data sets, understanding the contribution of ancestry, genotyping platform, and site bias are of vital importance.</p><p>Through the course of the eMERGE project, multiple genotyping platforms from both Illumina and Affymetrix were utilized (Gottesman et al., <xref rid="B7" ref-type="bibr">2013</xref>; Crawford et al., <xref rid="B3" ref-type="bibr">2014</xref>). Imputation using the BEAGLE software was then carried out to allow merging of the diverse data sets (Verma et al., <italic>Imputation and quality control steps for combining multiple genome-wide data sets</italic>. Manuscript submitted for publication).</p><p>There were ancestry or racial/ethnic differences both within and across the eMERGE Network sites in addition to the platform heterogeneity. The majority of eMERGE study sites based race/ethnicity on self-report while Vanderbilt University's BioVU used third-party or administratively assigned race/ethnicity (Dumitrescu et al., <xref rid="B5" ref-type="bibr">2010</xref>). The major group for the entire eMERGE sample set is of European-descent. eMERGE also includes a sizeable African-descent and Hispanic sample (Gottesman et al., <xref rid="B7" ref-type="bibr">2013</xref>). The latter represents a three-way admixture event (Manichaikul et al., <xref rid="B13" ref-type="bibr">2012</xref>) that further contributes to expected ancestral differences within and across eMERGE. There are also both cryptic and known related participants, especially in Marshfield Clinic Research Foundation (Gottesman et al., <xref rid="B7" ref-type="bibr">2013</xref>; Crawford et al., <xref rid="B3" ref-type="bibr">2014</xref>).</p><p>We present an example of integrating the diverse genetic data sets from the eMERGE Network in a systematic fashion and provide guidance for other investigators in large research networks. We outline a general approach for selecting variants for input into a sample variance-covariance matrix on the adult participants in eMERGE, the conventional principal component analysis (PCA) approach in human genetics research (Patterson et al., <xref rid="B16" ref-type="bibr">2006</xref>). We also describe how we categorized genetic ancestry based on self-reported race, framed in terms of continental origin, in line with standard protocol in human genetic research (NHGRI, <xref rid="B15" ref-type="bibr">2005</xref>; Ali-Khan et al., <xref rid="B1" ref-type="bibr">2011</xref>).</p><p>Given our “sizeable” non-European sample in the presence of platform bias and imputation, the eMERGE Network took great care in not only assessing and adjusting for ancestry, but also exploring alternative methods to do so and increase power. To assess ancestry in related individuals, Zhu et al. (<xref rid="B19" ref-type="bibr">2008</xref>) introduced a method of generating principal components (PCs) by deriving SNP loadings from founders, and applying them to the entire sample. We introduce this concept of deriving SNP loadings from the BEAGLE imputation 1000 Genomes reference sample, and apply it to the entire imputed sample set of 57,000 genotyped individuals from the eMERGE Network as an alternative approach to control for site and platform bias in addition to ancestry differences for our large cohort.</p></sec><sec id="s2"><title>2. Materials and methods</title><p>The eMERGE Network comprises a multi-ethnic cohort of ~57,000 participants linked to EHRs for phenotype mining from nine participating sites (seven adult; two pediatric) in the United States (Gottesman et al., <xref rid="B7" ref-type="bibr">2013</xref>) with genotype and imputed data.</p><sec><title>2.1. Imputation</title><p>The imputation and merging were performed by the eMERGE Coordinating Center (CC) at Pennsylvania State University (PSU). Detailed quality assurance/quality control (QA/QC) measures are outlined in the imputation guide provided on the PSU eMERGE CC web site (see Web Resources). Before imputation, study site data were converted to the same build (Build 37) as the imputation reference data set. Next, strand flipping was employed to account for different strand alignments including Illumina TOP/BOT strand, plus(+) / minus(−), and forward/reverse (Nelson et al., <xref rid="B14" ref-type="bibr">2012</xref>). Finally, phasing and imputation were performed on randomized ancestry sub-samples against a “Cosmopolitan” reference set from the 1000 Genomes containing multiple ancestry groups provided by the BEAGLE software package (Browning and Browning, <xref rid="B2" ref-type="bibr">2009</xref>). While the imputation data presented are derived from using BEAGLE software (Browning and Browning, <xref rid="B2" ref-type="bibr">2009</xref>), it should be noted that IMPUTE2 software (Howie et al., <xref rid="B10" ref-type="bibr">2012</xref>) produced nearly identical results (see Supplementary Figure <xref ref-type="supplementary-material" rid="SM1">S1</xref>) (Howie et al., <xref rid="B11" ref-type="bibr">2011</xref>; Delaneau et al., <xref rid="B4" ref-type="bibr">2013</xref>).</p></sec><sec><title>2.2. PCA</title><p>There are multiple software packages for running PCA to estimate genomic ancestry, but we utilized the high-performance computing toolset SNPRelate R package (Zheng et al., <xref rid="B18" ref-type="bibr">2012</xref>) for multiple reasons. First, the increased computational performance allows for PCA analyses of a large number of participants such as eMERGE. Second, this tool allows the extraction of both sample and SNP loadings, which allows the correction of population stratification for related and unrelated participants (Zhu et al., <xref rid="B19" ref-type="bibr">2008</xref>). The two types of matrices are mathematically equivalent and can be derived from one another. Finally, SNPRelate allows for absolute genotype-PC correlation to assess whether a local region of the genome is driving the correlation structure (Zheng et al., <xref rid="B18" ref-type="bibr">2012</xref>).</p><p>We derived PCs using three general approaches, each applied to the overall set and to each ancestry group. First, we performed PCA on a combined data set (across sites) after imputation using the BEAGLE software package (Version 3.3.1) (Browning and Browning, <xref rid="B2" ref-type="bibr">2009</xref>). Second, we performed PCA on a pre-imputed merged version (across sites) of the data. Finally, we derived PCs for the entire set using SNP loadings generated from the BEAGLE imputation reference set (Browning and Browning, <xref rid="B2" ref-type="bibr">2009</xref>).</p><p>For all genotype data used to generate the variance-covariance matrices and to eliminate redundant SNPs in high linkage disequilibrium (LD), we applied the following thresholds. The autosomal variants were selected after LD pruning at <italic>r</italic> > 0.5 with a 500 kbp (kilo basepairs) sliding window, and a minor allele frequency (MAF) > 0.05. In addition, a variant missingness filter of 0.02 was applied. For both PCA on the combined imputed and the combined preimputed, which is basically the singular value decomposition on the sample covariance matrix as outlined in Patterson et al. (<xref rid="B16" ref-type="bibr">2006</xref>).</p><sec><title>2.2.1. Deriving PCA using reference sample loadings</title><p>We also assessed PCA using the Zhu et al. (<xref rid="B19" ref-type="bibr">2008</xref>) method by deriving SNP loadings from the BEAGLE imputation 1000 Genomes reference sample, and applying it to the entire sample set. As such, we utilized their nomenclature with respect to generating the components. This was implemented using the SNPRelate R package (Zheng et al., <xref rid="B18" ref-type="bibr">2012</xref>), specifically the <monospace>snpgdsPCASampLoading</monospace> and <monospace>snpgdsPCASNPLoading</monospace> functions (see Web Resources).</p><p>We treated the entire eMERGE cohort as one “related” family, and the imputation reference sample as (<italic>a</italic> = 1,2,…, <italic>B</italic>) unrelated. Because of this, the <italic>g<sub>ij</sub></italic> marker genotype value of the <italic>j</italic>th individual in the <italic>i</italic>th family as utilized by Zhu et al. (<xref rid="B19" ref-type="bibr">2008</xref>), simplified to <italic>g<sub>j</sub></italic>. The column vector <italic>X<sub>ij</sub></italic> = (<italic>x</italic><sub><italic>j</italic></sub><sub>1</sub>, <italic>x</italic><sub><italic>j</italic>2</sub>,…, <italic>x</italic><sub><italic>jM</italic></sub>)<sup><italic>T</italic></sup> of <italic>l</italic> = 1,2,…, <italic>M</italic> biallelic markers, and was coded as an additive model of inheritance.</p><p>The variance-covariance matrix for the marker data from the reference sample (unrelated), took on the form <inline-formula><mml:math id="M1"><mml:mrow><mml:mstyle mathvariant="bold" mathsize="normal"><mml:mi>Σ</mml:mi></mml:mstyle><mml:mo>=</mml:mo><mml:msubsup><mml:mi>Σ</mml:mi><mml:mrow><mml:mi>a</mml:mi><mml:mtext> </mml:mtext><mml:mo>=</mml:mo><mml:mtext> </mml:mtext><mml:mn>1</mml:mn></mml:mrow><mml:mi>B</mml:mi></mml:msubsup><mml:mo stretchy="false">(</mml:mo><mml:msub><mml:mi>X</mml:mi><mml:mi>a</mml:mi></mml:msub><mml:mo>−</mml:mo><mml:mover accent="true"><mml:mi>X</mml:mi><mml:mo>¯</mml:mo></mml:mover><mml:mo stretchy="false">)</mml:mo><mml:msup><mml:mrow><mml:mo stretchy="false">(</mml:mo><mml:msub><mml:mi>X</mml:mi><mml:mi>a</mml:mi></mml:msub><mml:mo>−</mml:mo><mml:mover accent="true"><mml:mi>X</mml:mi><mml:mo>¯</mml:mo></mml:mover><mml:mo stretchy="false">)</mml:mo></mml:mrow><mml:mi>T</mml:mi></mml:msup></mml:mrow></mml:math></inline-formula>, assuming <overline><italic>X</italic></overline> as the overall genotype mean for those samples. Following Zhu et al. (<xref rid="B19" ref-type="bibr">2008</xref>), we let <italic>e</italic><sub><italic>l</italic></sub> be the <italic>l</italic>th eigenvalue of Σ, where <italic>l</italic> = 1,2,…, <italic>M</italic>, which is a vector of the SNP loadings. We then derived the <italic>l</italic>th PC for the individual (<italic>j</italic>) of the entire cohort or “related” family by <italic>t</italic><sub><italic>jl</italic></sub> = (<italic>X</italic><sub><italic>j</italic></sub> − <overline><italic>X</italic></overline>)<sup><italic>T</italic></sup><italic>e<sub>l</sub></italic>.</p></sec></sec><sec><title>2.3. Venous thromboembolism association</title><p>The venous thromboembolism (VTE) phenotype was extracted using an EHR-driven algorithm from African ancestry participants (Pathak et al., personal communication), excluding patients with cancer. A total of 400 VTE cases and 5,065 controls were selected from 4 sites and 4 different genotype platforms (Illumina 660, 1M, and Omni; and Affymetrix 6.0). We performed two logistic regressions for association using the software PLINK v1.07 (Purcell et al., <xref rid="B17" ref-type="bibr">2007</xref>). The first was adjusted for age, sex, stroke, sickle cell genetic variant, site-platform, and conventional PC1 and PC2 and the second was adjusted for age, sex, stroke, sickle cell genetic variant and “loadings” PC1 and PC2.</p></sec></sec><sec id="s3"><title>3. Results</title><sec><title>3.1. Demographics</title><p>Table <xref ref-type="table" rid="T1">1</xref> outlines the breakdown of the 38,288 adult participants included in these analyses by eMERGE site, self-reported or administratively assigned ancestry, sex, and genotyping platform. Most sites were predominantly of European ancestry. Compared with most other eMERGE study sites, both Vanderbilt University and Northwestern University had a greater representation of African ancestry (26 and 12%, respectively). Mount Sinai School of Medicine had the greatest proportion of African ancestry (70%), followed by a sizeable proportion of Hispanic participants (19%). Overall, there were more females than males (57% vs. 43%). All sites followed this pattern, except for Geisinger Health System (53% male). Most of the genotyping across all sites was performed using Illumina arrays (610, 660, 1M and Omni), with the exception of Mount Sinai School of Medicine (Affymetrix 6.0).</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p><bold>Summary of eMERGE sample by self-reported ancestry, sex, and genotyping platform for the adult participants</bold>.</p></caption><table frame="hsides" rules="groups"><thead><tr><th rowspan="1" colspan="1"/><th align="center" rowspan="1" colspan="1"><bold>Geisinger (<italic>N</italic> = 3, 111) (%)</bold></th><th align="center" rowspan="1" colspan="1"><bold>Group Health (<italic>N</italic> = 3, 520) (%)</bold></th><th align="center" rowspan="1" colspan="1"><bold>Marshfield (<italic>N</italic> = 4, 193) (%)</bold></th><th align="center" rowspan="1" colspan="1"><bold>Mayo (<italic>N</italic> = 6, 836) (%)</bold></th><th align="center" rowspan="1" colspan="1"><bold>Mt. Sinai (<italic>N</italic> = 6, 290) (%)</bold></th><th align="center" rowspan="1" colspan="1"><bold>Northwestern (<italic>N</italic> = 4, 858) (%)</bold></th><th align="center" rowspan="1" colspan="1"><bold>Vanderbilt (<italic>N</italic> = 9, 480) (%)</bold></th><th align="center" rowspan="1" colspan="1"><bold>Combined (<italic>N</italic> = 38, 288) (%)</bold></th></tr></thead><tbody><tr><td align="left" colspan="9" rowspan="1"><bold>SELF-REPORTED ANCESTRY</bold></td></tr><tr><td align="left" rowspan="1" colspan="1">African</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">4</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">70</td><td align="center" rowspan="1" colspan="1">12</td><td align="center" rowspan="1" colspan="1">26<xref ref-type="table-fn" rid="TN1"><sup>†</sup></xref></td><td align="center" rowspan="1" colspan="1">20% (7, 651)</td></tr><tr><td align="left" rowspan="1" colspan="1">European</td><td align="center" rowspan="1" colspan="1">99</td><td align="center" rowspan="1" colspan="1">92</td><td align="center" rowspan="1" colspan="1">99</td><td align="center" rowspan="1" colspan="1">99</td><td align="center" rowspan="1" colspan="1">11</td><td align="center" rowspan="1" colspan="1">88</td><td align="center" rowspan="1" colspan="1">66<xref ref-type="table-fn" rid="TN1"><sup>†</sup></xref></td><td align="center" rowspan="1" colspan="1">74% (28, 469)</td></tr><tr><td align="left" rowspan="1" colspan="1">Hispanic</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">19</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">3% (1, 258)</td></tr><tr><td align="left" rowspan="1" colspan="1">Other</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">5</td><td align="center" rowspan="1" colspan="1">1</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">7<xref ref-type="table-fn" rid="TN1"><sup>†</sup></xref></td><td align="center" rowspan="1" colspan="1">2% (910)</td></tr><tr><td align="left" colspan="9" rowspan="1"><bold>SEX</bold></td></tr><tr><td align="left" rowspan="1" colspan="1">Female</td><td align="center" rowspan="1" colspan="1">47</td><td align="center" rowspan="1" colspan="1">57</td><td align="center" rowspan="1" colspan="1">58</td><td align="center" rowspan="1" colspan="1">45</td><td align="center" rowspan="1" colspan="1">59</td><td align="center" rowspan="1" colspan="1">83</td><td align="center" rowspan="1" colspan="1">53</td><td align="center" rowspan="1" colspan="1">57% (21, 802)</td></tr><tr><td align="left" rowspan="1" colspan="1">Male</td><td align="center" rowspan="1" colspan="1">53</td><td align="center" rowspan="1" colspan="1">43</td><td align="center" rowspan="1" colspan="1">41</td><td align="center" rowspan="1" colspan="1">55</td><td align="center" rowspan="1" colspan="1">41</td><td align="center" rowspan="1" colspan="1">17</td><td align="center" rowspan="1" colspan="1">47</td><td align="center" rowspan="1" colspan="1">43% (16, 486)</td></tr><tr><td align="left" colspan="9" rowspan="1"><bold>GENOTYPING PLATFORM</bold></td></tr><tr><td align="left" rowspan="1" colspan="1">Affymetrix 6</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">44</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">7% (2, 775)</td></tr><tr><td align="left" rowspan="1" colspan="1">Illumina 1M</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">12</td><td align="center" rowspan="1" colspan="1">21</td><td align="center" rowspan="1" colspan="1">7% (2, 634)</td></tr><tr><td align="left" rowspan="1" colspan="1">Illumina 610</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">45</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">8% (3, 081)</td></tr><tr><td align="left" rowspan="1" colspan="1">Illumina 660</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">89</td><td align="center" rowspan="1" colspan="1">100</td><td align="center" rowspan="1" colspan="1">55</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">27</td><td align="center" rowspan="1" colspan="1">42</td><td align="center" rowspan="1" colspan="1">43% (16, 362)</td></tr><tr><td align="left" rowspan="1" colspan="1">Illumina Omni</td><td align="center" rowspan="1" colspan="1">100</td><td align="center" rowspan="1" colspan="1">11</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">0</td><td align="center" rowspan="1" colspan="1">56</td><td align="center" rowspan="1" colspan="1">61</td><td align="center" rowspan="1" colspan="1">37</td><td align="center" rowspan="1" colspan="1">35% (13, 436)</td></tr></tbody></table><table-wrap-foot><fn id="TN1"><label>†</label><p><italic>Race/ethnicity is administratively assigned</italic>.</p></fn></table-wrap-foot></table-wrap><p>Eigenvectors 1 and 2 for the 38,288 adult eMERGE participants are illustrated in Figure <xref ref-type="fig" rid="F1">1</xref>, annotated by self-reported race (Figure <xref ref-type="fig" rid="F1">1A</xref>), genotyping platform (Figure <xref ref-type="fig" rid="F1">1B</xref>), and by eMERGE study site (Figure <xref ref-type="fig" rid="F1">1C</xref>). Genetically determined ancestry was assigned by creating subjective boundaries for the African, European and Hispanic groups. These boundaries were set using the respective medians (<italic>Q</italic><sub>2</sub>) and standard deviations (<italic>SD</italic>) calculated for each genetic ancestry group, as illustrated in Figures <xref ref-type="fig" rid="F2">2A–C</xref> for the African (<italic>Q</italic><sub>2<sub><italic>A</italic></sub></sub> ± 2<italic>SD</italic>), European (<italic>Q</italic><sub>2</sub><sub><italic>E</italic></sub> ± 4<italic>SD</italic>) and Hispanic (<italic>Q</italic><sub>2<sub><italic>H</italic></sub></sub> ± 1<italic>SD</italic>) groups, respectively.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p><bold>PC plots of PCs 1 and 2 for all adults of eMERGE by self-reported race. (A), genotyping platform <bold>(B)</bold>, and eMERGE study site <bold>(C)</bold>, using BEAGLE imputed data</bold>. (1) geis, Geisinger Health System, (2) ghuw, Group Health Research Institute/University of Washington; (3) mrsh, Marshfield Clinic Research Foundation; (4) mayo, Mayo Clinic; (5) mtsi, Mount Sinai School of Medicine; (6) nwun, Northwestern University; and (7) vand, Vanderbilt University.</p></caption><graphic xlink:href="fgene-05-00352-g0001"/></fig><fig id="F2" position="float"><label>Figure 2</label><caption><p><bold>PC plots of PCs 1 and 2 comparing eMERGE genetically determined and self-reported ancestry, using BEAGLE imputed data. (A)</bold> African ancestry assigned using (<italic>Q</italic><sub>2<sub><italic>A</italic></sub></sub> ± 2<italic>SD</italic>) of eigenvectors 1 and 2 for self-reported as African ancestry. <bold>(B)</bold> European ancestry assigned using (<italic>Q</italic><sub>2<sub><italic>E</italic></sub></sub> ± 4<italic>SD</italic>) of eigenvectors 1 and 2 for self-reported as European ancestry. <bold>(C)</bold> Hispanic assigned using (<italic>Q</italic><sub>2<sub><italic>H</italic></sub></sub> ± 1<italic>SD</italic>) of eigenvectors 1 and 2 for self-reported as Hispanics.</p></caption><graphic xlink:href="fgene-05-00352-g0002"/></fig></sec><sec><title>3.2. Examination of the variance explained per PC using scree plots</title><p>To assess the variance explained from each PCA, we plotted the first ten PCs against the variance explained as illustrated in Figure <xref ref-type="fig" rid="F3">3</xref>. Across the columns of the trellis we show scree plots of joint, African ancestry, European ancestry, and Hispanic groups. Across each row, we have scree plots representing PC analyses on the imputed merged set, pre-imputed merged set, and on the PC analyses using the “loadings” method outlined in Subsection 2.2.1. As expected, eigenvector 1 explains most of the variance for the joint ancestry imputed (~7%), pre-imputed (~4%), and “loadings” (~7%). When we stratified by ancestry (across the trellis), the variance explained by eigenvector 1 for the imputed and pre-imputed data sets was <1%. For the “loadings” approach with the African and European genetic ancestry data sets, the variance explained <1%, and >2% for the Hispanic group. In all scenarios (joint and all ancestry groups) the variance explained approached 0 for eigenvectors 2 through 10 for the imputed and pre-imputed data sets. Interestingly, the “loadings” approach allows for more variance explained for eigenvectors 2 and beyond, especially for the Hispanics. For the joint loadings approach, the variance explained by eigenvector 2 approached ~4%, while the genetic ancestry groups approached 1%.</p><fig id="F3" position="float"><label>Figure 3</label><caption><p><bold>Scree plots illustrating variance explained for PCA outlined in this manuscript</bold>.</p></caption><graphic xlink:href="fgene-05-00352-g0003"/></fig></sec><sec><title>3.3. Evaluation of the effect of ancestry on PC plots—joint and stratified ancestry</title><p>We evaluated the population structure by plotting eigenvectors 1 and 2 for the joint data set (Figure <xref ref-type="fig" rid="F4">4</xref>) as well as for the African (Figure <xref ref-type="fig" rid="F5">5</xref>), European (Figure <xref ref-type="fig" rid="F6">6</xref>) and Hispanic (Figure <xref ref-type="fig" rid="F7">7</xref>) ancestry groups, separately. In each case of ancestry analysis, we plotted the imputed and pre-imputed merged data set, and the data set derived from the “loadings” method. Figures <xref ref-type="fig" rid="F4">4A,B</xref> illustrate the imputation and pre-imputation data sets, respectively, and are generally opposites with respect to eigenvector 1 due to different projections for that component. Figure <xref ref-type="fig" rid="F4">4C</xref> illustrates the “loadings” data set, which offers a different characterization of the joint data set, with the African and European genetic ancestry groups largely represented by two ellipses.</p><fig id="F4" position="float"><label>Figure 4</label><caption><p><bold>PC plots of eMERGE joint ancestry. (A)</bold> Plot of eigenvectors 1 and 2 for the joint imputed data set. <bold>(B)</bold> Plot of eigenvectors 1 and 2 for the joint pre-imputed data set. <bold>(C)</bold> Plot of eigenvectors 1 and 2 for the joint imputed data set using the “loadings” method.</p></caption><graphic xlink:href="fgene-05-00352-g0004"/></fig><fig id="F5" position="float"><label>Figure 5</label><caption><p><bold>PC plots of eMERGE participants geneticaly determined to be of African ancestry. (A)</bold> Plot of eigenvectors 1 and 2 for the imputed data set African ancestry participants, annotated by self-reported ancestry. <bold>(B)</bold> Plot of eigenvectors 1 and 2 for the imputed data set African ancestry participants, annotated by genotyping platform. <bold>(C)</bold> Plot of eigenvectors 1 and 2 for the imputed data set African ancestry participants, annotated by eMERGE site. <bold>(D)</bold> Plot of eigenvectors 1 and 2 for the pre-imputed data set African ancestry participants. <bold>(E)</bold> Plot of eigenvectors 1 and 2 for the imputed data set African ancestry participants using the “loadings” method.</p></caption><graphic xlink:href="fgene-05-00352-g0005"/></fig><fig id="F6" position="float"><label>Figure 6</label><caption><p><bold>PC plots of eMERGE participants genetically determined to be of European ancestry. (A)</bold> Plot of eigenvectors 1 and 2 for the imputed data set Hispanic participants. <bold>(B)</bold> Plot of eigenvectors 1 and 2 for the pre-imputed data set Hispanic participants. <bold>(C)</bold> Plot of eigenvectors 1 and 2 for the imputed data set Hispanic participants using the “loadings” method.</p></caption><graphic xlink:href="fgene-05-00352-g0006"/></fig><fig id="F7" position="float"><label>Figure 7</label><caption><p><bold>PC plots of eMERGE participants genetically determined to be Hispanic</bold>. <bold>(A)</bold> Plot of eigenvectors 1 and 2 for the imputed data set Hispanic participants. <bold>(B)</bold> Plot of eigenvectors 1 and 2 for the pre-imputed data set Hispanic participants. <bold>(C)</bold> Plot of eigenvectors 1 and 2 for the imputed data set Hispanic participants using the “loadings” method.</p></caption><graphic xlink:href="fgene-05-00352-g0007"/></fig><p>Figures <xref ref-type="fig" rid="F5">5A–C</xref> illustrate the African ancestry imputation data set, annotated by self-reported race, genotyping platform, and site, respectively. As illustrated in Figures <xref ref-type="fig" rid="F5">5B,C</xref>, there are batch effects by platform and study site. The pre-imputed data set (Figure <xref ref-type="fig" rid="F5">5D</xref>) has two distinct bands for both eigenvectors 1 and 2. The “loadings” approach (Figure <xref ref-type="fig" rid="F5">5E</xref>) produces an ellipse, indicating no effect due to platform or study site. Figures <xref ref-type="fig" rid="F6">6A–C</xref> illustrate the European ancestry imputed and pre-imputed data set, and the “loadings” data set, respectively. Eigenvectors 1 and 2 for the imputed data set (Figure <xref ref-type="fig" rid="F6">6A</xref>) produce separation much like the joint ancestry analyses, while the pre-imputed data set produces two separate bands (Figure <xref ref-type="fig" rid="F6">6B</xref>). Like the African genetic ancestry “loadings” set, the European set produces an ellipse. Finally, the Hispanic data sets are illustrated in Figures <xref ref-type="fig" rid="F7">7A–C</xref>. With only 994 participants, most of the variance seems to be explained by eigenvector 1 for both the imputed (Figure <xref ref-type="fig" rid="F7">7A</xref>) and pre-imputed (Figure <xref ref-type="fig" rid="F1">7B</xref>). The “loadings” approach (Figure <xref ref-type="fig" rid="F7">7C</xref>) produces the familiar ellipse, with the mixed ancestry in the middle, most likely representing the Hispanic sample.</p></sec><sec><title>3.4. Examination of SNP-PC correlation</title><p>We also illustrate component-genotype absolute correlation plots generated using the SNPRelate R package for the imputed and pre-imputed data sets. Ideally, a component will be driven by genome-wide correlation patterns, as illustrated by eigenvector 3 of the pre-imputed data in Figure <xref ref-type="fig" rid="F8">8A</xref>. However, many times chromosomal artifacts will drive local regions of correlation, resulting in components dominated by that pattern. Examples of this include Figures <xref ref-type="fig" rid="F8">8B,C</xref>. Figure <xref ref-type="fig" rid="F8">8B</xref> illustrates a known chromosome 8 inversion (Feuk et al., <xref rid="B6" ref-type="bibr">2006</xref>) driving the correlation patterns for eigenvector 9 in the imputed data. Figure <xref ref-type="fig" rid="F8">8C</xref> illustrates the correlation pattern driven by the HLA region for eigenvector 10 of the pre-imputed data.</p><fig id="F8" position="float"><label>Figure 8</label><caption><p><bold>Eigenvector-genotype correlation plots from the joint ancestry PCA analyses representing genome-wide correlation (A), correlation driven the chromosome 8 inversion (B), and correlation driven by the HLA region (C)</bold>.</p></caption><graphic xlink:href="fgene-05-00352-g0008"/></fig></sec><sec><title>3.5. Venous thromboembolism association</title><p>We applied our approach using the eMERGE VTE African ancestry cohort that consists of four adult sites and four genotyping platforms that had previously been analyzed controlling for site, platform and genomic ancestry (Heit et al., <xref rid="B8" ref-type="bibr">2013</xref>). For clarity, the original analysis' first two eigenvectors along with site and platform will be referred to as “PCs.” The principal components derived from the imputed data set by the conventional approach will be referred as normal eigenvectors (normal “EIGs”), and derived by the “loadings” approach as “loading” eigenvectors (“loadings EIGs”). We first compared the two first PCs obtained using the eMERGE African ancestry from the original analysis with the two first eigenvectors (PCs) using the “loadings” method (Figure <xref ref-type="fig" rid="F9">9</xref>). We observed that the PCs used in the analysis had similar pattern as the standard eigenvectors (Figures <xref ref-type="fig" rid="F9">9A,B</xref>, first row), but just in a different direction for the projections. Figure <xref ref-type="fig" rid="F9">9C</xref> illustrates a bivariate normal distribution with low variance of the African genetic ancestry when using the “loadings” eigenvectors.</p><fig id="F9" position="float"><label>Figure 9</label><caption><p><bold>PC comparisons derived from the “loadings” method and PCs derived from the equivalent of the imputation method for venous thromboembolism association in African ancestry participants</bold>.</p></caption><graphic xlink:href="fgene-05-00352-g0009"/></fig><p>We observed dispersion between the first PC and the first “loading” eigenvector (Figure <xref ref-type="fig" rid="F9">9D</xref>), demonstrating that the “loadings” approach captured a different aspect of variance. The first PC showed an inverse correlation with the first PC and first normal eigenvector (Figure <xref ref-type="fig" rid="F9">9E</xref>). Such an inversion is a consequence of the arbitrary nature of mathematical sign in the computation of PCs resulting in opposite projections. Figure <xref ref-type="fig" rid="F9">9F</xref> illustrates the second PC compared to the second “loadings” PC, which shows no correlation and some outliers in the PC projection.</p><p>Figure <xref ref-type="fig" rid="F9">9G</xref> depicts the comparison between the second PC with the second normal eigenvector that showed the same outliers observed previously but in a different scale. Thus, by using the BEAGLE loadings we have a more parsimonious model, and the association results in <italic>P</italic>-values and −<italic>log</italic><sub>10</sub>(<italic>P</italic>) are tighter for chromosome 22 (Figures <xref ref-type="fig" rid="F9">9H,I</xref>). Finally, Figures <xref ref-type="fig" rid="F10">10A,B</xref> represent the QQ plots for the conventional PC adjusting for site and platform method (λ = 1.01) and the “loadings” approach (λ = 1.02), respectively.</p><fig id="F10" position="float"><label>Figure 10</label><caption><p><bold>QQ plots of the venous thromboembolism (VTE) association in African ancestry participants</bold>. PC comparisons derived from the “loadings” method and PCs derived from the equivalent of the imputation method. <bold>(A)</bold> QQ plots of the VTE association in African ancestry participants using PCs derived from the equivalent of the imputation method. <bold>(B)</bold> QQ plots of the VTE association in African ancestry participants using PCs derived from the equivalent of the “loadings” method.</p></caption><graphic xlink:href="fgene-05-00352-g0010"/></fig></sec></sec><sec id="s4"><title>4. Discussion</title><p>Imputation depends on how well the genotype data (the observed LD) capture the true underlying LD. The more completely LD is represented, the more accurately the imputation will extend the LD to non-genotyped markers. There is always an inherent risk that the imputed genotypes will not represent the true state of nature accurately; this risk increases as the genotyped density decreases and the genotypes do not capture the underlying LD. We detected effects from the genotyping platform when performing the PCA (here we use platform to indicate the design as well as the method). The effect was most evident when a low-density platform such as the MetaboChip (data not shown) were combined with high-density platforms: the MetaboChip data set was an outlier even at overview scale. Platform differences re-appear when PCA is performed on apparently homogeneous subsets, e.g., African and European genetic ancestry subsets. These platform differences in homogeneous racial groups are amplified as the overall variance in the data set diminishes. Some of the differences might actually reflect subtle differences in LD in the populations due to ethnic stratification correlated with platform, because the populations were not randomly represented in the Biobank and therefore not randomized to platform.</p><p>In addition to difference of LD capture by platform, genotype encoding remains problematic when combining large data sets genotyped at different sites and on different platforms. A number of tools, e.g., liftOver (Hinrichs et al., <xref rid="B9" ref-type="bibr">2006</xref>), can be used to standardize the allele states between data sets. Nevertheless, coding remains fraught with problems (Nelson et al., <xref rid="B14" ref-type="bibr">2012</xref>). One data set was initially submitted with non-standard coding resulting in the data set being an outlier even with respect to other data sets on the same platform and chip. Such miscoding results in an extreme form of platform bias, as the LD is misrepresented. Other potential source of bias could be induced by the sites or genotyping center.</p><p>It is likely that the imputed data can exaggerate some underlying features. Any chromosomal variation that is poorly represented in the reference set can lead to more uniformity around the variation that causes that chromosome to be selected. Some regions that are promoted (occur prominently in a lower number PC), probably are reflecting rare chromosomes in the reference panel.</p><p>We have outlined a general checklist for filtering variants to be utilized with PCA: (1) Ensure uniformity of strand representation among different platforms to avoid the bias induced by site; (2) Select variants on autosomal chromosomes only, no sex chromosomes; (3) Filter variants with LD pruning (<italic>r</italic> = 0.50 − 0.84), in a sliding window of 500 kbp; (4) Filter variants on MAF >0.05, and for missingness <0.02; and (5) Examine plots of absolute correlation between PC and genotype as illustrated in Figure <xref ref-type="fig" rid="F10">10</xref> and remove regions where chromosome artifacts (e.g., HLA, chromosome 8 inversion) are driving the correlation pattern for a given component (Laurie et al., <xref rid="B12" ref-type="bibr">2010</xref>). However, in many cases removing the HLA region will not completely eliminate the correlation pattern in that region (data not shown). Normally the first ten eigenvectors are appropriate, but this depends on the proportion of variance explained and the specific analysis conducted.</p><p>As a proof of concept, we repeated a previously presented genome-wide association for VTE in participants of African ancestry (Heit et al., <xref rid="B8" ref-type="bibr">2013</xref>). We compared the performance of the two approaches described above: (a) PCs derived from the “loadings” method and (b) PCs derived from the equivalent of the conventional method. Our results showed that using the “loadings” approach provided similar association results and controlled for inflation while controlling for fewer covariates and consequently fewer degrees of freedom. This method will need further validation using simulated data, but does seem promising nonetheless.</p><p>We have demonstrated that analysis of data across sites in research networks can expose subtle biases and stratification effects. The conventional approach of adjusting for the first number of PCs does not adequately adjust for the bias of platform and site. We recognize that in comparison to most meta analyses which use summary statistics for aggregation, we have both individual subject genotypes as well as information on genotyping platform and site. We hope our research study will serve as a reference for similar projects that attempt to control for confounders and ancestry in large genetic association studies.</p></sec><sec id="s5"><title>5. Conclusion</title><p>In summary, we outline a general checklist for filtering genetic variants for conventional PCA to avoid the bias induced by platform and site as well as to avoid false-positive results due to the correlation between the PCs and the SNP genotypes. We have also proposed the “loadings” method as an alternative to the conventional method to derive PCs that control for bias due to the site and platform. Furthermore, we demonstrated the applicability of this new approach for the VTE genome-wide association analysis in genetic African ancestry eMERGE participants.</p></sec><sec><title>Web resources</title><list list-type="simple"><list-item><p>– eMERGE Coordinating Center genotyping data: <ext-link ext-link-type="uri" xlink:href="http://emerge.mc.vanderbilt.edu/genotyping-data-released">http://emerge.mc.vanderbilt.edu/genotyping-data-released</ext-link></p></list-item><list-item><p>– R package SNPRelate: <ext-link ext-link-type="uri" xlink:href="https://github.com/zhengxwen/SNPRelate">https://github.com/zhengxwen/SNPRelate</ext-link></p></list-item></list></sec><sec><title>Funding</title><p>This study was supported by the following U01 grants from the National Human Genome Research Institute (NHGRI), a component of the National Institutes of Health (NIH), Bethesda, MD, USA: (1) U01HG006375 (Group Health/University of Washington); (2) U01HG006382 (Geisinger Health System); (3) U01HG006379 (Mayo Clinic); (4) U01HG006389 (Essentia Health, Marshfield Clinic Research Foundation, and Pennsylvania State University); (5) U01HG006388 (Northwestern University); (6) HG004438 (Center for Inherited Disease Research, Johns Hopkins University); (7) HG004424 (Broad Institute of Harvard and MIT); (8) U01HG006378, U01HG006385, U01HG006385 (Vanderbilt University and Pennsylvania State University); (9) U01HG006380 (The Mt. Sinai Hospital); (10) U01HG006828 (Cincinnati Children's Hospital Medical Center/Harvard); (11) U01HG006830 (Childrens Hospital of Philadelphia). Additional support was provided by a State of Washington Life Sciences Discovery Fund award to the Northwest Institute of Genetic Medicine (Gail P. Jarvik).</p><sec><title>Conflict of interest statement</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></sec> |
Cause of death during 2009–2012, using a probabilistic model (InterVA-4): an experience from Ballabgarh Health and Demographic Surveillance System in India | <sec id="st1"><title>Objectives</title><p>The present study aimed to estimate the age and cause-specific mortality in Ballabgarh Health and Demographic Surveillance System (HDSS) site for the years 2009 to 2012, using a probabilistic model (InterVA-4).</p></sec><sec id="st2"><title>Methods</title><p>All Deaths in Ballabgarh HDSS from January 1, 2009, to December 31, 2012, were included in the study. InterVA-4 model (version 4.02) was used for assigning cause of death (COD). Data from the verbal autopsy (VA) tool were extracted and processed with the InterVA-4 model. Cause-specific mortality rate (CSMR) per 1,000 person-years was calculated.</p></sec><sec id="st3"><title>Results</title><p>A total of 2,459 deaths occurred in the HDSS during the year 2009 to 2012. Among them, 2,174 (88.4%) valid VA interviews were conducted. Crude death rate ranged from 7.1 (2009) to 6.4 (2012) per 1,000 population. The CSMR per 1,000 person-years over the years (2009–2012) for non-communicable diseases, communicable diseases, trauma, neoplasm, and maternal and neonatal diseases were 1.78, 1.68, 0.68, 0.49, and 0.48, respectively. The most common causes of death among children, adults, and the elderly were infectious diseases, trauma, and non-communicable diseases, respectively.</p></sec><sec id="st4"><title>Conclusions</title><p>Overall, non-communicable diseases constituted the largest proportion of mortality, whereas trauma was the most common COD among adults at Ballabgarh HDSS. Policy-makers ought to focus on prevention of premature CODs, especially prevention of infectious diseases in children, and intentional self-harm and road traffic accidents in the adult population.</p></sec> | <contrib contrib-type="author"><name><surname>Rai</surname><given-names>Sanjay K.</given-names></name><xref ref-type="aff" rid="AF0001">1</xref><xref ref-type="aff" rid="AF0002">2</xref></contrib><contrib contrib-type="author"><name><surname>Kant</surname><given-names>Shashi</given-names></name><xref ref-type="aff" rid="AF0001">1</xref><xref ref-type="aff" rid="AF0002">2</xref></contrib><contrib contrib-type="author"><name><surname>Misra</surname><given-names>Puneet</given-names></name><xref ref-type="aff" rid="AF0001">1</xref><xref ref-type="aff" rid="AF0002">2</xref></contrib><contrib contrib-type="author"><name><surname>Srivastava</surname><given-names>Rahul</given-names></name><xref ref-type="aff" rid="AF0001">1</xref><xref ref-type="aff" rid="AF0002">2</xref><xref ref-type="corresp" rid="cor1">*</xref></contrib><contrib contrib-type="author"><name><surname>Pandav</surname><given-names>Chandrakant S.</given-names></name><xref ref-type="aff" rid="AF0002">2</xref></contrib> | Global Health Action | <p>Death recording by age, sex, and cause, and calculating mortality rates and differentials are central to evidence-based health policy, monitoring, and evaluation (<xref rid="CIT0001" ref-type="bibr">1</xref>). Mortality levels are significant indicators of population health, and are of extreme importance to prioritize the goals of health systems and efficient resource allocation (<xref rid="CIT0002" ref-type="bibr">2</xref>–<xref rid="CIT0004" ref-type="bibr">4</xref>). About 46 million of the estimated 60 million deaths worldwide occur in developing countries (<xref rid="CIT0005" ref-type="bibr">5</xref>). Unfortunately, up to 90% of them die at home. These deaths remain uncertified and unregistered due to weak civil registration system. Only 10 to 40% deaths occur in hospitals for which information on causes of death are available (<xref rid="CIT0006" ref-type="bibr">6</xref>). India reported about 9 million deaths per year. Of these, more than three quarters occurred at home. Majority of these deaths did not have a medically certified cause of death (COD) (<xref rid="CIT0005" ref-type="bibr">5</xref>).</p><p>Magnitude and trends in cause-specific mortality provide critical insights to both evolving and ignored diseases, and the efficiency of present disease control policy. Understanding of changes in leading causes of deaths is essential to modify the strategy for addressing current needs. It is widely recognized that where deaths are not routinely certified for their cause, verbal autopsy (VA) is the interim method of choice for documenting cause-specific mortality patterns (<xref rid="CIT0007" ref-type="bibr">7</xref>).</p><p>Physician-assigned COD using VA has certain known limitations. Disagreement between the physicians (concordance between two physicians is required for assigning COD) lead to large proportion of causes of death being described as indeterminate (<xref rid="CIT0008" ref-type="bibr">8</xref>, <xref rid="CIT0009" ref-type="bibr">9</xref>). Since the diagnostic criteria are not standardized for the physicians assigning COD, reproducibility of VAs over time, and in different settings, becomes one of the major limitations (<xref rid="CIT0009" ref-type="bibr">9</xref>–<xref rid="CIT0011" ref-type="bibr">11</xref>). Involvement of physicians is a costly and time-consuming process as they are already an overstretched resource in low-income countries (<xref rid="CIT0009" ref-type="bibr">9</xref>, <xref rid="CIT0012" ref-type="bibr">12</xref>).</p><p>InterVA-4 model, a computer-based probabilistic model, can overcome these limitations, though it has its own limitations. Present study aimed to estimate the age and cause-specific mortality in Ballabgarh Health and Demographic Surveillance System (HDSS) site over the years 2009 to 2012 using a probabilistic model (InterVA-4).</p><sec sec-type="methods" id="S0002"><title>Methods</title><p>The Ballabgarh HDSS, also known as Comprehensive Rural Health Services Project, Ballabgarh, is situated in the northern part of India, and is around 22 miles from New Delhi (<xref ref-type="fig" rid="F0001">Fig. 1</xref>). At Ballabgarh HDSS, the population has been under surveillance since 1967 (<xref rid="CIT0013" ref-type="bibr">13</xref>). The project is a collaboration between the All India Institute of Medical Sciences and the State Government of Haryana. The total population under surveillance was 92,070 (residing in 28 villages) as of December 31, 2012. The population was served by two Primary Health Center (PHCs) and 12 sub-center. The population of the villages ranged from 3,000 to 8,000. The villages were almost similar in terms of socio-economic status and cultural habits.</p><fig id="F0001" position="float"><label>Fig. 1</label><caption><p>Map of Ballabgarh HDSS.</p></caption><graphic xlink:href="GHA-7-25573-g001"/></fig><p>Multipurpose health workers visited all households covered under the HDSS twice a month, and provided basic health services such as immunization, family planning, and so on, along with the collection of information on vital events. VAs were conducted for all the deaths occurring in the HDSS area since 2002. Trained health workers administered the VA tool within 2 to 4 weeks of death. Physician-coded COD was assigned to all these VAs. All deaths that occurred between January 1, 2009, and December 31, 2012, were included in the study. Data were extracted for InterVA-4 application from the VA records.</p><sec id="S0002-S20001"><title>VA tool</title><p>The VA tool used in the present study was developed at Ballabgarh HDSS in 2002. This VA tool is based on the WHO VA tool but is short and takes less time to complete when compared with WHO VA tool. There were three types of VA tools for different age groups, namely 0–28 days, 29 days to 5 years, and more than 5 years. All the tools were validated, and had a good agreement with the WHO tool (<xref rid="CIT0014" ref-type="bibr">14</xref>, <xref rid="CIT0015" ref-type="bibr">15</xref>). The VA tool contained a symptom checklist and a narrative portion.</p></sec><sec id="S0002-S20002"><title>COD ascertainment: InterVA-4model</title><p>Data were extracted from the VA tool in accordance with the WHO 2012 VA standard, and processed with the InterVA-4 model (version 4.02), a probabilistic approach to COD determination (<xref rid="CIT0016" ref-type="bibr">16</xref>). The data were entered into the already specified batchin.csv file format of InterVA-4, and a readable text output in log file format was chosen to assign the possible COD of each individual. Based on Bayes’ theorem (<xref rid="CIT0017" ref-type="bibr">17</xref>), the InterVA-4 model calculates the probability of a set of causes of death given the presence of circumstances, signs, and symptoms (collectively called ‘indicators’) reported in VA interviews. The method is described in detail elsewhere (<xref rid="CIT0013" ref-type="bibr">13</xref>, <xref rid="CIT0018" ref-type="bibr">18</xref>). Briefly, a finite number of COD are assigned to a predefined matrix of estimated probabilities of occurrence. The presence of indicators modifies the predefined probabilities of each COD upward or downward using Bayes’ theorem.</p></sec><sec id="S0002-S20003"><title>Analysis</title><p>Causes of deaths were classified into six broad categories, namely non-communicable diseases, communicable diseases, trauma, indeterminate, neonatal/maternal, and neoplasm. These broad categories were further sub-classified. The classification was in-built in the InterVA-4 software and was based on ICD-10. Cause-specific mortality rate (CSMR) (standardized to INDEPTH standard population in terms of age group, sex, and year) was calculated over time. Person-years observed was calculated for all years and age group independently from the prospective database maintained at Ballabgarh HDSS. Person-years observed, and cause- and age-specific deaths were used to calculate CSMR, per 1,000 person-years. The age criteria selected for the present study were: children (0–14 years); adult (15–49 years); elderly (≥50 years). Yearly aggregated COD analysis was done so that the output could be compared with the mortality rates of other HDSSs using InterVA-4 model (<xref rid="CIT0019" ref-type="bibr">19</xref>). All statistical analyses were performed with STATA release 11.1 software (Stata Corp., College Station, TX, USA).</p></sec></sec><sec sec-type="results" id="S0003"><title>Results</title><p>A total of 2,459 deaths occurred in the HDSS during the period 2009 to 2012. Among them, 2,285 (93%) VAs were conducted. In the remaining 7% of the deaths, VAs could not be conducted mainly due to the unavailability of the informant, for example, family migrated after the death of the individual or refused to provide information needed for the VA tool. Among the VA interviews conducted, 2,174 (88.4%) forms were valid (completely filled) while the remaining 111 (4.5%) forms did not have sufficient information that was required for InterVA-4 to assign COD. The incomplete VA forms were rejected by the InterVA-4 during assignment of COD. The availability of adequately filled VA was 85.6, 87.5, and 89.9% among children (0–14 years), adults (15–49 years), and elderly (≥50 years), respectively.</p><p>A declining trend of crude death rate (7.1 in 2009 to 6.4 in 2012 per 1,000 population) and infant mortality rate (53.7 in 2009 to 45.4 in 2012 per 1,000 live births) was observed in the Ballabgarh HDSS population (<xref ref-type="table" rid="T0001">Table 1</xref>). A similar declining trend was also observed in child mortality rate (0 to 14 years) and adult mortality rate (15 to 49 years) whereas elderly mortality rate (>50 years) had a rising trend. The aggregated (2009–2012) CSMR in descending order is as follows: non-communicable diseases (1.78), communicable diseases (1.68), trauma (0.68), neoplasm (0.49), and maternal and neonatal diseases (0.48) (<xref ref-type="fig" rid="F0002">Fig. 2</xref>).</p><fig id="F0002" position="float"><label>Fig. 2</label><caption><p>Cause-specific mortality rate (2009–2012) at Ballabgarh HDSS.</p></caption><graphic xlink:href="GHA-7-25573-g002"/></fig><fig id="F0003" position="float"><label>Fig. 3</label><caption><p>Cause-specific mortality rates (2009–2012) among males and females.</p></caption><graphic xlink:href="GHA-7-25573-g003"/></fig><table-wrap id="T0001" position="float"><label>Table 1</label><caption><p>Crude death rate and mortality trends at Ballabgarh HDSS, 2009–2012</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="center" rowspan="1" colspan="1"/><th align="center" rowspan="1" colspan="1"/><th align="center" rowspan="1" colspan="1"/><th align="center" rowspan="1" colspan="1"/><th align="center" rowspan="1" colspan="1"/><th align="center" colspan="3" rowspan="1">Mortality rate (deaths/1,000 person-years)</th></tr><tr><th align="center" rowspan="1" colspan="1"/><th align="center" rowspan="1" colspan="1"/><th align="center" rowspan="1" colspan="1"/><th align="center" rowspan="1" colspan="1"/><th align="center" rowspan="1" colspan="1"/><th align="center" colspan="3" rowspan="1">
<hr/>
</th></tr><tr><th align="center" rowspan="1" colspan="1">Year</th><th align="center" rowspan="1" colspan="1">Person-years</th><th align="center" rowspan="1" colspan="1">Number of deaths</th><th align="center" rowspan="1" colspan="1">Crude death rate (per 1,000 population)</th><th align="center" rowspan="1" colspan="1">IMR<xref ref-type="table-fn" rid="TF0001">a</xref> (per 1,000 live births)</th><th align="center" rowspan="1" colspan="1">Children (0–14 years)</th><th align="center" rowspan="1" colspan="1">Adult (15–49 years)</th><th align="center" rowspan="1" colspan="1">Elderly (≥50 years)</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">2009</td><td align="center" rowspan="1" colspan="1">87,502</td><td align="center" rowspan="1" colspan="1">622</td><td align="center" rowspan="1" colspan="1">7.1</td><td align="center" rowspan="1" colspan="1">53.7</td><td align="center" rowspan="1" colspan="1">5.8</td><td align="center" rowspan="1" colspan="1">3.0</td><td align="center" rowspan="1" colspan="1">27.8</td></tr><tr><td align="left" rowspan="1" colspan="1">2010</td><td align="center" rowspan="1" colspan="1">88,830</td><td align="center" rowspan="1" colspan="1">635</td><td align="center" rowspan="1" colspan="1">7.0</td><td align="center" rowspan="1" colspan="1">56.1</td><td align="center" rowspan="1" colspan="1">5.5</td><td align="center" rowspan="1" colspan="1">2.6</td><td align="center" rowspan="1" colspan="1">29.9</td></tr><tr><td align="left" rowspan="1" colspan="1">2011</td><td align="center" rowspan="1" colspan="1">90,479</td><td align="center" rowspan="1" colspan="1">610</td><td align="center" rowspan="1" colspan="1">6.7</td><td align="center" rowspan="1" colspan="1">48.2</td><td align="center" rowspan="1" colspan="1">5.0</td><td align="center" rowspan="1" colspan="1">2.4</td><td align="center" rowspan="1" colspan="1">29.0</td></tr><tr><td align="left" rowspan="1" colspan="1">2012</td><td align="center" rowspan="1" colspan="1">90,976</td><td align="center" rowspan="1" colspan="1">592</td><td align="center" rowspan="1" colspan="1">6.4</td><td align="center" rowspan="1" colspan="1">45.4</td><td align="center" rowspan="1" colspan="1">3.8</td><td align="center" rowspan="1" colspan="1">2.4</td><td align="center" rowspan="1" colspan="1">31.2</td></tr></tbody></table><table-wrap-foot><fn id="TF0001"><label>a</label><p>Infant mortality rate.</p></fn></table-wrap-foot></table-wrap><p>Over the years, CSMR (both male and female – all age group) for communicable diseases had declined (2.04 in 2009 to 1.45 in 2012) while that of non-communicable diseases had remained almost static. A declining trend was observed for communicable diseases among children (both male and female), and elderly male, whereas CSMR of communicable diseases fluctuated for adults (both male and female), and elderly females (<xref ref-type="fig" rid="F0003">Fig. 3</xref>). Among the elderly population, neoplasm was more common among males and also had an increasing trend. CSMR for neoplasm among elderly females was fluctuating. Trend of trauma was high among adult population but was fluctuating over the years for both males and females. Acute respiratory infection (ARI) (18.7%) was the commonest COD among infants and was followed by prematurity (18.0%). Among infants, Prematurity was more common among males (20.4%) when compared to females (15.1%). For children aged 1 to 4, diarrheal diseases was the commonest COD (22.5%) followed by ARI (19.2%) (<xref ref-type="table" rid="T0002">Table 2</xref>). Trauma (37.3%) was the most common COD observed in the adult population followed by infectious diseases (18.9%) and non-communicable diseases (18.5%) (<xref ref-type="table" rid="T0003">Table 3</xref>). Among trauma-associated deaths, the most common cause was transport accidents (13.3%), followed by intentional self-harm (12.4%). Intentional self-harm was slightly more common among females (13.6%) when compared to males (12.4%). Acute cardiac diseases and stroke were the most common causes among the non-communicable group. ‘Indeterminate’ was assigned as the COD in about 18% of all COD. It was highest for the elderly age group (19.8%) and lowest for children (15.0%).</p><table-wrap id="T0002" position="float"><label>Table 2</label><caption><p>Cause of deaths in infant and 1 to 4 year-old children at Ballabgarh HDSS, 2009 to 2012</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1"/><th align="center" colspan="3" rowspan="1">Infants (%)</th><th align="center" colspan="3" rowspan="1">Children (1–4 years) (%)</th></tr><tr><th align="left" rowspan="1" colspan="1"/><th align="center" colspan="3" rowspan="1">
<hr/>
</th><th align="center" colspan="3" rowspan="1">
<hr/>
</th></tr><tr><th align="left" rowspan="1" colspan="1">ICD-10 codes and cause of deaths</th><th align="center" rowspan="1" colspan="1">Male
(109)</th><th align="center" rowspan="1" colspan="1">Female
(86)</th><th align="center" rowspan="1" colspan="1">Total
(195)</th><th align="center" rowspan="1" colspan="1">Male
(85)</th><th align="center" rowspan="1" colspan="1">Female
(80)</th><th align="center" rowspan="1" colspan="1">Total
(165)</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">01.01 Sepsis (non-obstetric)</td><td align="center" rowspan="1" colspan="1">0.9</td><td align="center" rowspan="1" colspan="1">0.8</td><td align="center" rowspan="1" colspan="1">0.9</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.9</td><td align="center" rowspan="1" colspan="1">0.5</td></tr><tr><td align="left" rowspan="1" colspan="1">01.02 ARI, including pneumonia</td><td align="center" rowspan="1" colspan="1">18.9</td><td align="center" rowspan="1" colspan="1">18.4</td><td align="center" rowspan="1" colspan="1">18.7</td><td align="center" rowspan="1" colspan="1">11.2</td><td align="center" rowspan="1" colspan="1">27.4</td><td align="center" rowspan="1" colspan="1">19.2</td></tr><tr><td align="left" rowspan="1" colspan="1">01.03 HIV-/AIDS-related death</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">2.9</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">1.5</td></tr><tr><td align="left" rowspan="1" colspan="1">01.04 Diarrheal diseases</td><td align="center" rowspan="1" colspan="1">7.4</td><td align="center" rowspan="1" colspan="1">10.4</td><td align="center" rowspan="1" colspan="1">8.8</td><td align="center" rowspan="1" colspan="1">30.2</td><td align="center" rowspan="1" colspan="1">14.6</td><td align="center" rowspan="1" colspan="1">22.5</td></tr><tr><td align="left" rowspan="1" colspan="1">01.05 Malaria</td><td align="center" rowspan="1" colspan="1">1.3</td><td align="center" rowspan="1" colspan="1">1.7</td><td align="center" rowspan="1" colspan="1">1.5</td><td align="center" rowspan="1" colspan="1">18.3</td><td align="center" rowspan="1" colspan="1">18.6</td><td align="center" rowspan="1" colspan="1">18.4</td></tr><tr><td align="left" rowspan="1" colspan="1">01.07 Meningitis and encephalitis</td><td align="center" rowspan="1" colspan="1">1.2</td><td align="center" rowspan="1" colspan="1">2.2</td><td align="center" rowspan="1" colspan="1">1.6</td><td align="center" rowspan="1" colspan="1">1.8</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.9</td></tr><tr><td align="left" rowspan="1" colspan="1">01.10 Pertussis</td><td align="center" rowspan="1" colspan="1">0.2</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.1</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">1.9</td><td align="center" rowspan="1" colspan="1">0.9</td></tr><tr><td align="left" rowspan="1" colspan="1">01.99 Other and unspecified infect diseases</td><td align="center" rowspan="1" colspan="1">0.3</td><td align="center" rowspan="1" colspan="1">0.5</td><td align="center" rowspan="1" colspan="1">0.4</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td></tr><tr><td align="left" rowspan="1" colspan="1">02.99 Other and unspecified neoplasms</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.9</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.4</td></tr><tr><td align="left" rowspan="1" colspan="1">03.01 Severe anemia</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">1.1</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.6</td></tr><tr><td align="left" rowspan="1" colspan="1">03.02 Severe malnutrition</td><td align="center" rowspan="1" colspan="1">0.9</td><td align="center" rowspan="1" colspan="1">0.7</td><td align="center" rowspan="1" colspan="1">0.8</td><td align="center" rowspan="1" colspan="1">0.6</td><td align="center" rowspan="1" colspan="1">1.8</td><td align="center" rowspan="1" colspan="1">1.2</td></tr><tr><td align="left" rowspan="1" colspan="1">03.03 Diabetes mellitus</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.3</td><td align="center" rowspan="1" colspan="1">0.1</td><td align="center" rowspan="1" colspan="1">1.9</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">1.0</td></tr><tr><td align="left" rowspan="1" colspan="1">04.99 Other and unspecified cardiac diseases</td><td align="center" rowspan="1" colspan="1">0.4</td><td align="center" rowspan="1" colspan="1">0.4</td><td align="center" rowspan="1" colspan="1">0.4</td><td align="center" rowspan="1" colspan="1">1.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.5</td></tr><tr><td align="left" rowspan="1" colspan="1">06.01 Acute abdomen</td><td align="center" rowspan="1" colspan="1">1.3</td><td align="center" rowspan="1" colspan="1">0.6</td><td align="center" rowspan="1" colspan="1">1.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td></tr><tr><td align="left" rowspan="1" colspan="1">07.01 Renal failure</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">2.0</td><td align="center" rowspan="1" colspan="1">1.0</td></tr><tr><td align="left" rowspan="1" colspan="1">08.01 Epilepsy</td><td align="center" rowspan="1" colspan="1">1.0</td><td align="center" rowspan="1" colspan="1">0.9</td><td align="center" rowspan="1" colspan="1">1.0</td><td align="center" rowspan="1" colspan="1">1.8</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.9</td></tr><tr><td align="left" rowspan="1" colspan="1">10.01 Prematurity</td><td align="center" rowspan="1" colspan="1">20.4</td><td align="center" rowspan="1" colspan="1">15.1</td><td align="center" rowspan="1" colspan="1">18.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td></tr><tr><td align="left" rowspan="1" colspan="1">10.02 Birth asphyxia</td><td align="center" rowspan="1" colspan="1">7.8</td><td align="center" rowspan="1" colspan="1">7.2</td><td align="center" rowspan="1" colspan="1">7.5</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td></tr><tr><td align="left" rowspan="1" colspan="1">10.03 Neonatal pneumonia</td><td align="center" rowspan="1" colspan="1">10.9</td><td align="center" rowspan="1" colspan="1">9.0</td><td align="center" rowspan="1" colspan="1">10.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td></tr><tr><td align="left" rowspan="1" colspan="1">10.04 Neonatal sepsis</td><td align="center" rowspan="1" colspan="1">4.0</td><td align="center" rowspan="1" colspan="1">5.8</td><td align="center" rowspan="1" colspan="1">4.9</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td></tr><tr><td align="left" rowspan="1" colspan="1">10.06 Congenital malformation</td><td align="center" rowspan="1" colspan="1">1.7</td><td align="center" rowspan="1" colspan="1">1.9</td><td align="center" rowspan="1" colspan="1">1.8</td><td align="center" rowspan="1" colspan="1">2.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">1.0</td></tr><tr><td align="left" rowspan="1" colspan="1">10.99 Other and unspecified neonatal COD</td><td align="center" rowspan="1" colspan="1">6.2</td><td align="center" rowspan="1" colspan="1">7.1</td><td align="center" rowspan="1" colspan="1">6.6</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td></tr><tr><td align="left" rowspan="1" colspan="1">12.01 Road traffic accident</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">2.0</td><td align="center" rowspan="1" colspan="1">4.1</td><td align="center" rowspan="1" colspan="1">3.0</td></tr><tr><td align="left" rowspan="1" colspan="1">12.03 Accidental fall</td><td align="center" rowspan="1" colspan="1">0.5</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.3</td><td align="center" rowspan="1" colspan="1">4.0</td><td align="center" rowspan="1" colspan="1">4.1</td><td align="center" rowspan="1" colspan="1">4.0</td></tr><tr><td align="left" rowspan="1" colspan="1">12.04 Accidental drowning and submersion</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">1.2</td><td align="center" rowspan="1" colspan="1">0.6</td><td align="center" rowspan="1" colspan="1">2.0</td><td align="center" rowspan="1" colspan="1">2.1</td><td align="center" rowspan="1" colspan="1">2.1</td></tr><tr><td align="left" rowspan="1" colspan="1">12.05 Accidental expos to smoke fire and flame</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.3</td><td align="center" rowspan="1" colspan="1">0.1</td><td align="center" rowspan="1" colspan="1">1.8</td><td align="center" rowspan="1" colspan="1">1.6</td><td align="center" rowspan="1" colspan="1">1.7</td></tr><tr><td align="left" rowspan="1" colspan="1">12.06 Contact with venomous plant/animal</td><td align="center" rowspan="1" colspan="1">0.3</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.2</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">2.1</td><td align="center" rowspan="1" colspan="1">1.0</td></tr><tr><td align="left" rowspan="1" colspan="1">12.99 Other and unspecified external COD</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">2.8</td><td align="center" rowspan="1" colspan="1">1.4</td></tr><tr><td align="left" rowspan="1" colspan="1">99 Indeterminate</td><td align="center" rowspan="1" colspan="1">14.2</td><td align="center" rowspan="1" colspan="1">15.5</td><td align="center" rowspan="1" colspan="1">14.8</td><td align="center" rowspan="1" colspan="1">16.3</td><td align="center" rowspan="1" colspan="1">16.0</td><td align="center" rowspan="1" colspan="1">16.1</td></tr></tbody></table></table-wrap><table-wrap id="T0003" position="float"><label>Table 3</label><caption><p>Cause of deaths at Ballabgarh HDSS by age and sex, 2009 to 2012</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1"/><th align="center" colspan="3" rowspan="1">Children (%): 0–14 years</th><th align="center" colspan="3" rowspan="1">Adult (%): 15–49 years</th><th align="center" colspan="3" rowspan="1">Elderly (%): ≥50 years</th></tr><tr><th align="left" rowspan="1" colspan="1"/><th align="center" colspan="3" rowspan="1">
<hr/>
</th><th align="center" colspan="3" rowspan="1">
<hr/>
</th><th align="center" colspan="3" rowspan="1">
<hr/>
</th></tr><tr><th align="left" rowspan="1" colspan="1">Cause of death and ICD-10 codes</th><th align="center" rowspan="1" colspan="1">Male (267)</th><th align="center" rowspan="1" colspan="1">Female (233)</th><th align="center" rowspan="1" colspan="1">Total (500)</th><th align="center" rowspan="1" colspan="1">Male (271)</th><th align="center" rowspan="1" colspan="1">Female (172)</th><th align="center" rowspan="1" colspan="1">Total (443)</th><th align="center" rowspan="1" colspan="1">Male (728)</th><th align="center" rowspan="1" colspan="1">Female (503)</th><th align="center" rowspan="1" colspan="1">Total (1,213)</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">1-Infectious diseases</td><td align="center" rowspan="1" colspan="1">36.7</td><td align="center" rowspan="1" colspan="1">39.0</td><td align="center" rowspan="1" colspan="1">37.7</td><td align="center" rowspan="1" colspan="1">19.1</td><td align="center" rowspan="1" colspan="1">18.6</td><td align="center" rowspan="1" colspan="1">18.9</td><td align="center" rowspan="1" colspan="1">19.9</td><td align="center" rowspan="1" colspan="1">24.4</td><td align="center" rowspan="1" colspan="1">21.7</td></tr><tr><td align="left" rowspan="1" colspan="1"> 01.01 Sepsis (non-obstetric)</td><td align="center" rowspan="1" colspan="1">0.8</td><td align="center" rowspan="1" colspan="1">0.8</td><td align="center" rowspan="1" colspan="1">0.8</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.2</td><td align="center" rowspan="1" colspan="1">0.3</td><td align="center" rowspan="1" colspan="1">0.3</td></tr><tr><td align="left" rowspan="1" colspan="1"> 01.02 ARI, including pneumonia</td><td align="center" rowspan="1" colspan="1">16.0</td><td align="center" rowspan="1" colspan="1">18.7</td><td align="center" rowspan="1" colspan="1">17.3</td><td align="center" rowspan="1" colspan="1">0.7</td><td align="center" rowspan="1" colspan="1">1.1</td><td align="center" rowspan="1" colspan="1">0.9</td><td align="center" rowspan="1" colspan="1">5.1</td><td align="center" rowspan="1" colspan="1">5.4</td><td align="center" rowspan="1" colspan="1">5.2</td></tr><tr><td align="left" rowspan="1" colspan="1"> 01.04 Diarrheal diseases</td><td align="center" rowspan="1" colspan="1">11.4</td><td align="center" rowspan="1" colspan="1">10.4</td><td align="center" rowspan="1" colspan="1">11.0</td><td align="center" rowspan="1" colspan="1">0.7</td><td align="center" rowspan="1" colspan="1">1.0</td><td align="center" rowspan="1" colspan="1">0.8</td><td align="center" rowspan="1" colspan="1">1.1</td><td align="center" rowspan="1" colspan="1">1.7</td><td align="center" rowspan="1" colspan="1">1.4</td></tr><tr><td align="left" rowspan="1" colspan="1"> 01.07 Meningitis and encephalitis</td><td align="center" rowspan="1" colspan="1">1.2</td><td align="center" rowspan="1" colspan="1">2.1</td><td align="center" rowspan="1" colspan="1">1.6</td><td align="center" rowspan="1" colspan="1">0.8</td><td align="center" rowspan="1" colspan="1">0.8</td><td align="center" rowspan="1" colspan="1">0.8</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td></tr><tr><td align="left" rowspan="1" colspan="1"> Other and Unspecified</td><td align="center" rowspan="1" colspan="1">7.2</td><td align="center" rowspan="1" colspan="1">6.9</td><td align="center" rowspan="1" colspan="1">7.1</td><td align="center" rowspan="1" colspan="1">16.9</td><td align="center" rowspan="1" colspan="1">15.7</td><td align="center" rowspan="1" colspan="1">16.4</td><td align="center" rowspan="1" colspan="1">13.4</td><td align="center" rowspan="1" colspan="1">16.9</td><td align="center" rowspan="1" colspan="1">14.8</td></tr><tr><td align="left" rowspan="1" colspan="1">2-Neoplasms</td><td align="center" rowspan="1" colspan="1">0.2</td><td align="center" rowspan="1" colspan="1">1.0</td><td align="center" rowspan="1" colspan="1">0.6</td><td align="center" rowspan="1" colspan="1">5.8</td><td align="center" rowspan="1" colspan="1">6.3</td><td align="center" rowspan="1" colspan="1">6.0</td><td align="center" rowspan="1" colspan="1">11.7</td><td align="center" rowspan="1" colspan="1">12.2</td><td align="center" rowspan="1" colspan="1">11.9</td></tr><tr><td align="left" rowspan="1" colspan="1"> 02.01 Oral neoplasms</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.5</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.3</td><td align="center" rowspan="1" colspan="1">0.1</td><td align="center" rowspan="1" colspan="1">0.6</td><td align="center" rowspan="1" colspan="1">0.3</td></tr><tr><td align="left" rowspan="1" colspan="1"> 02.02 Digestive neoplasms</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">2.2</td><td align="center" rowspan="1" colspan="1">4.0</td><td align="center" rowspan="1" colspan="1">2.9</td><td align="center" rowspan="1" colspan="1">6.3</td><td align="center" rowspan="1" colspan="1">6.3</td><td align="center" rowspan="1" colspan="1">6.3</td></tr><tr><td align="left" rowspan="1" colspan="1"> 02.03 Respiratory neoplasms</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.4</td><td align="center" rowspan="1" colspan="1">0.1</td><td align="center" rowspan="1" colspan="1">0.3</td><td align="center" rowspan="1" colspan="1">1.8</td><td align="center" rowspan="1" colspan="1">2.0</td><td align="center" rowspan="1" colspan="1">1.9</td></tr><tr><td align="left" rowspan="1" colspan="1"> 02.04 Breast neoplasms</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.7</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.5</td><td align="center" rowspan="1" colspan="1">0.2</td><td align="center" rowspan="1" colspan="1">0.2</td><td align="center" rowspan="1" colspan="1">0.2</td></tr><tr><td align="left" rowspan="1" colspan="1"> 02.05 and 02.06 Reproductive neoplasm</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.5</td><td align="center" rowspan="1" colspan="1">0.5</td><td align="center" rowspan="1" colspan="1">0.5</td><td align="center" rowspan="1" colspan="1">2.8</td><td align="center" rowspan="1" colspan="1">2.2</td><td align="center" rowspan="1" colspan="1">2.6</td></tr><tr><td align="left" rowspan="1" colspan="1"> 02.99 Other and unspecified neoplasms</td><td align="center" rowspan="1" colspan="1">0.2</td><td align="center" rowspan="1" colspan="1">1.0</td><td align="center" rowspan="1" colspan="1">0.6</td><td align="center" rowspan="1" colspan="1">1.6</td><td align="center" rowspan="1" colspan="1">1.7</td><td align="center" rowspan="1" colspan="1">1.6</td><td align="center" rowspan="1" colspan="1">0.5</td><td align="center" rowspan="1" colspan="1">0.8</td><td align="center" rowspan="1" colspan="1">0.6</td></tr><tr><td align="left" rowspan="1" colspan="1">3-Non-communicable diseases</td><td align="center" rowspan="1" colspan="1">5.1</td><td align="center" rowspan="1" colspan="1">3.9</td><td align="center" rowspan="1" colspan="1">4.5</td><td align="center" rowspan="1" colspan="1">19.8</td><td align="center" rowspan="1" colspan="1">16.5</td><td align="center" rowspan="1" colspan="1">18.5</td><td align="center" rowspan="1" colspan="1">43.1</td><td align="center" rowspan="1" colspan="1">39.7</td><td align="center" rowspan="1" colspan="1">41.7</td></tr><tr><td align="left" rowspan="1" colspan="1"> 3.01 and 3.02 Severe Anemia and
Malnutrition</td><td align="center" rowspan="1" colspan="1">1.2</td><td align="center" rowspan="1" colspan="1">0.9</td><td align="center" rowspan="1" colspan="1">1.1</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.4</td><td align="center" rowspan="1" colspan="1">0.2</td><td align="center" rowspan="1" colspan="1">0.3</td></tr><tr><td align="left" rowspan="1" colspan="1"> 03.03 Diabetes mellitus</td><td align="center" rowspan="1" colspan="1">0.4</td><td align="center" rowspan="1" colspan="1">0.2</td><td align="center" rowspan="1" colspan="1">0.3</td><td align="center" rowspan="1" colspan="1">0.6</td><td align="center" rowspan="1" colspan="1">0.5</td><td align="center" rowspan="1" colspan="1">0.6</td><td align="center" rowspan="1" colspan="1">2.3</td><td align="center" rowspan="1" colspan="1">2.3</td><td align="center" rowspan="1" colspan="1">2.3</td></tr><tr><td align="left" rowspan="1" colspan="1"> 04.01 Acute cardiac disease</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">4.0</td><td align="center" rowspan="1" colspan="1">7.0</td><td align="center" rowspan="1" colspan="1">5.2</td><td align="center" rowspan="1" colspan="1">6.3</td><td align="center" rowspan="1" colspan="1">4.5</td><td align="center" rowspan="1" colspan="1">5.6</td></tr><tr><td align="left" rowspan="1" colspan="1"> 04.02 Stroke</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">2.9</td><td align="center" rowspan="1" colspan="1">1.4</td><td align="center" rowspan="1" colspan="1">2.3</td><td align="center" rowspan="1" colspan="1">10.8</td><td align="center" rowspan="1" colspan="1">11.3</td><td align="center" rowspan="1" colspan="1">11.0</td></tr><tr><td align="left" rowspan="1" colspan="1"> 5.01 and 5.02 COPD and Asthma</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">1.5</td><td align="center" rowspan="1" colspan="1">0.3</td><td align="center" rowspan="1" colspan="1">1.1</td><td align="center" rowspan="1" colspan="1">10.4</td><td align="center" rowspan="1" colspan="1">10.6</td><td align="center" rowspan="1" colspan="1">10.5</td></tr><tr><td align="left" rowspan="1" colspan="1"> 06.01 Acute abdomen</td><td align="center" rowspan="1" colspan="1">2.0</td><td align="center" rowspan="1" colspan="1">0.8</td><td align="center" rowspan="1" colspan="1">1.4</td><td align="center" rowspan="1" colspan="1">3.9</td><td align="center" rowspan="1" colspan="1">4.1</td><td align="center" rowspan="1" colspan="1">4.0</td><td align="center" rowspan="1" colspan="1">3.8</td><td align="center" rowspan="1" colspan="1">4.3</td><td align="center" rowspan="1" colspan="1">4.0</td></tr><tr><td align="left" rowspan="1" colspan="1"> 06.02 Liver cirrhosis</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.3</td><td align="center" rowspan="1" colspan="1">0.1</td><td align="center" rowspan="1" colspan="1">1.6</td><td align="center" rowspan="1" colspan="1">0.9</td><td align="center" rowspan="1" colspan="1">1.3</td><td align="center" rowspan="1" colspan="1">0.9</td><td align="center" rowspan="1" colspan="1">0.6</td><td align="center" rowspan="1" colspan="1">0.8</td></tr><tr><td align="left" rowspan="1" colspan="1"> 07.01 Renal failure</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.4</td><td align="center" rowspan="1" colspan="1">0.2</td><td align="center" rowspan="1" colspan="1">1.9</td><td align="center" rowspan="1" colspan="1">0.5</td><td align="center" rowspan="1" colspan="1">1.3</td><td align="center" rowspan="1" colspan="1">2.4</td><td align="center" rowspan="1" colspan="1">1.0</td><td align="center" rowspan="1" colspan="1">1.8</td></tr><tr><td align="left" rowspan="1" colspan="1"> 08.01 Epilepsy</td><td align="center" rowspan="1" colspan="1">1.1</td><td align="center" rowspan="1" colspan="1">1.1</td><td align="center" rowspan="1" colspan="1">1.1</td><td align="center" rowspan="1" colspan="1">1.7</td><td align="center" rowspan="1" colspan="1">0.6</td><td align="center" rowspan="1" colspan="1">1.3</td><td align="center" rowspan="1" colspan="1">0.4</td><td align="center" rowspan="1" colspan="1">0.2</td><td align="center" rowspan="1" colspan="1">0.3</td></tr><tr><td align="left" rowspan="1" colspan="1"> Others and unspecified NCDs</td><td align="center" rowspan="1" colspan="1">0.5</td><td align="center" rowspan="1" colspan="1">0.3</td><td align="center" rowspan="1" colspan="1">0.4</td><td align="center" rowspan="1" colspan="1">1.9</td><td align="center" rowspan="1" colspan="1">1.1</td><td align="center" rowspan="1" colspan="1">1.6</td><td align="center" rowspan="1" colspan="1">5.4</td><td align="center" rowspan="1" colspan="1">4.7</td><td align="center" rowspan="1" colspan="1">5.1</td></tr><tr><td align="left" rowspan="1" colspan="1">4- Maternal and neonatal</td><td align="center" rowspan="1" colspan="1">37.5</td><td align="center" rowspan="1" colspan="1">32.9</td><td align="center" rowspan="1" colspan="1">35.3</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">2.3</td><td align="center" rowspan="1" colspan="1">0.9</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td></tr><tr><td align="left" rowspan="1" colspan="1"> 09.04 Obstetric hemorrhage</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">1.4</td><td align="center" rowspan="1" colspan="1">0.6</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td></tr><tr><td align="left" rowspan="1" colspan="1"> Other and unspecified maternal COD</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.9</td><td align="center" rowspan="1" colspan="1">0.3</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td></tr><tr><td align="left" rowspan="1" colspan="1"> 10.01 Prematurity</td><td align="center" rowspan="1" colspan="1">14.9</td><td align="center" rowspan="1" colspan="1">10.8</td><td align="center" rowspan="1" colspan="1">13.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td></tr><tr><td align="left" rowspan="1" colspan="1"> 10.02 Birth asphyxia</td><td align="center" rowspan="1" colspan="1">5.7</td><td align="center" rowspan="1" colspan="1">5.1</td><td align="center" rowspan="1" colspan="1">5.4</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td></tr><tr><td align="left" rowspan="1" colspan="1"> 10.03 Neonatal pneumonia</td><td align="center" rowspan="1" colspan="1">7.9</td><td align="center" rowspan="1" colspan="1">6.4</td><td align="center" rowspan="1" colspan="1">7.2</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td></tr><tr><td align="left" rowspan="1" colspan="1"> 10.04 Neonatal sepsis</td><td align="center" rowspan="1" colspan="1">2.9</td><td align="center" rowspan="1" colspan="1">4.2</td><td align="center" rowspan="1" colspan="1">3.5</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td></tr><tr><td align="left" rowspan="1" colspan="1"> 10.06 Congenital malformation</td><td align="center" rowspan="1" colspan="1">1.6</td><td align="center" rowspan="1" colspan="1">1.4</td><td align="center" rowspan="1" colspan="1">1.5</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td></tr><tr><td align="left" rowspan="1" colspan="1"> 10.99 Other and unspecified neonatal</td><td align="center" rowspan="1" colspan="1">4.5</td><td align="center" rowspan="1" colspan="1">5.1</td><td align="center" rowspan="1" colspan="1">4.8</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td></tr><tr><td align="left" rowspan="1" colspan="1">5-Trauma</td><td align="center" rowspan="1" colspan="1">6.7</td><td align="center" rowspan="1" colspan="1">7.0</td><td align="center" rowspan="1" colspan="1">6.8</td><td align="center" rowspan="1" colspan="1">34.8</td><td align="center" rowspan="1" colspan="1">41.2</td><td align="center" rowspan="1" colspan="1">37.3</td><td align="center" rowspan="1" colspan="1">5.1</td><td align="center" rowspan="1" colspan="1">4.5</td><td align="center" rowspan="1" colspan="1">4.9</td></tr><tr><td align="left" rowspan="1" colspan="1"> RTAs and other transport Accidents<xref ref-type="table-fn" rid="TF0002">a</xref>
</td><td align="center" rowspan="1" colspan="1">1.4</td><td align="center" rowspan="1" colspan="1">0.8</td><td align="center" rowspan="1" colspan="1">1.1</td><td align="center" rowspan="1" colspan="1">13.5</td><td align="center" rowspan="1" colspan="1">13.1</td><td align="center" rowspan="1" colspan="1">13.3</td><td align="center" rowspan="1" colspan="1">1.8</td><td align="center" rowspan="1" colspan="1">1.7</td><td align="center" rowspan="1" colspan="1">1.7</td></tr><tr><td align="left" rowspan="1" colspan="1"> 12.03 Accidental fall</td><td align="center" rowspan="1" colspan="1">2.5</td><td align="center" rowspan="1" colspan="1">1.6</td><td align="center" rowspan="1" colspan="1">2.1</td><td align="center" rowspan="1" colspan="1">2.5</td><td align="center" rowspan="1" colspan="1">1.3</td><td align="center" rowspan="1" colspan="1">2.0</td><td align="center" rowspan="1" colspan="1">1.6</td><td align="center" rowspan="1" colspan="1">1.4</td><td align="center" rowspan="1" colspan="1">1.5</td></tr><tr><td align="left" rowspan="1" colspan="1"> 12.04 Accident drowning and submersion</td><td align="center" rowspan="1" colspan="1">1.1</td><td align="center" rowspan="1" colspan="1">1.7</td><td align="center" rowspan="1" colspan="1">1.4</td><td align="center" rowspan="1" colspan="1">1.4</td><td align="center" rowspan="1" colspan="1">3.9</td><td align="center" rowspan="1" colspan="1">2.4</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.2</td><td align="center" rowspan="1" colspan="1">0.1</td></tr><tr><td align="left" rowspan="1" colspan="1"> 12.05 Accidental exposure to smoke fire</td><td align="center" rowspan="1" colspan="1">0.3</td><td align="center" rowspan="1" colspan="1">0.9</td><td align="center" rowspan="1" colspan="1">0.6</td><td align="center" rowspan="1" colspan="1">0.7</td><td align="center" rowspan="1" colspan="1">3.0</td><td align="center" rowspan="1" colspan="1">1.6</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.2</td><td align="center" rowspan="1" colspan="1">0.1</td></tr><tr><td align="left" rowspan="1" colspan="1"> 12.08 Intentional self-harm</td><td align="center" rowspan="1" colspan="1">0.7</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.4</td><td align="center" rowspan="1" colspan="1">11.6</td><td align="center" rowspan="1" colspan="1">13.6</td><td align="center" rowspan="1" colspan="1">12.4</td><td align="center" rowspan="1" colspan="1">0.9</td><td align="center" rowspan="1" colspan="1">0.5</td><td align="center" rowspan="1" colspan="1">0.7</td></tr><tr><td align="left" rowspan="1" colspan="1"> 12.09 Assault</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">2.7</td><td align="center" rowspan="1" colspan="1">4.6</td><td align="center" rowspan="1" colspan="1">3.4</td><td align="center" rowspan="1" colspan="1">0.4</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.2</td></tr><tr><td align="left" rowspan="1" colspan="1"> 12.10 Exposure to force of nature</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.4</td><td align="center" rowspan="1" colspan="1">0.2</td><td align="center" rowspan="1" colspan="1">0.4</td><td align="center" rowspan="1" colspan="1">0.6</td><td align="center" rowspan="1" colspan="1">0.5</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td><td align="center" rowspan="1" colspan="1">0.0</td></tr><tr><td align="left" rowspan="1" colspan="1"> Other and unspecified external COD</td><td align="center" rowspan="1" colspan="1">0.6</td><td align="center" rowspan="1" colspan="1">1.4</td><td align="center" rowspan="1" colspan="1">1.0</td><td align="center" rowspan="1" colspan="1">2.0</td><td align="center" rowspan="1" colspan="1">1.1</td><td align="center" rowspan="1" colspan="1">1.7</td><td align="center" rowspan="1" colspan="1">0.5</td><td align="center" rowspan="1" colspan="1">0.5</td><td align="center" rowspan="1" colspan="1">0.5</td></tr><tr><td align="left" rowspan="1" colspan="1">6-Indeterminate</td><td align="center" rowspan="1" colspan="1">13.9</td><td align="center" rowspan="1" colspan="1">16.2</td><td align="center" rowspan="1" colspan="1">15.0</td><td align="center" rowspan="1" colspan="1">20.5</td><td align="center" rowspan="1" colspan="1">15.1</td><td align="center" rowspan="1" colspan="1">18.4</td><td align="center" rowspan="1" colspan="1">20.2</td><td align="center" rowspan="1" colspan="1">19.3</td><td align="center" rowspan="1" colspan="1">19.8</td></tr></tbody></table><table-wrap-foot><fn id="TF0002"><label>a</label><p>Road traffic accident.</p></fn></table-wrap-foot></table-wrap></sec><sec sec-type="discussion" id="S0004"><title>Discussion</title><p>The findings of the present study suggest a declining CSMR of communicable diseases, and an increasing CSMR of non-communicable diseases. Further, non-communicable diseases were the most common COD in this population. This trend was in agreement with various other studies that revealed the transition of the burden of disease from communicable to non-communicable diseases in India (<xref rid="CIT0020" ref-type="bibr">20</xref>–<xref rid="CIT0022" ref-type="bibr">22</xref>). The Global Burden of Disease 2010 estimates suggest that there is a shift from communicable diseases toward non-communicable diseases in adults in India (<xref rid="CIT0023" ref-type="bibr">23</xref>).</p><p>ARI and diarrhea continued to be the most common causes of deaths among children (0 to 4 years). Prevailing cultural practices and absence of routine pneumococcal and rotavirus vaccination might be contributing to high mortality due to ARI and diarrhea (<xref rid="CIT0024" ref-type="bibr">24</xref>).</p><p>Deaths in an economically productive age group adversely affect households as well as society as a whole and therefore prevention of premature deaths should be prioritized (<xref rid="CIT0025" ref-type="bibr">25</xref>). In the present study, the commonest COD among adults was trauma, which accounted for one third of all adult deaths. One third of the trauma cases among adults were due to intentional self-harm, and another one third was due to transport accidents. In adult population, intentional self-harm was more common among females (13.6% of all adult female deaths) as compared to males (11.6% of all adult male deaths). Globally, it has been reported that attempted intentional self-harm is commoner in women but intentional self-harm causing mortality is more common in men (<xref rid="CIT0026" ref-type="bibr">26</xref>). Ballabgarh HDSS (Salve et al.) reported that the major cause of intentional self-harm was marital conflicts followed by financial constraint, parental conflict, and educational failure (<xref rid="CIT0027" ref-type="bibr">27</xref>).</p><p>We had assigned COD by a computerized probabilistic model, InterVA-4. Level of agreement of this method with what is considered to be a gold standard for assigning COD using VA (medical death certificate) has ranged from 40 to 83% in different settings. The level of agreement was found to be better when the diseases were classified under broader groups rather than when compared to more specific CODs and also agreement varied for different groups (<xref rid="CIT0013" ref-type="bibr">13</xref>, <xref rid="CIT0019" ref-type="bibr">19</xref>, <xref rid="CIT0028" ref-type="bibr">28</xref>, <xref rid="CIT0029" ref-type="bibr">29</xref>). Since we had used a broader classification of diseases, we expect a high degree of agreement with the physician CODs. Present classification also allowed a comparison with the cause- and age-specific mortality rates from other developing countries, especially from other INDEPTH-associated HDSS sites. In our setting, there was a probability of inter-observer bias as the COD was assigned by a number of physicians. With the use of InterVA-4, more consistency and standardization was expected in assigning of COD.</p><p>There were certain limitations attached to the study. The proportion of ‘indeterminate’ as a COD was high in the present study, accounting for about one fifth of the total COD. This could be due to the fact that data extracted from the Ballabgarh VA tool was less when compared to the WHO 2012 VA tool, for which InterVA-4 is actually programmed. Second, we do not know whether the use of InterVA-4 was appropriate, since it has not been validated for Indian settings so far. A period of 4 years is very small to document any trend and hence the trend observed in the present study must be very cautiously interpreted. Cause of death may vary in different geographical locations as there are many factors (environmental, social, cultural, etc.) which are restricted to certain geographical location. This might even affect the probability of the IntervA-4 model. Geographical pattern of cause-specific mortality risk was not done in the present study, but it is a potential subject for future research. However, from the previous studies conducted in Ballabgarh HDSS, it is evident that geographical risk is homogenous at Ballabgarh HDSS (<xref rid="CIT0025" ref-type="bibr">25</xref>).</p></sec><sec sec-type="conclusions" id="S0005"><title>Conclusions</title><p>A declining trend in communicable diseases and a rising trend in non-communicable diseases were observed at Ballabgarh HDSS. Most common COD among children, adults, and the elderly were communicable diseases, trauma, and non-communicable diseases. Policy-makers ought to focus on prevention of premature CODs, especially prevention of infectious diseases in children, and intentional self-harm and road traffic accidents in the adult population.</p></sec> |
Multi-omic landscape of rheumatoid arthritis: re-evaluation of drug adverse effects | <p><bold>Objective:</bold> To provide a frame to estimate the systemic impact (side/adverse events) of (novel) therapeutic targets by taking into consideration drugs potential on the numerous districts involved in rheumatoid arthritis (RA) from the inflammatory and immune response to the gut-intestinal (GI) microbiome.</p><p><bold>Methods:</bold> We curated the collection of molecules from high-throughput screens of diverse (multi-<italic>omic</italic>) biochemical origin, experimentally associated to RA. Starting from such collection we generated RA-related protein-protein interaction (PPI) networks (<italic>interactomes)</italic> based on experimental PPI data. Pharmacological treatment simulation, topological and functional analyses were further run to gain insight into the proteins most affected by therapy and by multi-<italic>omic</italic> modeling.</p><p><bold>Results:</bold> Simulation on the administration of MTX results in the activation of expected (apoptosis) and adverse (nitrogenous metabolism alteration) effects. Growth factor receptor-bound protein 2 (GRB2) and Interleukin-1 Receptor Associated Kinase-4 (IRAK4, already an RA target) emerge as relevant nodes. The former controls the activation of inflammatory, proliferative and degenerative pathways in host and pathogens. The latter controls immune alterations and blocks innate response to pathogens.</p><p><bold>Conclusions:</bold> This multi-omic map properly recollects in a single analytical picture known, yet complex, information like the adverse/side effects of MTX, and provides a reliable platform for <italic>in silico</italic> hypothesis testing or recommendation on novel therapies. These results can support the development of RA translational research in the design of validation experiments and clinical trials, as such we identify GRB2 as a robust potential new target for RA for its ability to control both synovial degeneracy and dysbiosis, and, conversely, warn on the usage of IRAK4-inhibitors recently promoted, as this involves potential adverse effects in the form of impaired innate response to pathogens.</p> | <contrib contrib-type="author"><name><surname>Tieri</surname><given-names>Paolo</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref><xref ref-type="author-notes" rid="fn001"><sup>*</sup></xref><xref ref-type="author-notes" rid="fn004"><sup>†</sup></xref><uri xlink:type="simple" xlink:href="http://community.frontiersin.org/people/u/20242"/></contrib><contrib contrib-type="author"><name><surname>Zhou</surname><given-names>XiaoYuan</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref><xref ref-type="author-notes" rid="fn004"><sup>†</sup></xref><uri xlink:type="simple" xlink:href="http://community.frontiersin.org/people/u/182152"/></contrib><contrib contrib-type="author"><name><surname>Zhu</surname><given-names>Lisha</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref><uri xlink:type="simple" xlink:href="http://community.frontiersin.org/people/u/188580"/></contrib><contrib contrib-type="author"><name><surname>Nardini</surname><given-names>Christine</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref><xref ref-type="author-notes" rid="fn002"><sup>*</sup></xref><uri xlink:type="simple" xlink:href="http://community.frontiersin.org/people/u/115484"/></contrib> | Frontiers in Cell and Developmental Biology | <sec sec-type="introduction" id="s1"><title>Introduction</title><p>Rheumatoid arthritis (RA) is a multifaceted autoimmune, chronic and inflammatory disease with, to date, unclear etiology. As a consequence of its complexity, the definition of efficient and effective therapies remains a remarkable challenge due to the difficulties in controlling side effects and adverse events in relation to known (like genetic susceptibility, Stahl et al., <xref rid="B84" ref-type="bibr">2010</xref>) and emergent (epigenomic factors, Nakano et al., <xref rid="B61" ref-type="bibr">2012</xref>, dysbiosis, Scher and Abramson, <xref rid="B75" ref-type="bibr">2011</xref>) RA-associated con-causes.</p><p>Recently, translational research has welcomed into medicine a number of novel perspectives. Among these, sequencing technologies (<italic>omic</italic> screens) and computational intensive approaches (systems biology) now coagulate into a practice where technology and mathematical modeling serve basic research in the production of selected hypotheses, which testing <italic>in vitro, in vivo</italic> and ultimately in clinical studies can support medical research and practice (Okada et al., <xref rid="B65" ref-type="bibr">2014</xref>; You et al., <xref rid="B98" ref-type="bibr">2014</xref>). The recent acknowledgment of the importance and complexity of the gut intestinal (GI) microbiome in the onset, progression and regression of RA (Scher and Abramson, <xref rid="B75" ref-type="bibr">2011</xref>; Scher et al., <xref rid="B77" ref-type="bibr">2012</xref>, <xref rid="B76" ref-type="bibr">2013</xref>) and other autoimmune diseases, requires to incorporate the effects on the GI microbiome for any novel therapy. While protocols and medical best practice recommendations become mature in this direction, we propose the use of network approaches and <italic>omics</italic> from diverse origins (i.e., different biochemical districts/compartments/layers) including genomics, epigenomics, transcriptomics, post-transcriptomics, proteomics, and host-microbiome interface to GI metagenomics, to appropriately monitor the complexity of the disease. The novelty of the present work, therefore, lies not only in its application to RA, but also in the number of <italic>omic</italic> layers we have used, from genomic to proteomic and including the host-microbiome interface. These novelties allow to draw a single analytical picture of the fragmented molecular information available to date on RA, an easily consultable and extendable reference map for the researchers in the field, and—importantly—a systemic evaluation on the impact of a recently proposed RA therapeutic target (IRAK4), valuable <italic>per se</italic> and as an exemplar application of this approach. Overall, this work contributes to the general debate about data integration by offering details on our methodology, and to the area of complex inflammatory diseases, by providing specific examples of data choice and operational results.</p></sec><sec sec-type="methods" id="s2"><title>Methods</title><sec><title>Map construction</title><p>The datasets used to construct the map are gathered from 13 different sources from databases and literature (Table <xref ref-type="table" rid="T1">1</xref>). We included molecules experimentally associated to RA from manual curation of literature sources (<italic>core</italic> dataset, <italic>CD</italic>, 377 proteins, Data Sheet 1, Tables <xref ref-type="supplementary-material" rid="SM1">S1–S6</xref>), and additional molecules and pathways strongly yet not explicitly associated to RA (<italic>extended</italic> dataset, <italic>ED</italic>, 4709 proteins, Data Sheet 1, Tables <xref ref-type="supplementary-material" rid="SM1">S3A–E</xref>, <xref ref-type="supplementary-material" rid="SM1">S7–S13</xref>). A summary of all datasets and proteins' Uniprot IDs is provided in Data Sheet 1, Table <xref ref-type="supplementary-material" rid="SM1">S14</xref>. While the <italic>core</italic> set constitutes a more specific RA map, its extension offers a more systemic and practically usable map, notably in terms of the significance of the statistics that can be run on the extended map. The map presented here assembles genomic, epigenomic, transcriptomic, post-transcriptomic, proteomic, and host-microbiome interface data related to RA, as detailed below, and integrates such information at the functional level of protein-protein interactions (PPIs). The PPI framework is an assessed integrative approach (Hodgman, <xref rid="B24" ref-type="bibr">2007</xref>; Dittrich et al., <xref rid="B15" ref-type="bibr">2008</xref>; Jin et al., <xref rid="B33" ref-type="bibr">2008</xref>; Kim et al., <xref rid="B37" ref-type="bibr">2010</xref>; Iskar et al., <xref rid="B32" ref-type="bibr">2012</xref>) that has already been used in computational biology to understand diseases' pathogenesis (Huang et al., <xref rid="B27" ref-type="bibr">2009b</xref>); to implement tools for the interpretation of inferred gene and protein lists (Berger et al., <xref rid="B4" ref-type="bibr">2007</xref>; Antonov et al., <xref rid="B1" ref-type="bibr">2009</xref>); to prioritize cancer-associated genes (Wu et al., <xref rid="B96" ref-type="bibr">2012</xref>); to predict functional linkages among genes (Lehner and Lee, <xref rid="B44" ref-type="bibr">2008</xref>); to show the implication of protein networks topology in genetics, personal genomics, and therapy (Lee et al., <xref rid="B43" ref-type="bibr">2013</xref>); to implement data integration workflows showcased in obstructive nephropathy in children (Moulos et al., <xref rid="B59" ref-type="bibr">2011</xref>).</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p><bold>Data sources, subsets and number of elements of the RA map</bold>.</p></caption><table frame="hsides" rules="groups"><thead><tr><th valign="top" align="left" rowspan="1" colspan="1"><bold>Subset Id</bold>.</th><th valign="top" align="left" rowspan="1" colspan="1"><bold>Source of subset</bold></th><th valign="top" align="left" rowspan="1" colspan="1"><bold>Main dataset destination</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>No. of proteins in subset</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>Total no. of proteins in main dataset</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>No. of proteins (and PPIs) in the interactome map</bold></th><th valign="top" align="center" rowspan="1" colspan="1"><bold>No. of proteins (and PPIs) in the interactome map: main cluster</bold></th></tr></thead><tbody><tr><td valign="top" align="left" rowspan="1" colspan="1">1</td><td valign="top" align="left" rowspan="1" colspan="1">GWAS</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>Core</italic></td><td valign="top" align="center" rowspan="1" colspan="1">223</td><td valign="middle" align="center" rowspan="6" colspan="1">377</td><td valign="middle" align="center" rowspan="6" colspan="1">303 (597)</td><td valign="middle" align="center" rowspan="6" colspan="1">161 (542)</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">2</td><td valign="top" align="left" rowspan="1" colspan="1">UNIPROT</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>Core</italic></td><td valign="top" align="center" rowspan="1" colspan="1">49</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">3</td><td valign="top" align="left" rowspan="1" colspan="1">Literature review</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>Core</italic></td><td valign="top" align="center" rowspan="1" colspan="1">53</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">4</td><td valign="top" align="left" rowspan="1" colspan="1">Methylation</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>Core</italic></td><td valign="top" align="center" rowspan="1" colspan="1">37</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">5</td><td valign="top" align="left" rowspan="1" colspan="1">Exp. valid. micriob. interface</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>Core</italic></td><td valign="top" align="center" rowspan="1" colspan="1">54</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">6</td><td valign="top" align="left" rowspan="1" colspan="1">NF-κB consensus</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>Core</italic></td><td valign="top" align="center" rowspan="1" colspan="1">16</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">3A</td><td valign="top" align="left" rowspan="1" colspan="1">T cell activation pathways</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>Extended</italic></td><td valign="top" align="center" rowspan="1" colspan="1">1248</td><td valign="middle" align="center" rowspan="13" colspan="1">4709</td><td valign="middle" align="center" rowspan="13" colspan="1">3783 (24457)</td><td valign="middle" align="center" rowspan="13" colspan="1">3466 (24364)</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">3B</td><td valign="top" align="left" rowspan="1" colspan="1">Other pathways</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>Extended</italic></td><td valign="top" align="center" rowspan="1" colspan="1">283</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">3C</td><td valign="top" align="left" rowspan="1" colspan="1">Cytokines</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>Extended</italic></td><td valign="top" align="center" rowspan="1" colspan="1">1536</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">3D</td><td valign="top" align="left" rowspan="1" colspan="1">Growth and differentiation</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>Extended</italic></td><td valign="top" align="center" rowspan="1" colspan="1">472</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">3E</td><td valign="top" align="left" rowspan="1" colspan="1">Intracell signaling and TFs</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>Extended</italic></td><td valign="top" align="center" rowspan="1" colspan="1">1837</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">7</td><td valign="top" align="left" rowspan="1" colspan="1">Transcriptional RA map</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>Extended</italic></td><td valign="top" align="center" rowspan="1" colspan="1">212</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">8</td><td valign="top" align="left" rowspan="1" colspan="1">RA-miRNA reg. proteins</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>Extended</italic></td><td valign="top" align="center" rowspan="1" colspan="1">1652</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">9A</td><td valign="top" align="left" rowspan="1" colspan="1">Downreg. genes in RA</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>Extended</italic></td><td valign="top" align="center" rowspan="1" colspan="1">451</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">9B</td><td valign="top" align="left" rowspan="1" colspan="1">Upreg. genes in RA</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>Extended</italic></td><td valign="top" align="center" rowspan="1" colspan="1">210</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">10</td><td valign="top" align="left" rowspan="1" colspan="1">Inflammasomes</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>Extended</italic></td><td valign="top" align="center" rowspan="1" colspan="1">152</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">11</td><td valign="top" align="left" rowspan="1" colspan="1">Adenosine receptors</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>Extended</italic></td><td valign="top" align="center" rowspan="1" colspan="1">569</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">12</td><td valign="top" align="left" rowspan="1" colspan="1">GPCRs</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>Extended</italic></td><td valign="top" align="center" rowspan="1" colspan="1">364</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">13</td><td valign="top" align="left" rowspan="1" colspan="1">Microbiome interface</td><td valign="top" align="left" rowspan="1" colspan="1"><italic>Extended</italic></td><td valign="top" align="center" rowspan="1" colspan="1">171</td></tr></tbody></table></table-wrap></sec><sec><title>Core dataset</title><p>The CD is composed of 377 proteins retrieved from six data sources (Data Sheet 1, Tables <xref ref-type="supplementary-material" rid="SM1">S1–S6</xref>):
<list list-type="simple"><list-item><p>1) RA genome-wide association studies (GWAS) gathered and integrated from five different databases (BioGPS (Wu et al., <xref rid="B95" ref-type="bibr">2009</xref>), HuGE (Yu et al., <xref rid="B99" ref-type="bibr">2008</xref>), NHGRI, OMIM, PharmGKB (Klein et al., <xref rid="B38" ref-type="bibr">2001</xref>); see Data Sheet 1, Table <xref ref-type="supplementary-material" rid="SM1">S1</xref> for the specific query processes);</p></list-item><list-item><p>2) RA-associated proteins from the Universal Protein Resource (Uniprot) (Consortium, <xref rid="B12" ref-type="bibr">2010</xref>), retrieved using as search parameters “<italic>rheumatoid arthritis</italic>” and “<italic>human</italic>” and then manually screened (Data Sheet 1, Table <xref ref-type="supplementary-material" rid="SM1">S2</xref>);</p></list-item><list-item><p>3) Genes and proteins retrieved from a comprehensive review of the literature, in particular genes appearing in Tables <xref ref-type="table" rid="T1">1</xref>, <xref ref-type="table" rid="T2">2</xref> of Review (Mcinnes and Schett, <xref rid="B53" ref-type="bibr">2011</xref>) and cited references (Data Sheet 1, Table <xref ref-type="supplementary-material" rid="SM1">S3</xref>);</p></list-item><list-item><p>4) Genes that show epigenetic changes in relation to RA, as specified in Trenkmann et al. (<xref rid="B89" ref-type="bibr">2010</xref>); Karouzakis et al. (<xref rid="B35" ref-type="bibr">2011</xref>) (Data Sheet 1, Table <xref ref-type="supplementary-material" rid="SM1">S4</xref>);</p></list-item><list-item><p>5) Proteins that are at the interface between the host and the oral microbiome, in particular proteins experimentally known to be differentially expressed in presence of <italic>Porphyromonas Gingivalis</italic> (Zhou and Amar, <xref rid="B100" ref-type="bibr">2006</xref>), a periodontitis-causing bacterium that has been strongly linked to the insurgence of RA (Mikuls et al., <xref rid="B57" ref-type="bibr">2012</xref>; Scher et al., <xref rid="B77" ref-type="bibr">2012</xref>; Smit et al., <xref rid="B82" ref-type="bibr">2012</xref>; Bingham and Moni, <xref rid="B5" ref-type="bibr">2013</xref>; Ogrendik, <xref rid="B63" ref-type="bibr">2013</xref>; Okada et al., <xref rid="B64" ref-type="bibr">2013</xref>) (Data Sheet 1, Table <xref ref-type="supplementary-material" rid="SM1">S5</xref>);</p></list-item><list-item><p>6) The key elements of the NF-κB system, the master regulator of inflammation (Oeckinghaus et al., <xref rid="B62" ref-type="bibr">2011</xref>; Smale, <xref rid="B81" ref-type="bibr">2011</xref>; Hayden and Ghosh, <xref rid="B22" ref-type="bibr">2012</xref>) at the center of a complex regulatory interactome (Tieri et al., <xref rid="B87" ref-type="bibr">2012</xref>) prominently implicated in the onset and development of RA (Miagkov et al., <xref rid="B55" ref-type="bibr">1998</xref>; Makarov, <xref rid="B50" ref-type="bibr">2001</xref>; Feldmann et al., <xref rid="B19" ref-type="bibr">2002</xref>; Okamoto, <xref rid="B66" ref-type="bibr">2006</xref>; Roman-Blas and Jimenez, <xref rid="B72" ref-type="bibr">2006</xref>, <xref rid="B73" ref-type="bibr">2008</xref>; Simmonds and Foxwell, <xref rid="B80" ref-type="bibr">2008</xref>; Van Loo and Beyaert, <xref rid="B90" ref-type="bibr">2011</xref>): we included 16 “consensus” proteins that appear at the intersection of the three main NF-κB-related datasets described in Tieri et al. (<xref rid="B87" ref-type="bibr">2012</xref>) (Data Sheet 1, Table <xref ref-type="supplementary-material" rid="SM1">S6</xref>).</p></list-item></list></p><table-wrap id="T2" position="float"><label>Table 2</label><caption><p><bold>RA-associated proteins significantly modified upon MTX therapy release and functional annotation clustering in DAVID</bold>.</p></caption><graphic xlink:href="fcell-02-00059-i0001"/><table-wrap-foot><p>Thirty-two proteins were identified to be significantly changed by the 20 MTX target proteins' deletion (1000 permutations, adjusted p-value = 0.01). The topological measures of betweenness and stress centrality were shown to be significantly altered increased (black arrow <bold>↑</bold>) or decreased (red arrow <inline-graphic xlink:href="fcell-02-00059-i0002.jpg"/>) after knocking out the MTX target proteins. Among the listed proteins, enriched for the shown GO categories, STAT3 was found to belong to the host-microbiome interface as defined in Methods. The top 2 functional annotation clusters run on the changed proteins identified enrichment for cell death and biosynthetic process as well as nitrogen compound metabolic process (Functional Annotation Clustering Classification stringency: high, see Supplementary Data Sheet 2, Table <xref ref-type="supplementary-material" rid="SM2">S18</xref>; BC, betweenness centrality; S, stress centrality).</p></table-wrap-foot></table-wrap></sec><sec><title>Extended dataset</title><p>The <italic>extended</italic> dataset (ED, that includes CD) is composed of 4709 proteins, which are involved in a broader sense in the onset and development of RA, such as proteins participating in signaling pathways or cascades of recognized importance for RA. This extension provides a more general setting for the molecular framing of RA, and offers a larger network to operate on, with more relevant statistics and analyses, giving account for contributions coming from entities that may have been neglected or that are not experimentally related to RA, but that participate to the inception of the disease. In addition to the proteins of the <italic>core</italic> dataset, we added eight main subsets, as follows (Data Sheet 1, Tables <xref ref-type="supplementary-material" rid="SM1">S3A–E</xref>, <xref ref-type="supplementary-material" rid="SM1">S7–S13</xref>):</p><list list-type="simple"><list-item><p>3A-B-C-D-E) in retrieving data from Mcinnes and Schett (<xref rid="B53" ref-type="bibr">2011</xref>) and references cited there, we considered that some of the key proteins can be “hidden” inside the signaling pathways involved in the disease. In order to take into account such potentially important and usually neglected elements, we expanded subset 3 of CD by a pathway enrichment analysis process, using the genes listed in Mcinnes and Schett (<xref rid="B53" ref-type="bibr">2011</xref>) Tables <xref ref-type="table" rid="T1">1</xref>, <xref ref-type="table" rid="T2">2</xref>. To populate these five subsets, the selected genes have been input in the <italic>pathway over-representation analysis</italic> (ORA) tool of InnateDB, one of the most comprehensive sources of pathways data available (Lynn et al., <xref rid="B49" ref-type="bibr">2008</xref>; Breuer et al., <xref rid="B7" ref-type="bibr">2013</xref>). Pathway ORA has been performed on InnateDB using hypergeometric distribution for <italic>p</italic>-value computation and Benjamini–Hochberg correction method for multiple hypothesis testing. All the proteins participating to such over-represented pathways were then included. We retrieved respectively: 39 enriched pathways accounting for 1248 proteins (subset 3A), 14 pathways and 283 proteins (3B), 46 pathways and 1536 proteins (3C), 5 pathways and 472 proteins (3D), and 92 pathways and 1837 proteins (3E), all collected in Data Sheet 1, Tables <xref ref-type="supplementary-material" rid="SM1">S3A–E</xref>;</p></list-item><list-item><p>7) Genes derived from the transcriptional RA map in Wu et al. (<xref rid="B97" ref-type="bibr">2010</xref>) (Data Sheet 1, Table <xref ref-type="supplementary-material" rid="SM1">S7</xref>);</p></list-item><list-item><p>8) RA-related miRNA-regulated genes: experimentally validated target genes of all miRNAs that are associated to RA in the database miRWalk (Dweep et al., <xref rid="B16" ref-type="bibr">2011</xref>) (search mode: <italic>holistic view of validated disease-miRNA interactions</italic>; web reference: <ext-link ext-link-type="uri" xlink:href="http://www.umm.uni-heidelberg.de/apps/zmf/mirwalk/disease.html">http://www.umm.uni-heidelberg.de/apps/zmf/mirwalk/disease.html</ext-link>; query keywords: <italic>Arthritis</italic> AND <italic>Rheumatic diseases</italic>) (Data Sheet 1, Table <xref ref-type="supplementary-material" rid="SM1">S8</xref>);</p></list-item><list-item><p>9A,B) gene expression profiles of RA patients and healthy controls were searched on Gene Expression Omnibus (GEO, (Barrett et al., <xref rid="B3" ref-type="bibr">2011</xref>) <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/geo/">http://www.ncbi.nlm.nih.gov/geo/</ext-link>) with the query [“rheumatoid arthritis” AND “(synovi<sup>*</sup> OR blood)”] (i.e., in synovial tissue and/or blood). In order to include only highly consistent information, datasets without pre-treatment samples, with no details about the therapy and no raw data were filtered out. Human PBMCs collected and processed by Affymetrix technology were selected, leaving only one dataset out of the initial 61, GSE7524, which contains transcriptomic profiles of 2 healthy controls, 2 before and 2 after anti-TNFα treatment samples. Affymetrix Human Genome U133A Array was used to measure the expression levels of ~14,500 well-characterized human genes. The raw data were pre-processed using <italic>affy</italic> package (Gautier et al., <xref rid="B21" ref-type="bibr">2004</xref>) in R (<ext-link ext-link-type="uri" xlink:href="http://www.r-project.org/">http://www.r-project.org/</ext-link>), normalized using robust multi-array average (<italic>rma</italic>) (Irizarry et al., <xref rid="B31" ref-type="bibr">2003</xref>) and for multiple probes corresponding to the same gene, the probe with the highest standard variation across all samples was used to represent the gene. Differentially expressed genes [fold-change (Murie et al., <xref rid="B60" ref-type="bibr">2009</xref>) =2] were identified with the comparison between the 2 healthy controls and the 2 before anti-TNFα treatment samples resulting in 646 genes differentially expressed, among which 440 genes (451 proteins) were down-regulated and 206 genes (210 proteins) were up-regulated (Data Sheet 1, Tables <xref ref-type="supplementary-material" rid="SM1">S9A,B</xref>);</p></list-item><list-item><p>10) Proteins related to the inflammasome, a multiprotein oligomer responsible for activation of inflammatory processes proteins, which is also known to be activated from the bacterium <italic>P. Gingivalis</italic>, among others, and recognized to play a relevant role in RA (Sidiropoulos et al., <xref rid="B79" ref-type="bibr">2008</xref>; Kolly et al., <xref rid="B41" ref-type="bibr">2010</xref>; Farquharson et al., <xref rid="B18" ref-type="bibr">2012</xref>; Mathews et al., <xref rid="B51" ref-type="bibr">2013</xref>) (Data Sheet 1, Table <xref ref-type="supplementary-material" rid="SM1">S10</xref>). This set was retrieved using <italic>ORA</italic> as described in 3A-B-C-D-E;</p></list-item><list-item><p>11) Adenosine receptors and related proteins, known to be involved in RA (Varani et al., <xref rid="B91" ref-type="bibr">2010</xref>, <xref rid="B92" ref-type="bibr">2011</xref>; Vincenzi et al., <xref rid="B93" ref-type="bibr">2013</xref>) and possibly at the basis of the mechanism of action of methotrexate, first-line therapy for the treatment of RA (Stamp et al., <xref rid="B85" ref-type="bibr">2012</xref>) (Data Sheet 1, Table <xref ref-type="supplementary-material" rid="SM1">S11</xref>). This set was retrieved using <italic>ORA</italic> as in 3A-B-C-D-E and 10;</p></list-item><list-item><p>12) The large family of G Protein Coupled Receptors (GPCRs) (Hutchings et al., <xref rid="B29" ref-type="bibr">2010</xref>; Lozupone et al., <xref rid="B48" ref-type="bibr">2012</xref>; Maynard et al., <xref rid="B52" ref-type="bibr">2012</xref>; Tremaroli and Backhed, <xref rid="B88" ref-type="bibr">2012</xref>), pertaining to host-microbiome interface proteins (grouped in a separate set from 13 due to their numerosity), retrieved from <ext-link ext-link-type="uri" xlink:href="http://www.iuphar-db.org/DATABASE/ReceptorFamiliesForward?type=GPCR">http://www.iuphar-db.org/DATABASE/ReceptorFamiliesForward?type=GPCR</ext-link> (Sharman et al., <xref rid="B78" ref-type="bibr">2013</xref>) (Data Sheet 1, Table <xref ref-type="supplementary-material" rid="SM1">S12</xref>);</p></list-item><list-item><p>13) The set of host-microbiome interacting proteins, manually curated from recent reviews (Lozupone et al., <xref rid="B48" ref-type="bibr">2012</xref>; Maynard et al., <xref rid="B52" ref-type="bibr">2012</xref>; Tremaroli and Backhed, <xref rid="B88" ref-type="bibr">2012</xref>), to describe the bridge between innate immunity (altered in RA) and the GI microbiome [known to be involved in immune diseases in general and in RA in particular (Scher and Abramson, <xref rid="B75" ref-type="bibr">2011</xref>)]. Globally this dataset accounts for the Toll-like Receptor family (TLRs), the mucin proteins family, selected Immunoglobulins (Ig) and their receptors, among others (Data Sheet 1, Table <xref ref-type="supplementary-material" rid="SM1">S13</xref>).</p></list-item></list><p>Datasets are integrated at the PPI level as peers to avoid introducing any bias <italic>a priori</italic> in the network construction and to warrant that these data are connected in a biologically meaningful way. Protein-protein interactions were retrieved in Cytoscape from the Agile Protein Interaction DataAnalyzer database (APID, Prieto and De Las Rivas, <xref rid="B71" ref-type="bibr">2006</xref>) that includes all known experimentally validated protein-protein interactions from BIND, BioGRID, DIP, HPRD, IntAct and MINT databases, accessed via the APID2NET (Hernandez-Toro et al., <xref rid="B23" ref-type="bibr">2007</xref>) plugin. This process lead to the definitions of, respectively, the core interactome (<italic>CI</italic>, 303 proteins, 597 interactions, high resolution Image <xref ref-type="supplementary-material" rid="SM4">S1</xref>) and the extended interactome (<italic>EI</italic>, 3783 proteins, 24457 interactions, high resolution Image <xref ref-type="supplementary-material" rid="SM5">S2</xref>). Discussion on caveats and choices of original sources can be found in Tieri and Nardini (<xref rid="B86" ref-type="bibr">2013</xref>).</p></sec><sec><title>Topological analysis</title><p>Topological analysis was run separately on the main <italic>connected component</italic> of each interactome (i.e., excluding the proteins for which no PPI was retrieved, i.e., that remained isolated) to evaluate a number of network parameters (Assenov et al., <xref rid="B2" ref-type="bibr">2008</xref>): <italic>degree</italic>, or <italic>connectivity</italic>, i.e., the number of nodes linked to the node of interest (number of edges); and <italic>betweenness centrality</italic> (BC), a measure of the amount of control that a node exerts over the interactions of other nodes in the network. This measure favors nodes that join communities such as dense subnetworks, rather than nodes that lie inside a community, and has been shown to characterize essential proteins (Platzer et al., <xref rid="B70" ref-type="bibr">2007</xref>). All calculated network parameters and rankings are listed in Data Sheet 2, Tables <xref ref-type="supplementary-material" rid="SM2">S15</xref>, <xref ref-type="supplementary-material" rid="SM2">S16</xref> or can be recalculated from the Cytoscape CI_EI.cys (Data Sheet <xref ref-type="supplementary-material" rid="SM3">3</xref>) file available at <ext-link ext-link-type="uri" xlink:href="http://www.picb.ac.cn/ClinicalGenomicNTW/RAmultiomic.html">http://www.picb.ac.cn/ClinicalGenomicNTW/RAmultiomic.html</ext-link>.</p></sec><sec><title>Pharmacological treatment simulation</title><p>To simulate the pharmacological treatment, a virtual node knockout experiment has been performed by controlling (manual removal of the nodes and Cytoscape plugin <italic>Interference</italic> (Scardoni et al., <xref rid="B42" ref-type="bibr">2014</xref>) 20 MTX controlled targets identified in literature (Cutolo et al., <xref rid="B13" ref-type="bibr">2001</xref>; Chan and Cronstein, <xref rid="B8" ref-type="bibr">2002</xref>) present in EI (Data Sheet 2, Table <xref ref-type="supplementary-material" rid="SM2">S17</xref>). Betweenness centrality (and, to add robustness to the analysis, stress, S, i.e., an alternative centrality functional form) were then re-calculated to assess the impact of such therapy on the topology and hence the functionality of the network. Manual node removal and pharmacological simulation plugin present overlapping results (<italic>betweenness</italic>: 95.9%, <italic>stress</italic>: 98.2%, Data Sheet 2, Table <xref ref-type="supplementary-material" rid="SM2">S17</xref>). The <italic>p</italic>-values, corrected for multiple testing (threshold 0.05), have been calculated after constructing null betweenness centrality distributions by 1000 random deletions of 20 nodes, as many as the MTX targets (Efron and Tibshirani, <xref rid="B17" ref-type="bibr">1993</xref>). Functional clustering analysis has been then performed (Data Sheet 2, Table <xref ref-type="supplementary-material" rid="SM2">S18</xref>).</p></sec><sec><title>Comparative analysis</title><p>We further run a comparative analysis between our newly constructed multi-<italic>omic</italic> map, EI, and TR, that represent an earlier transcriptional-only version (Wu et al., <xref rid="B97" ref-type="bibr">2010</xref>), to highlight the biological mechanisms that have been better emphasized from the usage of multilayer <italic>omic</italic> data.</p><p><italic>Degree</italic> was evaluated as the number of edges attached to a node for the undirected networks as EI (and CI) are (i.e., connections among nodes do not indicate <italic>directional</italic> cause-effect nor temporal relationship). For TR (directed network) proteins and their modified instances (such as MAPKs and phosphorylated-MAPKs) were first considered as one (complex) node, then in-degrees (edges <italic>to</italic> the node) and out-degrees (edges <italic>from</italic> the node) of the components (MAPK and phosphorylated-MAPK) were summed up to obtain the undirected degree, after subtracting the number of edges connecting the members of the complex node. To complete the compatibility of the degree defined for undirected maps (and namely EI), given the different sizes of EI and TR, the percentrank of the degree was also computed. The nodes which degree rank was modified by more than 10% between the two networks, were considered as nodes undergoing a <italic>transition</italic>. A node was defined as <italic>accomplished</italic> when its % rank degree was preserved, <italic>loser</italic> when the ranking reduced from TR to EI, <italic>climber</italic> when it increased from TR to EI (Data Sheet 2, Table <xref ref-type="supplementary-material" rid="SM2">S19</xref>). From a strictly topological point of view, the threshold that defines a node as <italic>accomplished</italic> can be set to zero, and hence this definition identifies only the nodes with the same exact degree. From a biological standpoint, and for an informative biological interpretation of the results, it is not necessary to impose the exact matching of the ranking. For this reason we relaxed the threshold and defined as accomplished the nodes that present the same, higher or lower % rank of the degree with ±10% tolerance, as a reasonable compromise.</p><p>Biological meaning for <italic>climbers</italic> and <italic>accomplished</italic> nodes in the transition TR to EI was assessed by enrichment analysis Enrichr (Chen et al., <xref rid="B10" ref-type="bibr">2013</xref>) see Data Sheet 2, Table <xref ref-type="supplementary-material" rid="SM2">S20</xref>.</p></sec></sec><sec><title>Results and discussion</title><p>After curating all molecular information (Table <xref ref-type="table" rid="T1">1</xref>) we inferred the network from the reconstructed lists with the PPI approach, which consists of connecting nodes (molecules) based on their interactions at the protein level, a broadly assessed approach in computational biology, and already used for RA in both already cited (Okada et al., <xref rid="B65" ref-type="bibr">2014</xref>; You et al., <xref rid="B98" ref-type="bibr">2014</xref>). All following results pertain to the analysis on the extended interactome (EI), more informative for its larger size.</p><p>To validate the ability of our network to model the biomolecular aspects of RA, we first simulated a therapeutic approach with MTX (see methods) and compared the results with the major known effects reported in literature (Figure <xref ref-type="fig" rid="F1">1A</xref>). As a result of the control on 20 MTX targets removal, the network changes its topology (Figure <xref ref-type="fig" rid="F1">1B</xref>; Data Sheet 2, Table <xref ref-type="supplementary-material" rid="SM2">S17</xref>), and the functional analysis indicates that 32 molecules which BC significantly altered (Data Sheet 2, Table <xref ref-type="supplementary-material" rid="SM2">S17</xref>, col. 2) pertain to two main functions [Data Sheet 2, Table <xref ref-type="supplementary-material" rid="SM2">S18</xref>, DAVID (Huang et al., <xref rid="B26" ref-type="bibr">2009a</xref>)]: regulation of programmed cell death, a known effect of MTX (Spurlock et al., <xref rid="B83" ref-type="bibr">2011</xref>); and metabolic and biosynthetic processes, an alteration known to constitute a side effect of the treatment (Phillips et al., <xref rid="B69" ref-type="bibr">2003</xref>), as well as an area of synergy between host and microbiome (Tremaroli and Backhed, <xref rid="B88" ref-type="bibr">2012</xref>; Devaraj et al., <xref rid="B14" ref-type="bibr">2013</xref>; Winter et al., <xref rid="B94" ref-type="bibr">2013</xref>). Moving down to the gene level, as illustrated in Table <xref ref-type="table" rid="T2">2</xref>, Signal Transducers and Activators of Transcription 3 (STAT3) deserves particular attention, as it is a crucial player in the JAK/STAT signaling cascade, at the basis of the signal transduction mechanism for many cytokine receptors, highly activated in RA (Paunovic et al., <xref rid="B68" ref-type="bibr">2008</xref>), and an important member of the host-microbiome interface (Zhou and Amar, <xref rid="B100" ref-type="bibr">2006</xref>), being involved in the host susceptibility/defense against intestinal infections at the mucosal level (Miettinen et al., <xref rid="B56" ref-type="bibr">2000</xref>).</p><fig id="F1" position="float"><label>Figure 1</label><caption><p><bold>(A)</bold> Snapshot of the extended interactome (EI) with nodes highlighted by betweenness centrality (BC), high resolution browsable figure provided in Supplementary Files (Image <xref ref-type="supplementary-material" rid="SM5">S2</xref>). <bold>(B)</bold> Zoom on the top ranking BC node (GRB2) and its closer interactome. Pathways relevant in the indication of GRB2 as an RA target, able to control inflammation TGF-β (TGFB1-3), TNF-α (TNF, TNFRS10C), MAPK (MAP4K1, MAPK3), degeneracy EMT (TWIST1-2, CDH1), and dysbiosis (TRL4) are also highlighted. <bold>(C)</bold> Visual summary of the influence of GRB2 on the RA-affected districts highlight a homeostatic (blue) influence on inflammation, GI microbiome, growth, differentiation. The pie-chart slices' size is proportional to the number of molecules considered in each district. Districts were merged from the total 13 datasets according to biochemical homogeneity in the following 8 categories: Genomic (DNA, Dataset 1); Epigenomic (mDNA, Dataset 4); Transcriptomic (mRNA, Datasets 7, 9A, 9B); Post-transcriptomic (miRNA, Dataset 8); Proteomic (proteins, Dataset 2); Microbiome (Host-microbiome proteins interface, Oral microbiome Datasets 5, 10, 12, 13); Inflammation (6, 3A, 3B, 3C); Others, i.e., Growth, Differentiation (Datasets 3, 11, 3D, 3E).</p></caption><graphic xlink:href="fcell-02-00059-g0001"/></fig><p>From a topological point of view, STAT3 presents enhanced <italic>betweenness</italic> and reduced <italic>stress centralities</italic> after virtual MTX treatment. This is an unusual topological condition—since there is commonly correlation between stress and betweenness—where, upon perturbation (MTX) a higher fraction of shortest paths converges on STAT3 (gain in <italic>betweenness centrality</italic>) despite a decrease in their absolute number (loss of <italic>stress centrality</italic>). This indicates that the networks shrinks and STAT3 becomes more important, a fact that can be translated in biological terms as the compensatory mechanisms induced by the loss of some molecules' presence/activity (MTX targets), which globally force STAT3 to become the molecule through which more numerous (higher <italic>betweenness</italic>) but less efficient molecular reactions (longer paths, lower <italic>stress</italic>) occur.</p><p>Overall, STAT3, which is already considered a crucial target in RA for its critical role in the T regulatory/helper 17 lymphoid cells [T<sub>reg</sub>/Th<sub>17</sub> balance overabundant in RA (Leipe et al., <xref rid="B45" ref-type="bibr">2010</xref>)] is coherently shown as an indirectly controlled target by MTX explaining the ability of the therapy to rebalance Th17/IL17 ratio (Li et al., <xref rid="B46" ref-type="bibr">2012</xref>).</p><p>In conclusion, our map is able to recollect known and yet complex information about the effects of MTX, this represents an important validation of our frame for further simulations. Additionally, our map indicates a clear link between MTX and dysbiosis, which to date has not been explicitly unrevealed, although enterocolitis is a known toxic effect of MTX, linked to the induced nitroxidative stress (Kolli et al., <xref rid="B39" ref-type="bibr">2008</xref>, <xref rid="B40" ref-type="bibr">2013</xref>). This is a critical fact as the known adverse effects of MTX, generally described as immunodepressive, appear to be composed not only by the known oxidative organ stress, but also by an added dysbiosis, possibly mediated by an overload on STAT3.</p><p>The topological analysis highlights the striking relevance of Growth factor receptor-bound protein 2 (GRB2) with values of <italic>BC</italic> more than two-fold (Data Sheet 2, Table <xref ref-type="supplementary-material" rid="SM2">S16</xref>) compared to the second in rank, the Epidermal growth factor receptor (EGFR). Based on literature, GRB2 is an effective target (Phase I clinical trial, <ext-link ext-link-type="uri" xlink:href="http://www.biopathholdings.com/">http://www.biopathholdings.com/</ext-link>) for Acute Myeloid Leukemia (AML), Chronic myelogenous leukemia (CML) and Myelodysplastic syndromes (MDS); an important mediator of the oncogenic activities of TGF-β, via epithelial mesenchymal transition (EMT) (Galliher-Beckley and Schiemann, <xref rid="B20" ref-type="bibr">2008</xref>); a crucial player in the host-microbiome interaction of <italic>Helicobacter pylori</italic>, able to induce host cell scattering and proliferation via the activation of the Ras/MEK/ERK pathway (Mimuro et al., <xref rid="B58" ref-type="bibr">2002</xref>); a marker of RA in synoviocites (Huh et al., <xref rid="B28" ref-type="bibr">2003</xref>). GRB2 is additionally activated by leptin (Pai et al., <xref rid="B67" ref-type="bibr">2005</xref>), abundant in RA (Bokarewa et al., <xref rid="B6" ref-type="bibr">2003</xref>) and able to increase <italic>Prevotella intermedia</italic> LPS-induced TNF-α production (Kim, <xref rid="B36" ref-type="bibr">2010</xref>). Moreover, another member of the <italic>Prevotella</italic> genus (<italic>P. copri</italic>) has recently been liaised to RA (Scher et al., <xref rid="B76" ref-type="bibr">2013</xref>), as a specific marker of GI microbiome dysbiosis associated to the disease. When observed from the network perspective this apparently scattered information fits in a connected map (Figure <xref ref-type="fig" rid="F1">1B</xref>) and hence builds a robust rationale for considering GRB2 as a target for RA. The activation of proliferative and inflammatory pathways as well as EMT, are hallmarks of RA (You et al., <xref rid="B98" ref-type="bibr">2014</xref>) suggesting that the control on GRB2 as a regulator of such mechanisms is appropriate. Additionally, the control on GRB2 exerted by <italic>H. pylori</italic> [already proposed in relation to RA (Melby et al., <xref rid="B54" ref-type="bibr">1999</xref>)] and by <italic>P. intermedia</italic> in the presence of leptin indicate that targeting of GRB2 is not only of relevance to control the phenotypic symptoms of RA (joints degeneracy) but also the recently highlighted dysbiosis that accompany the disease, via the control of the disruptive mechanisms by which pathogens can exert their action on the host (Figure <xref ref-type="fig" rid="F1">1C</xref>).</p><p>Given the relevance of RA as a paradigmatic autoimmune disease, a variety of <italic>in silico</italic> modeling approaches have been devised (Okada et al., <xref rid="B65" ref-type="bibr">2014</xref>; You et al., <xref rid="B98" ref-type="bibr">2014</xref>), and, among those, an early transcriptional only map (hereinafter TR, 302 nodes; Wu et al., <xref rid="B97" ref-type="bibr">2010</xref>). The previous compilation of this simplified version put us in the relatively unique position to be able to quantify the benefit, in terms of information content, of expanding from transcriptional to multi-omic the network modeling of RA. The molecules that gain importance (i.e., have a higher degree) in the multi-<italic>omic</italic> map versus the TR (<italic>climbers</italic>, see Methods and Figure <xref ref-type="fig" rid="F2">2A</xref>) pertain mostly to the MAPK Signaling Pathway (Figure <xref ref-type="fig" rid="F2">2B</xref> and Data Sheet 2, Table <xref ref-type="supplementary-material" rid="SM2">S19</xref>). This category is also highly enriched for <italic>accomplished</italic> nodes, thus validating the importance of this pathway in the disease. However, <italic>climbers</italic>, all representing genes shared between TR and EI, include molecules known to belong also to the GI interface (SFR, MAP2K4, MAP3K8), absent in the <italic>accomplished</italic>, implying the importance of the involvement of the host-microbiome interface, not taken into account in the TR map. In particular, Interleukin-1 Receptor Associated Kinase-4 (IRAK4, <italic>climber</italic>) is known to play a critical role in initiating response to foreign pathogens (Hofman and Vouret-Craviari, <xref rid="B25" ref-type="bibr">2012</xref>) and was recently presented to the American College of Rheumatology (ACR), based on promising results on the control of B-cell-like diffuse large B-cell lymphoma (DLBCL), as a potential treatment for RA (Chaudahry and Al, <xref rid="B9" ref-type="bibr">2012</xref>). In the network perspective, this choice calls for words of cautions. Indeed, while correlating with regression of some aspects of the disease, the control on IRAK4 affects the response to pathogens, and in particular IRAK4 inhibitors impacts on pDCs in RA patients (Chiang et al., <xref rid="B11" ref-type="bibr">2011</xref>), therefore limiting the appropriate and immediate innate host response in case of bacterial infections (Figure <xref ref-type="fig" rid="F2">2C</xref>).</p><fig id="F2" position="float"><label>Figure 2</label><caption><p><bold>(A)</bold> Multi-<italic>omic</italic> map (EI) nodes highlighted according to their role in comparison with a transcriptional-only map (TR). In orange, nodes that maintain their role and importance in both EI and TR (<italic>accomplished</italic>); in red, nodes that gain importance in the multi-<italic>omic</italic> context, (<italic>climbers</italic>). <bold>(B)</bold> Functional analysis of the climber hubs, which highlight the striking significance of MAPK signals. Panel <bold>(C)</bold> is built in the same way of Figure <xref ref-type="fig" rid="F1">1C</xref> to permit easy comparison of the two targets. It represents the summary of the influence of IRAK4 on the RA-affected districts, and highlights a homeostatic (blue) influence on inflammation, growth, differentiation as well as transcriptomic and post-transcriptomic districts. However, the microbiome interface response is impaired by IRAK4 inhibition of the innate immune response to pathogens. The pie-chart slices' size is proportional to the number of molecules considered in each district (as in Figure <xref ref-type="fig" rid="F1">1</xref>).</p></caption><graphic xlink:href="fcell-02-00059-g0002"/></fig></sec><sec sec-type="conclusion" id="s3"><title>Conclusion</title><p>The aim of the designed framework is to draw hypotheses that can support basic research and further clinical practice. In particular, we here highlight two major areas of application: support in the identification of novel drug targets (exemplified by GRB2); support in the identification of potential contraindication to novel therapies, i.e., support in the design of robust clinical trials (exemplified by IRAK4-inhibitors). While the former application joins other efforts in different clinical areas [such as on diabetes (Liu et al., <xref rid="B47" ref-type="bibr">2007</xref>; Santiago and Potashkin, <xref rid="B74" ref-type="bibr">2013</xref>), in cancer (Hwang et al., <xref rid="B30" ref-type="bibr">2013</xref>), and on glioblastoma (Junhua et al., <xref rid="B34" ref-type="bibr">2012</xref>)], the latter descends from the inclusion of numerous data types, including for the first time to our knowledge, the GI microbiome interface. The results discussed in this article are the output of the knowledge distilled from ~4000 selected molecules and ~15 public databases, a humongous amount of information carefully and often redundantly peer-reviewed by the scientific community. Future and ongoing research and the resulting discoveries will impact on the breadth and possibly on the topology of our map. To take into account these expected (and desirable) events, our map was drawn using open source programs and pathway molecules' standards to allow full map usability, editing and updating by the whole scientific community.</p></sec><sec><title>Author contribution</title><p>Paolo Tieri built and analyzed the map; XiaoYuan Zhou performed pharmacological simulation; XiaoYuan Zhou and Lisha Zhu run functional and comparative analyses; Christine Nardini analyzed the connection to the GI microbiome; Paolo Tieri and Christine Nardini designed the study, analyzed the results and wrote the manuscript; XiaoYuan Zhou and Lisha Zhu contributed to write and revise the manuscript.</p></sec><sec><title>Data sharing statement</title><p>All data are available publicly, our map is publicly available here: <ext-link ext-link-type="uri" xlink:href="http://www.picb.ac.cn/ClinicalGenomicNTW/RAmultiomic.html">http://www.picb.ac.cn/ClinicalGenomicNTW/RAmultiomic.html</ext-link></p><sec><title>Conflict of interest statement</title><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p></sec></sec> |
T lymphocytes are not required for the development of fatty degeneration after rotator cuff tear | <sec><title>Objectives </title><p>Rotator cuff tears are among the most common and debilitating
upper extremity injuries. Chronic cuff tears result in atrophy and
an infiltration of fat into the muscle, a condition commonly referred
to as ‘fatty degeneration’. While stem cell therapies hold promise
for the treatment of cuff tears, a suitable immunodeficient animal
model that could be used to study human or other xenograft-based
therapies for the treatment of rotator cuff injuries had not previously
been identified.</p></sec><sec><title>Methods </title><p>A full-thickness, massive supraspinatus and infraspinatus tear
was induced in adult T-cell deficient rats. We hypothesised that,
compared with controls, 28 days after inducing a tear we would observe
a decrease in muscle force production, an accumulation of type IIB
fibres, and an upregulation in the expression of genes involved
with muscle atrophy, fibrosis and inflammation.</p></sec><sec><title>Results </title><p>Chronic cuff tears in nude rats resulted in a 30% to 40% decrease
in muscle mass, a 23% reduction in production of muscle force, and
an induction of genes that regulate atrophy, fibrosis, lipid accumulation,
inflammation and macrophage recruitment. Marked large lipid droplet
accumulation was also present.</p></sec><sec><title>Conclusions </title><p>The extent of degenerative changes in nude rats was similar to
what was observed in T-cell competent rats. T cells may not play
an important role in regulating muscle degeneration following chronic
muscle unloading. The general similarities between nude and T-cell
competent rats suggest the nude rat is likely an appropriate preclinical
model for the study of xenografts that have the potential to enhance
the treatment of chronically torn rotator cuff muscles.</p><p>Cite this article: <italic>Bone Joint Res </italic>2014;3:262–72.</p></sec> | <contrib id="c1" contrib-type="author"><name><surname>Gumucio</surname><given-names>J.</given-names></name><degrees>BS</degrees><role>PhD
Student</role><xref ref-type="aff" rid="a1">1</xref></contrib><contrib id="c2" contrib-type="author"><name><surname>Flood</surname><given-names>M.</given-names></name><degrees>BS</degrees><role>Research
Associate</role><xref ref-type="aff" rid="a1">1</xref></contrib><contrib id="c3" contrib-type="author"><name><surname>Harning</surname><given-names>J.</given-names></name><degrees>BS</degrees><role>Undergraduate
Student</role><xref ref-type="aff" rid="a1">1</xref></contrib><contrib id="c4" contrib-type="author"><name><surname>Phan</surname><given-names>A.</given-names></name><degrees>BS</degrees><role>Undergraduate
Student</role><xref ref-type="aff" rid="a1">1</xref></contrib><contrib id="c5" contrib-type="author"><name><surname>Roche</surname><given-names>S.</given-names></name><degrees>BSc</degrees><role>Research
Associate</role><xref ref-type="aff" rid="a1">1</xref></contrib><contrib id="c6" contrib-type="author"><name><surname>Lynch</surname><given-names>E.</given-names></name><degrees>MS</degrees><role>Research
Associate</role><xref ref-type="aff" rid="a1">1</xref></contrib><contrib id="c7" contrib-type="author"><name><surname>Bedi</surname><given-names>A.</given-names></name><degrees>MD</degrees><role>Assistant
Professor</role><xref ref-type="aff" rid="a1">1</xref></contrib><contrib id="c8" contrib-type="author"><name><surname>Mendias</surname><given-names>C.</given-names></name><degrees>PhD, ATC</degrees><role>Assistant
Professor</role><xref ref-type="aff" rid="a1">1</xref></contrib><aff id="a1"><label>1</label>University of Michigan, Department
of Orthopaedic Surgery, 109 Zina Pitcher Place, BSRB 2017, Ann
Arbor, Michigan, 48109-2200, USA.</aff> | Bone & Joint Research | <sec><title>Article focus </title><list list-type="bullet"><list-item><p>To assess the role of T lymphocytes in the development of fatty
degeneration following rotator cuff tear</p></list-item><list-item><p>To evaluate the viability of an immunodeficient rodent model
that could be used to study xenograft-based therapies for the treatment
of rotator cuff tears</p></list-item></list></sec><sec><title>Key messages </title><list list-type="bullet"><list-item><p>T lymphocytes are dispensable for the onset of lipid infiltration
and muscle atrophy following rotator cuff tear</p></list-item><list-item><p>The NIH nude rat is an attractive model for the study of xenograft-based
therapies for enhancing the treatment of rotator cuff tears</p></list-item></list></sec><sec><title>Strengths and limitations </title><list list-type="bullet"><list-item><p>Strength: Detailed analysis of changes in muscle contractility,
muscle fibre architecture, lipid infiltration, and macrophage infiltration
in a rodent model of rotator cuff tear.</p></list-item><list-item><p>Limitation: We only measured at one time point of one month and
did not evaluate changes at other time points</p></list-item></list></sec><sec><title>Introduction</title><p>Rotator cuff tears are among the most frequent and debilitating
upper extremity injuries, with over 250 000 surgical repairs performed
each year in the United States.<sup><xref rid="r1" ref-type="bibr">1</xref></sup> While
there have been notable improvements in surgical repair and rehabilitation
techniques, many patients continue to have symptoms after repair,
and re-tear rates for surgical repair of full-thickness tears remain unacceptably
high.<sup><xref rid="r2" ref-type="bibr">2</xref></sup> Tears
of the supraspinatus are most common, followed by the infraspinatus
tendon, and less involved are the subscapularis and teres minor
tendons.<sup><xref rid="r3" ref-type="bibr">3</xref></sup> A
set of common pathological changes occur in torn rotator cuff muscles
including atrophy of muscle fibres, fibrosis, and an accumulation
of lipid within and around muscle fibres.<sup><xref rid="r3" ref-type="bibr">3</xref></sup> These changes are commonly referred
to as ‘fatty degeneration’ and despite successful surgical repair
of the tear, fatty degeneration often does not improve after repair
and correlates with poor functional outcomes.<sup><xref rid="r4" ref-type="bibr">4</xref></sup> The cellular and
molecular aetiology of fatty degeneration is not fully understood,
and gaining greater insight into the physiological processes that
regulate muscle fibre atrophy, fibrosis and lipid accumulation will
likely improve the treatment of patients with chronic rotator cuff
tears.</p><p>The immune system plays an important role in the regeneration
of muscle after injury.<sup><xref rid="r5" ref-type="bibr">5</xref></sup> Macrophages,
which arise from monocytes produced in bone marrow, are the most
well studied members of the leukocyte family that participate in
muscle regeneration.<sup><xref rid="r5" ref-type="bibr">5</xref></sup> There
are two general classes of macrophages that participate in muscle
regeneration. After muscle injury, pro-inflammatory M1 macrophages
rapidly enter muscle tissue and participate in phagocytosis of muscle
debris, and also secrete cytokines and produce nitric oxide, which
promote muscle degeneration.<sup><xref rid="r6" ref-type="bibr">6</xref></sup> Anti-inflammatory
M2 macrophages have a slower entry into injured muscle, and secrete
anti-inflammatory cytokines that attenuate M1 macrophages and can
promote muscle fibre regeneration.<sup><xref rid="r6" ref-type="bibr">6</xref></sup> T lymphocytes, which arise from haematopoietic
stem cells in the bone marrow and mature in the thymus, are thought
to play an important role in coordinating the inflammation and regeneration
of muscle after injury, through direct interactions with muscle
fibres, as well as indirectly through the regulation of M1 and M2
macrophages.<sup><xref rid="r7" ref-type="bibr">7</xref>-<xref rid="r9" ref-type="bibr">9</xref></sup> T lymphocytes are
also recruited into adipose tissue in obese individuals and contribute
to obesity-related increases in local and systemic inflammation
through direct interactions with adipocytes or via the recruitment
of M1 macrophages.<sup><xref rid="r10" ref-type="bibr">10</xref></sup> A
similar response has been observed in atherosclerotic plaques, where
T lymphocytes directly promote local inflammation and lipid accumulation
through the secretion of pro-inflammatory cytokines and the activation
of M1 macrophages.<sup><xref rid="r9" ref-type="bibr">9</xref></sup> Additionally,
diseases that result in T cell depletion, like HIV, may increase
the susceptibility to developing a rotator cuff tear.<sup><xref rid="r11" ref-type="bibr">11</xref></sup> While T lymphocytes
have a clear role in promoting inflammation and lipid accumulation
in several tissues, the role that these cells play in orchestrating the
response of the rotator cuff to a tear was not known. To address
the role of T lymphocytes in fatty degeneration of a rotator cuff,
we used the NIH nude rat (NIH-<italic>Foxn1</italic><sup>rnu</sup>). This
outbred line of rats has a naturally occurring mutation in the <italic>Foxn1</italic> gene,
which results in the failure to form a mature thymus and subsequent
lack of mature T lymphocytes, although monocytes and B cells are
still present.<sup><xref rid="r12" ref-type="bibr">12</xref>-<xref rid="r14" ref-type="bibr">14</xref></sup>
</p><p>There has been much interest in the use of stem cell therapy
to augment the healing of torn rotator cuff muscles.<sup><xref rid="r15" ref-type="bibr">15</xref></sup> Mesenchymal stem
cells have the ability to differentiate into multiple cell types
that may be helpful for the treatment of chronic rotator cuff tears,
including skeletal muscle, tendon, cartilage and bone.<sup><xref rid="r16" ref-type="bibr">16</xref></sup> While animal studies
using autologous cells can be informative, evaluating human derived
stem cells in animal models of injury and disease is also important
in identifying safe and effective stem cell therapies.<sup><xref rid="r17" ref-type="bibr">17</xref></sup> The lack of T
lymphocytes allows nude rats to accept xenografts and to potentially
serve as models to study cell-based therapies in specific injuries and
disease states.<sup><xref rid="r14" ref-type="bibr">14</xref></sup></p><p>To gain a greater understanding of the role that T lymphocytes
play in fatty degeneration of a rotator cuff, and to evaluate the
suitability of an immune deficient rodent model that could be used
to study xenograft-based therapies for the treatment of rotator
cuff injuries, we used a well established chronic experimental technique
of a full-thickness rotator cuff tear<sup><xref rid="r18" ref-type="bibr">18</xref>-<xref rid="r20" ref-type="bibr">20</xref></sup> in
a group of adult NIH nude rats. We determined changes in muscle
fibre contractility, type of muscle fibre, distribution and size,
and the expression of messenger RNA (mRNA) and microRNAs (miRNAs)
involved in muscle atrophy, inflammation, lipid synthesis and storage, extracellular
matrix synthesis and fibrosis, and macrophage accumulation in nude
rats that underwent a full-thickness supraspinatus and infraspinatus
tear. We tested the hypothesis that 28 days following a rotator cuff
tear in nude rats, there would be a reduction in muscle-specific
fibre force production and an induction in the expression of mRNA
and miRNA transcripts that regulate atrophy, autophagy, inflammation,
lipid accumulation and fibrosis.</p></sec><sec sec-type="materials|methods"><title>Materials and Methods</title><sec><title content-type="h3">Animals</title><p>This study was approved by the University of Michigan Committee
for the Use and Care of Animals. Four-month-old male athymic nude
rats (NIH-<italic>Foxn1</italic><sup>rnu</sup>) (n = 5) were obtained
from Charles River Laboratories (Wilmington, Massachusetts). We
selected this sample size based upon a power analysis of specific
force data from a previous study.<sup><xref rid="r18" ref-type="bibr">18</xref></sup> Rats were housed in specific pathogen-free
conditions and provided food and water <italic>ad libidum</italic>.
Animal handling and care was performed in accordance with the NIH
Policy on Humane Care and Use of Laboratory Animals. </p></sec><sec><title content-type="h3">Surgeries</title><p>Full-thickness tears of the right supraspinatus and infraspinatus
were performed as previously described,<sup><xref rid="r18" ref-type="bibr">18</xref>,<xref rid="r21" ref-type="bibr">21</xref></sup> with
the left supraspinatus and infraspinatus muscles serving as sham-operated,
intact controls. We chose these muscles, rather than the subscapularis
and teres minor, due to the higher frequency of injury in humans.<sup><xref rid="r3" ref-type="bibr">3</xref></sup> The skin around
the shoulder was thoroughly scrubbed with ChloraPrep (CareFusion),
and a full-thickness tenectomy of the right supraspinatus and infraspinatus
tendons was performed through a deltoid-splitting transacromial
approach. This technique simulates a massive rotator cuff tear and
prevents scarring and healing of detached tendons that can occur
in rodent models.<sup><xref rid="r19" ref-type="bibr">19</xref></sup> The left
shoulder served as a sham-operated control in which a deltoid-splitting
surgery was performed, but the rotator cuff tendons were left intact.
A splash block of 0.5% bupivicaine with 1:100 000 epinephrine was
administered. The deltoid was closed using an absorbable 5-0 chromic
gut suture (Johnson and Johnson, New Brunswick, New Jersey) and
the skin was closed using 5-0 nylon (Johnson and Johnson) and GLUture
(Abbott Laboratories, Abbott Park, Illinois). Following surgery,
a single dose of ampicillin (20 mg/kg, subcutaneous) and carprofen
(5 mg/kg, subcutaneous) was administered. Buprenorphine (0.05 mg/kg)
was administered for analgesia during the post-operative period.
Free cage activity and weight-bearing were allowed, and rats were
closely monitored for signs of distress or infection. A total of
28 days after surgery, rats were anaesthetised with sodium pentobarbital
(50 mg/kg), and the supraspinatus and infraspinatus muscles were harvested.
The distal ends of all muscles were free and no signs of lateral
adhesion formation were present. Rats were then humanely euthanised
with pentobarbital overdose and induction of pneumothorax. Supraspinatus
muscles were weighed, finely minced, and immediately prepared for
RNA isolation. Infraspinatus muscles were weighed, and separated
at the mid-belly for histology and single fibre contractility.</p></sec><sec><title content-type="h3">Histology and immunohistochemistry</title><p>Distal segments of the infraspinatus were frozen in Tissue-Tek
(Sakura, Torrance, California) using isopentane cooled liquid nitrogen,
and stored at -80ºC until use. Muscles were cryosectioned at a thickness
of 10 µm, and prepared for staining with Oil red O and haematoxylin
or prepared for immunohistochemistry (IHC).<sup><xref rid="r17" ref-type="bibr">17</xref>,<xref rid="r18" ref-type="bibr">18</xref></sup> Distribution
of fibre type was determined by labeling sections with antibodies against
myosin heavy chain type I (DSHB, Iowa City, Iowa), myosin heavy
chain type IIA (DSHB) and myosin heavy chain type IIB (DSHB). The
extracellular matrix (ECM) was identified using wheat germ agglutinin
(WGA) lectin conjugated to AlexaFluor 488 (Invitrogen, Grand Island,
New York). Type IIX fibres were detected by the absence of fluorescent
signal. To identify macrophages and areas of lipid accumulation,
slides were incubated with antibodies against the macrophage surface
antigen F4/80 (AbCam, Cambridge, Massachusetts) and the neutral
lipid stain BODIPY 493/503 (Invitrogen). Primary antibodies were
detected using highly cross-adsorbed secondary antibodies conjugated
to AlexaFluor fluorescent probes (Invitrogen). Images were obtained
using an Axioplan 2 (Zeiss, Jena, Germany) microscope equipped with
AxioCam (Zeiss) cameras. Quantitative histomorphometry was performed
using ImageJ software (NIH, Bethesda, Maryland).</p></sec><sec><title content-type="h3">Muscle fibre contractility</title><p>Contractile measurements of permeabilised single muscle fibres
from the proximal infraspinatus were performed as described.<sup><xref rid="r18" ref-type="bibr">18</xref>,<xref rid="r22" ref-type="bibr">22</xref></sup> A total of ten to 20 type II fibres
were tested from each infraspinatus muscle.</p></sec><sec><title content-type="h3">Gene expression</title><p>Samples of supraspinatus muscle were homogenised in Qiazol (Qiagen,
Valencia, California) and separated using chloroform. Total RNA
was further purified from the aqueous phase of the homogenised solution
using a miRNeasy kit (Qiagen). DNAse I (Qiagen) was used to eliminate
genomic DNA. The concentration of isolated RNA was measured on a
NanoDrop (Thermo Scientific, Wilmington, Delaware). RNA (500 ng
per sample) was reverse transcribed to cDNA using the RT<sup>2</sup> First
strand kit (Qiagen) according to the protocol of the manufacturer.
RT<sup>2</sup> SYBR Green qPCR mix was used to amplify cDNA along
with a custom array of primers for specific mRNA and miRNA molecules
(Qiagen) in a real-time thermal cycler (CFX96, Bio-Rad, Hercules,
California). Expression of mRNA transcripts was normalised to the
stable housekeeping gene β-actin, and miRNA transcripts to the stable
housekeeping gene Rnu6 using the techniques of Schmittgen and Livak.<sup><xref rid="r23" ref-type="bibr">23</xref></sup> The list of RNA
transcripts and corresponding RefSeq and miRBase information is
listed in Tables I and II.</p><table-wrap id="t1" orientation="portrait" position="float"><label>Table I</label><caption><p>Messenger RNA transcripts evaluated
by qPCR</p></caption><table frame="hsides" rules="groups"><thead><tr><th rowspan="1" colspan="1"><bold>Gene </bold></th><th rowspan="1" colspan="1"><bold>RefSeq</bold></th><th rowspan="1" colspan="1"><bold>Name</bold></th></tr></thead><tbody><tr><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">Acat1</td><td rowspan="1" colspan="1">NM_017075</td><td rowspan="1" colspan="1">Acetyl-coenzyme A acetyltransferase 1</td></tr><tr><td rowspan="1" colspan="1">Actb</td><td rowspan="1" colspan="1">NM_031144</td><td rowspan="1" colspan="1">Actin, beta</td></tr><tr><td rowspan="1" colspan="1">Agpat6</td><td rowspan="1" colspan="1">NM_001047849</td><td rowspan="1" colspan="1">1-acylglycerol-3-phosphate O-acyltransferase 6 </td></tr><tr><td rowspan="1" colspan="1">Alox5</td><td rowspan="1" colspan="1">NM_012822</td><td rowspan="1" colspan="1">5-LOX, Arachidonate 5-lipoxygenase</td></tr><tr><td rowspan="1" colspan="1">Apoe</td><td rowspan="1" colspan="1">NM_138828</td><td rowspan="1" colspan="1">Apolipoprotein E</td></tr><tr><td rowspan="1" colspan="1">Atg16l1</td><td rowspan="1" colspan="1">NM_001108809</td><td rowspan="1" colspan="1">ATG16 autophagy related 16-like 1 (S. cerevisiae)</td></tr><tr><td rowspan="1" colspan="1">Atg5</td><td rowspan="1" colspan="1">NM_001014250</td><td rowspan="1" colspan="1">ATG5 autophagy related 5 homolog (S. cerevisiae)</td></tr><tr><td rowspan="1" colspan="1">Becn1</td><td rowspan="1" colspan="1">NM_053739</td><td rowspan="1" colspan="1">Beclin 1, autophagy related</td></tr><tr><td rowspan="1" colspan="1">Cd11b</td><td rowspan="1" colspan="1">NM_012711</td><td rowspan="1" colspan="1">Integrin, alpha M</td></tr><tr><td rowspan="1" colspan="1">Cd68</td><td rowspan="1" colspan="1">NM_001031638</td><td rowspan="1" colspan="1">Cd68 molecule</td></tr><tr><td rowspan="1" colspan="1">Cebpa</td><td rowspan="1" colspan="1">NM_012524</td><td rowspan="1" colspan="1">CCAAT/enhancer binding protein (C/EBP), alpha</td></tr><tr><td rowspan="1" colspan="1">Cgi58</td><td rowspan="1" colspan="1">NM_212524</td><td rowspan="1" colspan="1">Abhydrolase domain containing 5</td></tr><tr><td rowspan="1" colspan="1">Col1a2</td><td rowspan="1" colspan="1">NM_053356</td><td rowspan="1" colspan="1">Collagen, type I, alpha 2</td></tr><tr><td rowspan="1" colspan="1">Cox1</td><td rowspan="1" colspan="1">NM_017043</td><td rowspan="1" colspan="1">Prostaglandin-endoperoxide synthase 1</td></tr><tr><td rowspan="1" colspan="1">Cox2</td><td rowspan="1" colspan="1">NM_017232</td><td rowspan="1" colspan="1">Prostaglandin-endoperoxide synthase 2</td></tr><tr><td rowspan="1" colspan="1">Dgat1</td><td rowspan="1" colspan="1">NM_053437</td><td rowspan="1" colspan="1">Diacylglycerol O-acyltransferase homolog 1 (mouse)</td></tr><tr><td rowspan="1" colspan="1">Emr1</td><td rowspan="1" colspan="1">NM_001007557</td><td rowspan="1" colspan="1">EGF-like module containing, mucin-like, hormone receptor-like
1</td></tr><tr><td rowspan="1" colspan="1">Fbxo32</td><td rowspan="1" colspan="1">NM_133521</td><td rowspan="1" colspan="1">Atrogin-1</td></tr><tr><td rowspan="1" colspan="1">Fitm1</td><td rowspan="1" colspan="1">NM_001106037</td><td rowspan="1" colspan="1">Fat storage-inducing transmembrane protein 1</td></tr><tr><td rowspan="1" colspan="1">Fitm2</td><td rowspan="1" colspan="1">NM_001107799</td><td rowspan="1" colspan="1">Fat storage-inducing transmembrane protein 2</td></tr><tr><td rowspan="1" colspan="1">Fsp1</td><td rowspan="1" colspan="1">NM_012618</td><td rowspan="1" colspan="1">S100 calcium-binding protein A4</td></tr><tr><td rowspan="1" colspan="1">Fsp27</td><td rowspan="1" colspan="1">NM_001024333</td><td rowspan="1" colspan="1">Cell death-inducing DFFA-like effector c</td></tr><tr><td rowspan="1" colspan="1">Cd168</td><td rowspan="1" colspan="1">NM_012964</td><td rowspan="1" colspan="1">Hyaluronan mediated motility receptor (RHAMM)</td></tr><tr><td rowspan="1" colspan="1">Il10</td><td rowspan="1" colspan="1">NM_012854</td><td rowspan="1" colspan="1">Interleukin 10</td></tr><tr><td rowspan="1" colspan="1">Il1b</td><td rowspan="1" colspan="1">NM_031512</td><td rowspan="1" colspan="1">Interleukin 1 beta</td></tr><tr><td rowspan="1" colspan="1">Il6</td><td rowspan="1" colspan="1">NM_012589</td><td rowspan="1" colspan="1">Interleukin 6</td></tr><tr><td rowspan="1" colspan="1">Mmp14</td><td rowspan="1" colspan="1">NM_031056</td><td rowspan="1" colspan="1">Matrix metallopeptidase 14 (membrane-inserted)</td></tr><tr><td rowspan="1" colspan="1">Mmp2</td><td rowspan="1" colspan="1">NM_031054</td><td rowspan="1" colspan="1">Matrix metallopeptidase 2</td></tr><tr><td rowspan="1" colspan="1">Mmp9</td><td rowspan="1" colspan="1">NM_031055</td><td rowspan="1" colspan="1">Matrix metallopeptidase 9</td></tr><tr><td rowspan="1" colspan="1">eMHC</td><td rowspan="1" colspan="1">NM_012604</td><td rowspan="1" colspan="1">Myosin, heavy chain 3, skeletal muscle, embryonic</td></tr><tr><td rowspan="1" colspan="1">Pdgfra</td><td rowspan="1" colspan="1">NM_012802</td><td rowspan="1" colspan="1">Platelet derived growth factor receptor, alpha polypeptide</td></tr><tr><td rowspan="1" colspan="1">Pld1</td><td rowspan="1" colspan="1">NM_030992</td><td rowspan="1" colspan="1">Phospholipase D1</td></tr><tr><td rowspan="1" colspan="1">Plin</td><td rowspan="1" colspan="1">NM_013094</td><td rowspan="1" colspan="1">Perilipin 1</td></tr><tr><td rowspan="1" colspan="1">Plin2</td><td rowspan="1" colspan="1">NM_001007144</td><td rowspan="1" colspan="1">Perilipin 2, ADRP, ADFP, Adiophilin</td></tr><tr><td rowspan="1" colspan="1">Plin5</td><td rowspan="1" colspan="1">NM_001134637</td><td rowspan="1" colspan="1">Perilipin 5</td></tr><tr><td rowspan="1" colspan="1">Atgl</td><td rowspan="1" colspan="1">NM_001108509</td><td rowspan="1" colspan="1">Adipose triglyceride lipase</td></tr><tr><td rowspan="1" colspan="1">Pparg</td><td rowspan="1" colspan="1">NM_013124</td><td rowspan="1" colspan="1">Peroxisome proliferator-activated receptor gamma</td></tr><tr><td rowspan="1" colspan="1">Fat/Cd36</td><td rowspan="1" colspan="1">XM_575338</td><td rowspan="1" colspan="1">Similar to fatty acid translocase/CD36</td></tr><tr><td rowspan="1" colspan="1">Scx</td><td rowspan="1" colspan="1">NM_001130508</td><td rowspan="1" colspan="1">Scleraxis</td></tr><tr><td rowspan="1" colspan="1">Timp1</td><td rowspan="1" colspan="1">NM_053819</td><td rowspan="1" colspan="1">TIMP metallopeptidase inhibitor 1</td></tr><tr><td rowspan="1" colspan="1">Timp2</td><td rowspan="1" colspan="1">NM_021989</td><td rowspan="1" colspan="1">TIMP metallopeptidase inhibitor 2</td></tr><tr><td rowspan="1" colspan="1">Tnmd</td><td rowspan="1" colspan="1">NM_022290</td><td rowspan="1" colspan="1">Tenomodulin</td></tr><tr><td rowspan="1" colspan="1">Trim63</td><td rowspan="1" colspan="1">NM_080903</td><td rowspan="1" colspan="1">MuRF-1, tripartite motif-containing 63</td></tr><tr><td rowspan="1" colspan="1">Vps34</td><td rowspan="1" colspan="1">NM_022958</td><td rowspan="1" colspan="1">Phosphoinositide-3-kinase, class 3</td></tr></tbody></table></table-wrap><table-wrap id="t2" orientation="portrait" position="float"><label>Table II</label><caption><p>microRNA transcripts evaluated by
qPCR.</p></caption><table frame="hsides" rules="groups"><thead><tr><th rowspan="1" colspan="1"><bold>miRNA</bold></th><th rowspan="1" colspan="1"><bold>miRBase</bold> Accession ID</th></tr></thead><tbody><tr><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1">rno-miR-1</td><td rowspan="1" colspan="1">MIMAT0003125</td></tr><tr><td rowspan="1" colspan="1">rno-miR-103</td><td rowspan="1" colspan="1">MIMAT0000824</td></tr><tr><td rowspan="1" colspan="1">rno-miR-107</td><td rowspan="1" colspan="1">MIMAT0000826</td></tr><tr><td rowspan="1" colspan="1">rno-miR-126</td><td rowspan="1" colspan="1">MIMAT0000832</td></tr><tr><td rowspan="1" colspan="1">rno-mir-130a</td><td rowspan="1" colspan="1">MI0000903</td></tr><tr><td rowspan="1" colspan="1">rno-miR-133a</td><td rowspan="1" colspan="1">MIMAT0000839</td></tr><tr><td rowspan="1" colspan="1">rno-miR-133b</td><td rowspan="1" colspan="1">MIMAT0003126</td></tr><tr><td rowspan="1" colspan="1">rno-miR-138</td><td rowspan="1" colspan="1">MIMAT0000844</td></tr><tr><td rowspan="1" colspan="1">rno-miR-143</td><td rowspan="1" colspan="1">MIMAT0000849</td></tr><tr><td rowspan="1" colspan="1">rno-miR-196a</td><td rowspan="1" colspan="1">MIMAT0000871</td></tr><tr><td rowspan="1" colspan="1">rno-miR-206</td><td rowspan="1" colspan="1">MIMAT0000879</td></tr><tr><td rowspan="1" colspan="1">rno-miR-21</td><td rowspan="1" colspan="1">MIMAT0000790</td></tr><tr><td rowspan="1" colspan="1">rno-miR-214</td><td rowspan="1" colspan="1">MIMAT0000885</td></tr><tr><td rowspan="1" colspan="1">rno-miR-221</td><td rowspan="1" colspan="1">MIMAT0000890</td></tr><tr><td rowspan="1" colspan="1">rno-miR-222</td><td rowspan="1" colspan="1">MIMAT0000891</td></tr><tr><td rowspan="1" colspan="1">rno-mir-23a</td><td rowspan="1" colspan="1">MIMAT0000792</td></tr><tr><td rowspan="1" colspan="1">rno-miR-23b</td><td rowspan="1" colspan="1">MIMAT0000793</td></tr><tr><td rowspan="1" colspan="1">rno-mir-27a</td><td rowspan="1" colspan="1">MIMAT0000799</td></tr><tr><td rowspan="1" colspan="1">rno-miR-29b</td><td rowspan="1" colspan="1">MIMAT0000801</td></tr><tr><td rowspan="1" colspan="1">rno-miR-31</td><td rowspan="1" colspan="1">MIMAT0000810</td></tr><tr><td rowspan="1" colspan="1">rno-miR-338</td><td rowspan="1" colspan="1">MIMAT0000581</td></tr><tr><td rowspan="1" colspan="1">rno-miR-381</td><td rowspan="1" colspan="1">MIMAT0003199</td></tr><tr><td rowspan="1" colspan="1">rno-miR-499</td><td rowspan="1" colspan="1">MIMAT0003381</td></tr><tr><td rowspan="1" colspan="1">rno-let-7a</td><td rowspan="1" colspan="1">MIMAT0000774</td></tr><tr><td rowspan="1" colspan="1">rno-let-7b</td><td rowspan="1" colspan="1">MIMAT0000775</td></tr><tr><td rowspan="1" colspan="1">rno-let-7c</td><td rowspan="1" colspan="1">MIMAT0000776</td></tr><tr><td rowspan="1" colspan="1">rno-let-7d</td><td rowspan="1" colspan="1">MIMAT0000562</td></tr><tr><td rowspan="1" colspan="1">rno-let-7e</td><td rowspan="1" colspan="1">MIMAT0000777</td></tr></tbody></table></table-wrap></sec><sec><title content-type="h3">Statistical analysis</title><p>Data are presented as mean and standard error of the mean (<sc>sem</sc>).
Differences between the sham-operated shoulder and the shoulder
with a rotator cuff tear were tested using Student’s <italic>t</italic>-tests
(α = 0.05) in GraphPad Prism 6.0 (GraphPad, La Jolla, California).</p></sec></sec><sec><title>Results</title><p>A total of 28 days after inducing a full-thickness rotator cuff
tear, there was a 33% decrease in the mass of supraspinatus muscles
(sham 417 mg; <sc>sem</sc> 20.3, tear 279 mg; <sc>sem </sc>20.5,
p < 0.05) and a 40% decrease in the mass of infraspinatus muscles
(sham 394 mg; <sc>sem</sc> 19.7, tear 235 mg; <sc>sem</sc> 12.9
, p < 0.05). For single muscle contractility measurements, a
rotator cuff tear did not affect muscle fibre CSA, but a 16% decrease
in F<sub>o</sub> and a 23% decrease in sF<sub>o</sub> was observed
(Fig. 1). A rotator cuff tear increased the size and accumulation
of type IIB muscle fibres by 30% and 18%, respectively, and decreased
the amount of type I and IIA muscle fibres by approximately 10%
(Fig. 2). </p><fig id="f1" orientation="portrait" position="float"><label>Figs. 1a - 1c</label><caption><p>Graphs showing permeabilised
muscle fibre size and contractile force. Figure 1a - Permeabilised
fibre cross-sectional area (CSA); Figure 1b - maximum isometric
force (Fo) and figure 1c - specific force (sFo) of sham-operated
control and torn rotator cuff muscles. Values are mean and<sc> sem</sc>.
n = 5 muscles from each group. * different from sham-operated control
group (p < 0.05). </p></caption><graphic xlink:href="2000294-galleyfig1a"/><graphic xlink:href="2000294-galleyfig1b"/><graphic xlink:href="2000294-galleyfig1c"/></fig><fig id="f2" orientation="portrait" position="float"><label>Figs. 2a - 2c</label><caption><p>Muscle fibre type size and percent
composition. Figure 2a – Graphs showing cross-sectional area (CSA)
and b) percent distribution of myosin heavy chain isoform of muscle
fibres from sham-operated control and torn rotator cuff muscles.
Figure 2c - Representative immunohistochemistry demonstrating different
fibre types. White, extracellular matrix (WGA-lectin); blue, MHC I;
red, MHC IIA; green, MHC IIB; black, MHC IIX; blue-red, hybrid MHC
I/IIA; red-black, hybrid MHC IIA/IIX; green-black, hybrid MHC IIB/IIX.
Values are mean and <sc>sem</sc>. n = 5 muscles. * different from
sham-operated control group (p < 0.05).</p></caption><graphic xlink:href="2000294-galleyfig2a"/><graphic xlink:href="2000294-galleyfig2b"/><graphic xlink:href="2000294-galleyfig2c"/></fig><p>We next measured the expression of mRNAs and miRNAs involved
in adipogenesis and lipid storage, ECM synthesis, autophagy, inflammation
and macrophage accumulation. For genes involved with lipid synthesis
and storage, there was an increase in PPAR-γ, PLD1 and miR-27a, while
GPAT4 and FIT2 were downregulated (Figs. 3a and 3b, 4a and 4b).
A significant upregulation in several ECM synthesis and remodelling
genes were observed, including Type Iα2 collagen, MMP2, MMP9, MMP14,
TIMP1, TIMP2, FSP-1, tenomodulin, miR-21, miR-31, miR-214 and miR-221
(Fig. 3c and 4c). There was also an increase in several genes that
induce inflammation and muscle atrophy, including COX1, COX2, IL-1β,
IL-6 (Fig. 3e), while miRNAs involved with muscle growth and adaptation, miR-133a,
miR-133b and miR-499, were downregulated (Fig. 4a). Rotator cuff tear also increased the expression
of the pan-macrophage marker F4/80, as well as M1 markers CD11b,
CD68 and CCR7 (Fig. 3f). A decrease in the M2 marker CD168 was also
observed (Fig. 3f). No differences in autophagy-related genes or
the let-7 cluster of miRNAs were observed (Fig. 3d, 4d). Consistent
with gene expression data, increases in lipid deposition and deposits
of lipid-laden macrophages were also observed in torn rotator cuff
muscles (Fig. 5).</p><fig id="f3" orientation="portrait" position="float"><label>Figs. 3a - 3f</label><caption><p>Graphs showing mRNA
expression. Expression of genes associated with a) adipogenesis,
b) lipid storage, c) extracellular matrix synthesis and fibrosis,
d) autophagy, e) inflammation and atrophy and f) macrophage and
fatty macrophage accumulation. Target gene expression was normalised
to ß-actin. Sham-operated control values are plotted on the left,
and torn muscles are plotted on the right and shown in orange. Values
are mean and <sc>sem</sc>. n = 5 muscles from each group. * significantly different
from sham-operated control group (p < 0.05).</p></caption><graphic xlink:href="2000294-galleyfig3a"/><graphic xlink:href="2000294-galleyfig3b"/><graphic xlink:href="2000294-galleyfig3c"/><graphic xlink:href="2000294-galleyfig3d"/><graphic xlink:href="2000294-galleyfig3e"/><graphic xlink:href="2000294-galleyfig3f"/></fig><fig id="f4" orientation="portrait" position="float"><label>Figs. 4a - 4d</label><caption><p>Graphs showing miRNA expression.
Expression of miRNAs associated with a) muscle adaptation, b) lipid
storage and synthesis, c) fibrosis and extracellular matrix synthesis
and d) the let-7 cluster. Target miRNA expression was normalised
to Rnu6. Sham-operated control values are plotted on the left, and
torn muscles are plotted on the right and shown in orange. Values
are mean and <sc>sem</sc>; n = 5 muscles from each group. * significantly
different from sham-operated control group (p < 0.05).</p></caption><graphic xlink:href="2000294-galleyfig4a"/><graphic xlink:href="2000294-galleyfig4b"/><graphic xlink:href="2000294-galleyfig4c"/><graphic xlink:href="2000294-galleyfig4d"/></fig><fig id="f5" orientation="portrait" position="float"><label>Figs. 5a - 5d</label><caption><p>Representative Oil red O histology
sections from a) sham-operated control and b) torn rotator cuff
muscles. Blue represents haematoxylin; red, Oil red O. Representative
immunohistochemistry from c) sham-operated control and d) torn rotator
cuff muscles; blue represents nuclei (DAPI); red, extracellular
matrix (WGA lectin); green, lipid (BODIPY); white, macrophages (F4/
80).</p></caption><graphic xlink:href="2000294-galleyfig5a"/><graphic xlink:href="2000294-galleyfig5b"/><graphic xlink:href="2000294-galleyfig5c"/><graphic xlink:href="2000294-galleyfig5d"/></fig></sec><sec><title>Discussion</title><p>Chronic rotator cuff tears are a frequent and debilitating injury,
and options to treat fatty degeneration after rotator cuff tear
are currently limited. T lymphocytes are thought to play an important
role in regulating the inflammatory response and regeneration of
muscle after injury,<sup><xref rid="r7" ref-type="bibr">7</xref>,<xref rid="r8" ref-type="bibr">8</xref></sup> and this was the
first study, to our knowledge, to specifically evaluate the response
of skeletal muscle to a chronic injury in an animal that lacked
competent T cells. The sF<sub>o</sub> of sham-operated muscle fibres of nude rats, as well as the magnitude
of mass and sF<sub>o</sub> reduction and increase in the number
of type IIB muscle fibres that occurred after rotator cuff tear
in nude rats, was similar to what was previously observed in immune
competent adult rats.<sup><xref rid="r18" ref-type="bibr">18</xref>,<xref rid="r19" ref-type="bibr">19</xref></sup> A reduction in
sF<sub>o</sub> and accumulation of type IIB muscle fibres often
occurs in states of chronic injury,<sup><xref rid="r18" ref-type="bibr">18</xref>,<xref rid="r24" ref-type="bibr">24</xref></sup> and
these results suggest that T-cell deficient rats develop a reduction
in muscle function after a rotator cuff tear in a fashion that is
generally similar to what is observed in T-cell competent rats. </p><p>T cells, along with many other inflammatory cell populations,
secrete many different types of interleukins, prostaglandins, and
leukotrienes, and several of these molecules have important roles
in regulating muscle inflammation, fibrosis and atrophy after injury.<sup><xref rid="r5" ref-type="bibr">5</xref>,<xref rid="r25" ref-type="bibr">25</xref>,<xref rid="r26" ref-type="bibr">26</xref></sup> IL-1β and
IL-6 are pro-inflammatory cytokines that promote muscle atrophy<sup><xref rid="r5" ref-type="bibr">5</xref></sup> and were upregulated
following tear in T-cell deficient rats, although the expression
of the anti-inflammatory gene IL-10 was not different. COX-1 and COX-2
catalyse the conversion of arachidonic acid into pro-inflammatory
prostaglandins that inhibit muscle stem-cell proliferation and muscle
regeneration,<sup><xref rid="r25" ref-type="bibr">25</xref>,<xref rid="r27" ref-type="bibr">27</xref></sup> and were upregulated
after tear, although 5-LOX which converts arachidonic acid into
pro-inflammatory leukotrienes<sup><xref rid="r26" ref-type="bibr">26</xref></sup> was
not upregulated. Atrogin-1 and MuRF-1 are the major E3 ubiquitin
ligases that serve as key rate-limiting enzymes in skeletal muscle
protein degradation,<sup><xref rid="r28" ref-type="bibr">28</xref></sup> but
their expression was not different between sham-operated and torn
rotator cuff muscles, which is consistent with observations from
previous work in T-cell competent rats.<sup><xref rid="r18" ref-type="bibr">18</xref>,<xref rid="r29" ref-type="bibr">29</xref></sup> In
adult animals, eMHC is a marker of muscle fibres that have been
injured or damaged in some fashion and are undergoing regeneration,<sup><xref rid="r24" ref-type="bibr">24</xref></sup> and in nude rats,
torn rotator cuff muscles had an increase in eMHC expression. While
an induction in autophagy-related transcripts was observed in torn
rotator cuff muscles from T-cell competent rats,<sup><xref rid="r18" ref-type="bibr">18</xref></sup> no changes in
the autophagy-related transcripts Atg16L1, Atg5, Beclin-1 or Vps34
were observed in the current study. For miRNAs which play a role
in myogenesis and muscle regeneration,<sup><xref rid="r30" ref-type="bibr">30</xref>,<xref rid="r31" ref-type="bibr">31</xref></sup> miR-1,
-23a, -23b, -29b, -126 and -206 were unchanged following a tear, although
miR-133a, -133b and -499 were downregulated in torn rotator muscles.
The let-7 cluster of miRNAs play an important role in regulating
myogenesis,<sup><xref rid="r32" ref-type="bibr">32</xref></sup> but
no change in the expression of any let-7 cluster member was observed
in torn rotator cuff muscles. The results from this study suggest
that although several pro-inflammatory markers are upregulated,
similar to other chronic models of rotator cuff tear, persistent
activation of atrogin-1 and MuRF-1 and activation of autophagy pathways are
dispensable for chronic atrophy and weakness in torn rotator cuff
muscles of T-cell deficient rats.</p><p>Fibrosis is often observed in chronically torn rotator cuff muscles,<sup><xref rid="r3" ref-type="bibr">3</xref>,<xref rid="r18" ref-type="bibr">18</xref>,<xref rid="r19" ref-type="bibr">19</xref></sup> and
in the current study, torn rotator cuff muscles had an increase
in type I collagen gene expression and a robust accumulation of
connective tissue apparent from immunohistochemistry. Genes associated
with ECM degradation and remodelling, MMP2, MMP9 and MMP14<sup><xref rid="r33" ref-type="bibr">33</xref></sup> were elevated,
but we also observed increases in the MMP inhibitors TIMP1 and TIMP2.
FSP-1 and tenomodulin are markers of fibroblast cells,<sup><xref rid="r34" ref-type="bibr">34</xref>,<xref rid="r35" ref-type="bibr">35</xref></sup> and are elevated in torn muscles.
For other fibroblast markers, PDGFR-α was not different, and scleraxis
was downregulated. MiRNAs associated with fibrosis, miR-21, -214,
-221, and -31<sup><xref rid="r36" ref-type="bibr">36</xref>-<xref rid="r38" ref-type="bibr">38</xref></sup> were upregulated
following a chronic rotator cuff tear, however, miR-222, which is closely
associated with miR-221 functionally,<sup><xref rid="r36" ref-type="bibr">36</xref>-<xref rid="r38" ref-type="bibr">38</xref></sup> was
not different. Overall, the changes in mRNA and miRNA transcripts,
and the gross appearance of muscles via histology indicate that
the regulation of ECM production and fibroblast proliferation was
similar to what was seen in our previous study in T-cell competent
rats.<sup><xref rid="r18" ref-type="bibr">18</xref>,<xref rid="r19" ref-type="bibr">19</xref></sup></p><p>The deposition of fat in and around muscle fibres is commonly
observed after a chronic rotator cuff tear.<sup><xref rid="r3" ref-type="bibr">3</xref>,<xref rid="r18" ref-type="bibr">18</xref>,<xref rid="r19" ref-type="bibr">19</xref></sup> In the T-cell
deficient rats, the torn rotator cuff muscles from nude rats exhibited
lipid accumulation similar to that observed in T-cell competent
rats.<sup><xref rid="r18" ref-type="bibr">18</xref>,<xref rid="r19" ref-type="bibr">19</xref></sup> PPAR-γ and C/EBPα direct
the transport and storage of fatty acids, as well as the synthesis
of intracellular lipid droplets. In agreement with previous studies,<sup><xref rid="r18" ref-type="bibr">18</xref>,<xref rid="r39" ref-type="bibr">39</xref>-<xref rid="r41" ref-type="bibr">41</xref></sup> PPAR-γ expression
was elevated after rotator cuff tear in nude rats, although C/EBPα expression
was not different between sham-operated control and torn muscles. MIR-27a, which functions
to inhibit adipogenesis by binding to the 3’ regulatory region of
PPAR-γ,<sup><xref rid="r42" ref-type="bibr">42</xref></sup> was
upregulated in torn rotator cuff muscles. In our previous study,<sup><xref rid="r18" ref-type="bibr">18</xref></sup> there was a much greater
increase in PPAR-γ expression in torn rotator cuff muscles, but
no change in miR-27a was observed. The increase in miR-27a expression
in the current study may explain the smaller increase in PPAR-γ expression
that occurred after a rotator cuff tear. Although further study
is necessary, T cells may be responsible for the attenuation of
miR-27a expression after a rotator cuff tear, leading to a larger
increase in PPAR-γ in T-cell competent rats. </p><p>Lipid droplets in many tissues are dynamic organelles and direct
many cellular activities ranging from energy metabolism to inflammation
and apoptosis.<sup><xref rid="r43" ref-type="bibr">43</xref>,<xref rid="r44" ref-type="bibr">44</xref></sup> The biology of
lipid droplet formation in skeletal muscle tissue has been extensively
studied in the context of endurance training or diabetes, which
share many overlapping molecular pathways.<sup><xref rid="r45" ref-type="bibr">45</xref>,<xref rid="r46" ref-type="bibr">46</xref></sup> In
oil red O images, the size of intramyocellular lipid droplets in
T-cell deficient rats is roughly the same size as a typical observation
in T-cell competent animals after a rotator cuff tear,<sup><xref rid="r18" ref-type="bibr">18</xref>,<xref rid="r19" ref-type="bibr">19</xref></sup> but is several orders of magnitude
larger than what is typically observed when lipid accumulates in
skeletal muscle tissue of endurance-trained athletes or type 2 diabetics.<sup><xref rid="r45" ref-type="bibr">45</xref>,<xref rid="r46" ref-type="bibr">46</xref></sup> To gain greater insight into the
processes that regulate lipid droplet formation, we evaluated the
expression of several canonical genes that directly regulate the
size, number and mobilisation of lipid droplets both in muscle and
non-muscle cells, which are discussed in detail elsewhere.<sup><xref rid="r18" ref-type="bibr">18</xref>,<xref rid="r21" ref-type="bibr">21</xref>,<xref rid="r47" ref-type="bibr">47</xref></sup> These genes
included perilipin 1, 2 and 5, as well as FAT/CD36, DGAT1, GPAT,
FIT1, FIT2, FSP27, CGI58, ATGL and PLD1. Despite the presence of
extremely large lipid droplets, there were no changes in perilipin
1, 2 and 5, FSP27, FAT/CD36, DGAT1, and FIT1 in T-cell deficient
rats, and a decrease in GPAT4 and FIT2 expression. Although this
is in agreement with prior observations in T-cell competent rats,<sup><xref rid="r18" ref-type="bibr">18</xref>,<xref rid="r19" ref-type="bibr">19</xref></sup> these genes would otherwise be anticipated
to be upregulated in the context of lipid accumulation in endurance-trained
or diabetic muscle.<sup><xref rid="r48" ref-type="bibr">48</xref></sup> Only
one lipid accumulation gene, PLD1, was upregulated in the torn cuff muscles of T-cell deficient rats.
MiRNAs involved in adipogenesis,<sup><xref rid="r49" ref-type="bibr">49</xref></sup> miR-103,
-107, -130a, -138, -143 and -196a, were not different between sham-operated
control and torn rotator cuff muscles. However, miR-133a and -133b, which
are involved in inhibiting the conversion of satellite cells into
brown adipocytes,<sup><xref rid="r50" ref-type="bibr">50</xref>,<xref rid="r51" ref-type="bibr">51</xref></sup> were downregulated.
For intramyocellular lipid, many of the fat deposits in the current
study contained macrophages, which is consistent with our previous findings in T-cell competent
rats.<sup><xref rid="r18" ref-type="bibr">18</xref>,<xref rid="r19" ref-type="bibr">19</xref></sup> Together, these
results indicate that T cells are not required for the development
of fat accumulation after a rotator cuff tear. The lack of change
in the expression of many canonical lipid uptake and storage genes
after a rotator cuff tear further support the idea that the dramatic increase
in fat that occurs with fatty degeneration, is not being actively
used for metabolic purposes, and may be a pathological response
to a rotator cuff tear.</p><p>There are at least two populations of macrophages that are thought
to play a central role in regulating skeletal muscle degeneration
and regeneration following injury. Classically-activated M1 macrophages
promote muscle necrosis and degeneration, while alternately-activated
M2 macrophages help to reduce local inflammation and stimulate regeneration.<sup><xref rid="r6" ref-type="bibr">6</xref></sup> Following many muscle
injuries, M1 marker levels increase immediately following muscle
injury and return to baseline levels four to six days after injury, while
M2 macrophages increase during this time and return to baseline
levels ten to 14 days after injury.<sup><xref rid="r6" ref-type="bibr">6</xref></sup> We previously demonstrated a marked
accumulation of macrophages in torn rotator cuff muscles of T-cell
competent rats,<sup><xref rid="r18" ref-type="bibr">18</xref></sup> although
we did not assess markers of specific M1 and M2 macrophages. In
the current study, an increase in the expression of the pan-macrophage
marker F4/80 was observed, as there was for M1 markers CD11b, CD68
and CCR7. A downregulation in the M2 marker CD168 was observed.
The sustained elevation of M1 macrophage transcripts with a concurrent
decrease in M2 transcripts suggests a chronic inflammatory state
within torn rotator cuff muscles that is consistent with previous
findings.<sup><xref rid="r21" ref-type="bibr">21</xref></sup>
</p><p>While adipocytes accumulate after a rotator cuff tear and contribute
to the overall increase in lipid content in torn muscles,<sup><xref rid="r3" ref-type="bibr">3</xref>,<xref rid="r41" ref-type="bibr">41</xref></sup> we also identified a class of lipid-laden macrophages
that are found in a torn rotator cuff muscles<sub>,</sub><sup><xref rid="r18" ref-type="bibr">18</xref>,<xref rid="r21" ref-type="bibr">21</xref></sup> which resemble a class of fatty
macrophages seen in atherosclerosis. These fatty macrophages are often
referred to as ‘foam cells’ and function to promote local inflammation
in atherosclerotic plaques.<sup><xref rid="r48" ref-type="bibr">48</xref></sup> In atherosclerosis,
fatty macrophages can enhance lipid plaque formation which subsequently
exacerbates the severity of the disorder,<sup><xref rid="r48" ref-type="bibr">48</xref></sup> and these cells may also play an important
role in fatty degeneration in rotator cuff tears. Differentiation
of macrophages into fatty macrophages shares some overlap with genetic
programmes that regulate adipocyte differentiation and maturation.
PPAR-γ promotes the differentiation of macrophages into fatty macrophages
in atherosclerotic plaques, and once differentiated, these cells
highly express ApoE, FSP27 and ACAT1.<sup><xref rid="r52" ref-type="bibr">52</xref>,<xref rid="r53" ref-type="bibr">53</xref></sup> In
the current study, elevations in PPAR-γ and ApoE were observed in
torn rotator cuff muscles, although no changes in ACAT1 or FSP27
were present. Additionally, we observed fatty macrophages that accumulate
around lipid plaques in T-cell deficient rats via immunohistochemistry.
T cells are thought to play an important role in coordinating the inflammation
and regeneration of muscle after injury through direct interactions
with muscle fibres as well as through the regulation of M1 and M2
macrophages,<sup><xref rid="r7" ref-type="bibr">7</xref>-<xref rid="r9" ref-type="bibr">9</xref></sup> however, the results from
the current study indicate that T cells are not required for fatty
macrophage accumulation in skeletal muscle after injury. </p><p>There are several limitations to this study. Due to many shared
anatomical features with humans, the rat is an ideal animal model
for the study of rotator cuff tears, however, rats do not develop
the severity of fatty degeneration seen in human patients.<sup><xref rid="r3" ref-type="bibr">3</xref>,<xref rid="r19" ref-type="bibr">19</xref>,<xref rid="r20" ref-type="bibr">20</xref>,<xref rid="r41" ref-type="bibr">41</xref></sup> We chose to evaluate
chronic rotator cuff tears using a single time point of one month
to allow for comparisons to our previous studies,<sup><xref rid="r18" ref-type="bibr">18</xref>,<xref rid="r19" ref-type="bibr">19</xref></sup> but we did not evaluate changes
at other time points. We used the contralateral side as the sham-operated
control muscle, and animals were still able to use both forelimbs
for ambulation. While we did not notice any signs of muscle injury
in sham-operated control muscles, it is possible that these muscles
experienced a small amount of muscle hypertrophy. For single fibre
contractility measurements, we used type II muscle fibres, and did
not measure changes by specific type II myosin isoform. We selected
this approach due to the greater amount of type II fibres in the
cuff, and sF<sub>o</sub> does not change between fibres of different
type II myosin isoforms.<sup><xref rid="r54" ref-type="bibr">54</xref></sup> While
we measured the expression of multiple mRNA molecules, we did not
directly quantify protein levels, and changes in gene expression
may not reflect changes in protein abundance. We point to many similarities
between the current study and our previous study using immunocompetent
rats, and while we used slightly more mature rats in the previous
study, we do not believe this affects the ability to make these
comparisons with the current study. Despite the presence of these
limitations, this study provided insight into the cellular and molecular changes
in rotator cuff muscles of T-cell deficient rats. </p></sec><sec><title>Conclusion</title><p>T lymphocytes are thought to play an important role in modulating
the inflammatory and regeneration responses of injured skeletal
muscle, but their function in directing fatty degeneration in the
rotator cuff had not previously been studied. Compared with a similar
injury in T-cell competent rats,<sup><xref rid="r18" ref-type="bibr">18</xref>,<xref rid="r19" ref-type="bibr">19</xref></sup> nude rats displayed
a similar response to a chronic rotator cuff tear. These results
suggest that T cells may not play an important role in regulating
muscle degeneration or remodelling following chronic muscle unloading.
Furthermore, as there has been much interest in the use of stem-cell
therapy to enhance rotator cuff regeneration<sup>,<xref rid="r15" ref-type="bibr">15</xref></sup> and evaluating human-derived
stem cells in animal models of injury and disease is important in
identifying safe and effective stem-cell therapies,<sup><xref rid="r17" ref-type="bibr">17</xref></sup> the general similarities
between nude rats and T-cell competent rats indicate that the nude
rats are likely a favourable preclinical model for the study of human-derived
stem cells or other xenografts that may enhance the treatment of
chronically torn rotator cuff muscles. However, further work may
need to be undertaken to investigate this definitively.</p></sec> |
Validation of a model-based measurement
of the minimum insert thickness of knee prostheses | <sec><title>Introduction</title><p>Wear of polyethylene inserts plays an important role in failure
of total knee replacement and can be monitored <italic>in vivo</italic> by
measuring the minimum joint space width in anteroposterior radiographs.
The objective of this retrospective cross-sectional study was to
compare the accuracy and precision of a new model-based method with the
conventional method by analysing the difference between the minimum
joint space width measurements and the actual thickness of retrieved
polyethylene tibial inserts. </p></sec><sec><title>Method</title><p>Before revision, the minimum joint space width values and their
locations on the insert were measured in 15 fully weight-bearing
radiographs. These measurements were compared with the actual minimum
thickness values and locations of the retrieved tibial inserts after
revision. </p></sec><sec><title>Results</title><p>The mean error in the model-based minimum joint space width measurement
was significantly smaller than the conventional method for medial
condyles (0.50 <italic>vs</italic> 0.94 mm, p < 0.01) and for
lateral condyles (0.06 <italic>vs</italic> 0.34 mm, p = 0.02). The
precision (standard deviation of the error) of the methods was similar
(0.84 <italic>vs </italic>0.79 mm medially and both 0.46 mm laterally).
The distance between the true minimum joint space width locations
and the locations from the model-based measurements was less than
10 mm in the medial direction in 12 cases and less in the lateral direction
in 13 cases.</p></sec><sec><title>Conclusion</title><p>The model-based minimum joint space width measurement method
is more accurate than the conventional measurement with the same
precision.</p><p>Cite this article: <italic>Bone Joint Res</italic> 2014;3:289–96</p></sec> | <contrib id="c1" contrib-type="author"><name><surname>van IJsseldijk</surname><given-names>E. A.</given-names></name><degrees>MSc</degrees><role>PhD student</role><xref ref-type="aff" rid="a1">1</xref><uri content-type="homepage" xlink:type="simple" xlink:href="http://orthodox.boneandjoint.org.uk/viewprofileinfo.aspx?authorid=1010496">http://orthodox.boneandjoint.org.uk/viewprofileinfo.aspx?authorid=1010496</uri></contrib><contrib id="c2" contrib-type="author"><name><surname>Harman</surname><given-names>M. K.</given-names></name><degrees>PhD</degrees><role>Assistant Professor</role><xref ref-type="aff" rid="a2">2</xref></contrib><contrib id="c3" contrib-type="author"><name><surname>Luetzner</surname><given-names>J.</given-names></name><degrees>MD</degrees><role>Orthopaedic
Surgeon</role><xref ref-type="aff" rid="a3">3</xref></contrib><contrib id="c4" contrib-type="author"><name><surname>Valstar</surname><given-names>E. R.</given-names></name><degrees>PhD, MSc</degrees><role>Professor</role><xref ref-type="aff" rid="a1">1</xref><uri content-type="homepage" xlink:type="simple" xlink:href="http://orthodox.boneandjoint.org.uk/viewprofileinfo.aspx?authorid=1006735">http://orthodox.boneandjoint.org.uk/viewprofileinfo.aspx?authorid=1006735</uri></contrib><contrib id="c5" contrib-type="author"><name><surname>Stoel</surname><given-names>B. C.</given-names></name><degrees>PhD</degrees><role>Assistant Professor</role><xref ref-type="aff" rid="a5">4</xref></contrib><contrib id="c6" contrib-type="author"><name><surname>Nelissen</surname><given-names>R. G. H. H.</given-names></name><degrees>PhD, MD</degrees><role>Head
of Department</role><xref ref-type="aff" rid="a1">1</xref><uri content-type="homepage" xlink:type="simple" xlink:href="http://orthodox.boneandjoint.org.uk/viewprofileinfo.aspx?authorid=1007100">http://orthodox.boneandjoint.org.uk/viewprofileinfo.aspx?authorid=1007100</uri></contrib><contrib id="c7" contrib-type="author"><name><surname>Kaptein</surname><given-names>B. L.</given-names></name><degrees>PhD,
MSc</degrees><role>Senior Researcher</role><xref ref-type="aff" rid="a1">1</xref></contrib><aff id="a1"><label>1</label>Leiden University Medical Center, Department
of Orthopaedics, Biomechanics and Imaging Group, PO
9600, 2300 RC, Leiden, The
Netherlands.</aff><aff id="a2"><label>2</label>Clemson University, Department
of Bioengineering, 301 Rhodes Engineering Research Center, Clemson, 29634, USA.</aff><aff id="a3"><label>3</label>University Hospital Carl Gustav Carus, Department
of Orthopaedic and Trauma Surgery, TU Dresden, Fetscherstrasse
74, 01307 Dresden, Germany.</aff><aff id="a5"><label>4</label>Leiden University Medical Center, Department
of Radiology, Div. of Image Processing, PO 9600, 2300
RC, Leiden, The Netherlands.</aff> | Bone & Joint Research | <sec><title>Article Focus</title><p>Comparing the accuracy and precision of two minimum joint space
width measurement methods for assessing insert wear in total knee
prostheses</p><p>Investigating the relationship between locations of the minimum
joint space width measurement and minimum insert thickness</p></sec><sec><title>Key Messages</title><p>Model-based minimum joint space width measurement is more accurate
than the conventional measurement</p></sec><sec><title>Strengths and Limitations</title><p>Comparison with retrieval data delivers the best possible evidence
for evaluating the accuracy of minimum joint space width measurements</p><p>Validation is done with a range of implant designs and degrees
of wear</p><p>Limitation: Study is conducted with only 15 samples, due to which
only general conclusions can be drawn</p></sec><sec><title>Introduction</title><p>Polyethylene is used as bearing material in total knee replacements
(TKR) and its wear plays an important role in TKR failure.<sup><xref rid="r1" ref-type="bibr">1</xref></sup> Remarkably, standardised
(computer-assisted) tools for the <italic>in vivo</italic> assessment
of polyethylene wear in TKR do not exist. Rather, planar radiographs
are the medical standard for routine monitoring of TKR performance
and they are used to estimate changes in the thickness of polyethylene
inserts during clinical follow-up. This thickness is quantified
with the minimum joint space width (mJSW), which is the apparent
distance between the metal tibial tray and the femoral condyles
in standard frontal plane radiographs.<sup><xref rid="r2" ref-type="bibr">2</xref>-<xref rid="r4" ref-type="bibr">4</xref></sup> The
insert thickness and its change over time can predict TKR failure.<sup><xref rid="r5" ref-type="bibr">5</xref>,<xref rid="r6" ref-type="bibr">6</xref></sup> However, the conventional mJSW method
is applied to image projections, which are subject to parallax errors that
occur when the metal tibial baseplate surface is not aligned with
the X-ray beam during sequential radiological assessments. mJSW
errors in measurement of up to 2 mm are not exceptional and numerous
follow-up visits are required to obtain a reliable estimation of
the wear rate.<sup><xref rid="r3" ref-type="bibr">3</xref>,<xref rid="r7" ref-type="bibr">7</xref></sup></p><p>In our earlier work, a novel model-based method was presented
to measure the mJSW in standard anteroposterior radiographs using
highly accurate and precise model-based Roentgen stereophotogrammetric
analysis (RSA) software.<sup><xref rid="r8" ref-type="bibr">8</xref>,<xref rid="r9" ref-type="bibr">9</xref></sup> This method has
two advantages over the standard mJSW measurements: the effect of
parallax errors is reduced by applying a three-dimensional (3D)
reconstruction of the prosthesis components using surface models
and it gives insight into both the magnitude and location of the
mJSW. For a fixed-bearing prosthesis, <italic>in vitro</italic> validation
showed that the model-based method is superior in accuracy (mean
-0.03 mm <italic>vs</italic> 0.20 mm), precision (standard deviation
(<sc>sd</sc>) = 0.19 mm <italic>vs</italic> 0.40 mm) absolute error
(mean 0.14 mm <italic>vs</italic> 0.35 mm) compared with the conventional
method.<sup><xref rid="r8" ref-type="bibr">8</xref></sup> Thus,
this method has the potential to improve the accuracy of mJSW measurements,
enabling more accurate detection of wear-related complications and
improving the power of clinical studies evaluating differences in
wear rates between different TKR designs.</p><p>In this retrospective cross-sectional study, the actual minimum
insert thickness, measured in 3D laser scan data of retrieved polyethylene
tibial inserts, was compared with the mJSW measurements acquired
using the model-based and conventional methods applied to weight-bearing
pre-revision radiographs. The primary objective was to compare the
accuracy and precision of these mJSW measurement methods using the
insert thickness measured from TKR retrievals as a ‘gold standard’. The
secondary objective was to investigate whether the mJSW location
determined in the model-based method corresponds to wear locations
evident on the explanted polyethylene inserts. </p></sec><sec sec-type="materials|methods"><title>Materials and Methods</title><sec><title content-type="h3">Data</title><p>We searched a database of explanted TKRs catalogued in an Implant
Retrieval Program previously established with institutional review
board approval (clinical protocol number in Germany EK348112009;
retrieval analysis protocol number in USA IBC2011-26) and patient-informed
consent. Wear scars on polyethylene tibial inserts of 60 fixed-bearing
TKRs retrieved from a single clinic (University Hospital Carl Gustav
Carus, Dresden) were grossly assessed using optical microscopy to visualise
the damage modes and physical touch to detect changes in the articular
surface contour. A total of 15 posterior cruciate ligament-retaining
TKRs were selected to represent a wide range of articular wear scar
sizes and shapes, ensuring that the validation study was meaningful
for the extensive wear scar variations that can occur in clinical
practice.<sup><xref rid="r10" ref-type="bibr">10</xref></sup></p><p>Table I lists clinical information such as the TKR design, duration
of <italic>in vivo</italic> TKR function, the reasons for revision surgery
and the grade of the wear scar (mild, moderate or severe). Wear
scars were graded as mild if the damage modes visibly disrupted
the machine marks on the articular surface without causing a perceptible
change in the articular geometry (six TKRs); moderate if the damage modes
visibly disrupted the machine marks on the articular surface and
the wear scar was tangible when physically touching the articular
surface (five TKRs); and severe if there was visibly gross material
loss (e.g. delamination) and a notable tactile change in the articular
geometry due to gross disruption of the bearing surface (four TKRs).</p><table-wrap id="t1" orientation="portrait" position="float"><label>Table I</label><caption><p>Description of the 15 total knee
replacements (TKR) used in this study</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1"><bold>Case</bold></th><th align="left" rowspan="1" colspan="1"><bold>TKR design<sup>†</sup></bold></th><th align="left" rowspan="1" colspan="1"><bold>Wear degree</bold></th><th align="left" rowspan="1" colspan="1"><bold>TKR lifetime
(mths)</bold></th><th align="left" rowspan="1" colspan="1"><bold>Lifetime after
radiograph<sup>‡</sup> (mths)</bold></th><th align="left" rowspan="1" colspan="1"><bold>Reason for revision</bold></th></tr></thead><tbody><tr><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">K2004</td><td align="left" rowspan="1" colspan="1">TC-Plus</td><td align="left" rowspan="1" colspan="1">Mild</td><td align="left" rowspan="1" colspan="1">  41</td><td align="left" rowspan="1" colspan="1">  0.7</td><td align="left" rowspan="1" colspan="1">Infection</td></tr><tr><td align="left" rowspan="1" colspan="1">K2133</td><td align="left" rowspan="1" colspan="1">TC-Plus</td><td align="left" rowspan="1" colspan="1">Mild</td><td align="left" rowspan="1" colspan="1">  17</td><td align="left" rowspan="1" colspan="1">  0.2</td><td align="left" rowspan="1" colspan="1">Pain</td></tr><tr><td align="left" rowspan="1" colspan="1">K2145</td><td align="left" rowspan="1" colspan="1">TC-Plus</td><td align="left" rowspan="1" colspan="1">Mild</td><td align="left" rowspan="1" colspan="1">  24</td><td align="left" rowspan="1" colspan="1">  3.0</td><td align="left" rowspan="1" colspan="1">Infection</td></tr><tr><td align="left" rowspan="1" colspan="1">K2154</td><td align="left" rowspan="1" colspan="1">Zimmer NK</td><td align="left" rowspan="1" colspan="1">Mild</td><td align="left" rowspan="1" colspan="1">  50</td><td align="left" rowspan="1" colspan="1">12.1</td><td align="left" rowspan="1" colspan="1">Infection</td></tr><tr><td align="left" rowspan="1" colspan="1">K2171</td><td align="left" rowspan="1" colspan="1">TC-Plus</td><td align="left" rowspan="1" colspan="1">Mild</td><td align="left" rowspan="1" colspan="1">  34</td><td align="left" rowspan="1" colspan="1">  0.0</td><td align="left" rowspan="1" colspan="1">Painful flexion, infection</td></tr><tr><td align="left" rowspan="1" colspan="1">K2178</td><td align="left" rowspan="1" colspan="1">TC-Plus</td><td align="left" rowspan="1" colspan="1">Mild</td><td align="left" rowspan="1" colspan="1">  19</td><td align="left" rowspan="1" colspan="1">  2.5</td><td align="left" rowspan="1" colspan="1">Infection</td></tr><tr><td align="left" rowspan="1" colspan="1">K2035<sup>*</sup></td><td align="left" rowspan="1" colspan="1">TC-Plus</td><td align="left" rowspan="1" colspan="1">Moderate</td><td align="left" rowspan="1" colspan="1">  23</td><td align="left" rowspan="1" colspan="1">  0.0</td><td align="left" rowspan="1" colspan="1">Infection</td></tr><tr><td align="left" rowspan="1" colspan="1">K2132</td><td align="left" rowspan="1" colspan="1">TC-Plus</td><td align="left" rowspan="1" colspan="1">Moderate</td><td align="left" rowspan="1" colspan="1">  86</td><td align="left" rowspan="1" colspan="1">  0.2</td><td align="left" rowspan="1" colspan="1">Infection</td></tr><tr><td align="left" rowspan="1" colspan="1">K2137</td><td align="left" rowspan="1" colspan="1">TC-Plus</td><td align="left" rowspan="1" colspan="1">Moderate</td><td align="left" rowspan="1" colspan="1">130</td><td align="left" rowspan="1" colspan="1">23.6</td><td align="left" rowspan="1" colspan="1">Suspected osteolysis later diagnosed as metastasis</td></tr><tr><td align="left" rowspan="1" colspan="1">K2144</td><td align="left" rowspan="1" colspan="1">TC-Plus</td><td align="left" rowspan="1" colspan="1">Moderate</td><td align="left" rowspan="1" colspan="1">132</td><td align="left" rowspan="1" colspan="1">  3.1</td><td align="left" rowspan="1" colspan="1">Aseptic loosening</td></tr><tr><td align="left" rowspan="1" colspan="1">K2175</td><td align="left" rowspan="1" colspan="1">TC-Plus</td><td align="left" rowspan="1" colspan="1">Moderate</td><td align="left" rowspan="1" colspan="1">  60</td><td align="left" rowspan="1" colspan="1">  0.0</td><td align="left" rowspan="1" colspan="1">Infection</td></tr><tr><td align="left" rowspan="1" colspan="1">K2046<sup>*</sup></td><td align="left" rowspan="1" colspan="1">Encore foundation</td><td align="left" rowspan="1" colspan="1">Severe</td><td align="left" rowspan="1" colspan="1">144</td><td align="left" rowspan="1" colspan="1">  4.0</td><td align="left" rowspan="1" colspan="1">Aseptic loosening</td></tr><tr><td align="left" rowspan="1" colspan="1">K2156<sup>*</sup></td><td align="left" rowspan="1" colspan="1">Stryker 7000</td><td align="left" rowspan="1" colspan="1">Severe</td><td align="left" rowspan="1" colspan="1">  77</td><td align="left" rowspan="1" colspan="1">  0.2</td><td align="left" rowspan="1" colspan="1">Infection</td></tr><tr><td align="left" rowspan="1" colspan="1">K2159<sup>*</sup></td><td align="left" rowspan="1" colspan="1">Sulzer Protek</td><td align="left" rowspan="1" colspan="1">Severe</td><td align="left" rowspan="1" colspan="1">108</td><td align="left" rowspan="1" colspan="1">  1.6</td><td align="left" rowspan="1" colspan="1">Infection</td></tr><tr><td align="left" rowspan="1" colspan="1">K2161</td><td align="left" rowspan="1" colspan="1">TC-Plus</td><td align="left" rowspan="1" colspan="1">Severe</td><td align="left" rowspan="1" colspan="1">108</td><td align="left" rowspan="1" colspan="1">  0.2</td><td align="left" rowspan="1" colspan="1">Infection</td></tr><tr><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/></tr></tbody></table><table-wrap-foot><fn><p>
* For these TKRs, double leg standing radiographs
were used for measuring mJSW; for all other TKR a single leg standing
radiograph was used
† List of manufacturers: TC-Plus (Smith & Nephew, London, UK);
Zimmer NK (Zimmer, Warsaw, Indiana); Encore Foundation (DJO Surgical,
Vista, California); Stryker 7000 (Stryker, Kalamazoo, Michigan);
Sulzer Protek (Protek Medical Product Inc., Coralville, Iowa)
‡ The period between the radiograph acquisition and revision surgery
</p></fn></table-wrap-foot></table-wrap><p>For each TKR, the most recent anteroposterior planar radiograph
was selected from those acquired during routine clinical examination
prior to the revision surgery. The radiographs were acquired with
a Siemens Aristos FX Axiom imaging device (0.143 mm per pixel).
All patients were instructed to remain fully weight-bearing on both limbs.
The selected radiographs include unilateral (n = 11) and bilateral
(n = 4) exposures. The radiographs were transmitted in DICOM format
following a de-identification process to protect patient privacy
in preparation for the radiological assessments.</p><p>Individual 3D surface models (triangulated meshes) of the explanted
components (metal tibial baseplate, polyethylene tibial insert,
metal femoral component) were generated using reverse engineering
software and a 3D laser scanner (Next Engine, Santa Monica, California). These
scans had an accuracy of 0.1 mm.</p></sec><sec><title content-type="h3">Assessment methods</title><p>The mJSW was measured on the pre-revision radiograph using both
the conventional (C) and model-based (MB) methods; the true insert
thickness (d<sub>0</sub>) and position (p<sub>0</sub>) on the medial
and lateral compartments were measured from the scanned models of
the polyethylene inserts. The details of these assessments are described
below and depicted in Figure 1. Last, the articular wear scar on
the insert was identified by digitising the periphery of the worn
area.</p><fig id="f1" orientation="portrait" position="float"><label>Fig. 1</label><caption><p>Overview of the measurement methods
applied for a single total knee replacement (TKR). The rows in the
figure represent the measurement methods that were compared: 1)
the input radiograph; 2) the conventional insert thickness measurement;
3) 2D/3D matching of the component models; 4) model-based mJSW measurement;
5) the minimum insert thickness and location based on the 3D laser
scan of the insert.</p></caption><graphic xlink:href="2000304-galleyfig1"/></fig></sec><sec><title content-type="h3">Conventional mJSW method</title><p>In the conventional mJSW method, the insert thickness (d<sub>c</sub>)
was assessed directly in the radiological image, based on the metal-to-middle<italic/>method.<sup><xref rid="r7" ref-type="bibr">7</xref></sup> This assessment
was conducted by an experienced orthopaedic surgeon and an experienced
researcher (HvdL, EAvIJ) and the mean values of the observations
were used in the further analysis. Commercially available software was used (Digimizer, MedCalc Software,
Mariakerke, Belgium) for annotation,
image processing and measurement of distances. A reference line
was drawn that annotated the superior rim of the metal tibial baseplate
at its largest medial–lateral width. The shortest, tibiofemoral
distances between this line and the distal femoral condylar edges
were measured. The tibial component rim is used for the capture
mechanisms securing the polyethylene tibial inserts to the metal
baseplate. Therefore, the height of the rim above the tibial baseplate
surface was measured by one observer (EAvlJ) at three locations
using Magics (Materialize, Leuven, Belgium). The mean height was
added to the tibiofemoral distances, yielding the final estimate
of the insert thickness.</p><p>Image magnification was calculated using the ratio between the
tibial tray widths in the image silhouette and in the scanned model.
This was used to convert all image-based mJSW measurements to real-world
dimensions, recorded as the medial d<sub>c</sub> and lateral d<sub>c</sub>.</p></sec><sec><title content-type="h3">Model-based mJSW method</title><p>In the model-based method, the mJSW (d<sub>MB</sub>) was assessed
using triangulated surface models of the components (tibia, insert
and femur) and model-based RSA software (Version 3.34, RSAcore, Leiden,
The Netherlands).<sup><xref rid="r11" ref-type="bibr">11</xref></sup> The
tibial model and the insert model were aligned in such a way that
the insert’s inferior surface and the tibial baseplate’s superior
surface coincided with the 0 xz-plane of the model coordinate systems.</p><p>Assessment of the TKRs was initiated with an image focus calibration
step. The pixel size was obtained from the DICOM data and the focus
position was set at a distance of 115 cm from the centre of the
image, in accordance with the hospital’s imaging protocol. Next,
the tibial and femoral models were matched with the radiographs
using 2D image/3D model registration.</p><p>The mJSW was measured by detecting the femoral condylar model
with the shortest distance to the tibial baseplate (d<sub>MB</sub>).
The projection of the points (p<sub>MB</sub>)was stored and
expressed in anterioposterior (AP) and mediolateral (ML) coordinates
with respect to the centre of the tibial baseplate. The measurement
was repeated by two researchers (EAvIJ and BLK), who independently
conducted the registration and measurement processes. The mean values
of the observations were used in further analysis.</p></sec><sec><title content-type="h3">Insert measurements</title><p>Using the 3D laser scan of the explanted polyethylene inserts,
the minimum insert thickness in millimetres (d<sub>0</sub>) was
measured as the minimum perpendicular distance between the inferior
backside surface and the articular surface of the insert. The scans
were aligned with the tibial models and the locations of the minimum
insert thickness (p<sub>0</sub>) were expressed in the same coordinate
system as in the model-based mJSW method.</p><p>One experienced observer (MKH) analysed the wear scar area of
the inserts using the following approach: The wear scar areas were
visually identified using an optical stereomicroscope (model Z30L,
Cambridge Instruments, Cambridge, Massachusetts). Subsequently,
the circumference of both the insert periphery and the wear scars were
digitised on calibrated digital images of the articular surface
using published photogrammetry methods.<sup><xref rid="r12" ref-type="bibr">12</xref>,<xref rid="r13" ref-type="bibr">13</xref></sup> The
insert circumference was used to map these data to the tibial model
coordinate system.</p></sec><sec><title content-type="h3">Statistical analysis</title><p>The values ∆<sub>C</sub> and ∆<sub>MB</sub> were calculated as
the difference between the respective mJSW assessment d<sub>C</sub> and
d<sub>MB</sub> and the reference insert thickness d<sub>0 </sub>(∆<sub>C</sub> =
d<sub>C</sub> – d<sub>0</sub>, ∆<sub>MB</sub> = d<sub>MB</sub> –
d<sub>0</sub>). The mean and <sc>sd</sc> of these differences over
the 15 cases were calculated and compared (paired <italic>t</italic>-test).
In addition, the mean measurement errors were calculated as the
mean of the absolute difference ∆<sub>C </sub>and ∆<sub>MB</sub> and
the number of cases having an absolute difference less than 1 mm
was counted, similar to the analysis by Collier et al.<sup><xref rid="r7" ref-type="bibr">7</xref></sup> Inter-observer agreement was
analysed with the limits of agreement and Bland–Altman plots per
condyle and mJSW measurement method.<sup><xref rid="r14" ref-type="bibr">14</xref></sup></p><p>To investigate whether the model-based mJSW measurement can accurately
determine the location of the minimum insert thickness, the locations
of the model-based mJSW assessment (p<sub>MB</sub>) and minimum
insert thickness (p<sub>0</sub>) were compared. The accuracy of
the mJSW could be associated with the difference in these locations
and this was tested by computing the correlation between these outcomes.
Absolute error and accuracy were determined based on measurement
of calibrated images of shapes with known dimensions. The technique
had an absolute error of 0.4 mm for linear distances and 3.5 mm<sup>2</sup> for
areas and was 98.6% accurate. Precision was 0.4 mm for linear distances
and 3.9 mm<sup>2</sup> for areas based on repeated measurements
taken by one user. The number of TKRs was counted for which the
model-based measurement points (p<sub>MB</sub>) were within the
wear scar periphery.</p></sec></sec><sec><title>Results</title><p>After enduring functional lifetimes of approximately 1.5 to 12
years, the actual minimum insert thickness measured on these explanted
polyethylene bearings ranged from d<sub>0</sub> = 1.99 mm to 7.86
mm medially and 4.97 mm to 7.92 mm laterally (Fig. 2). The mean
difference between the mJSW (d<sub>MB</sub> or d<sub>C</sub>) and
insert thickness (d<sub>0</sub>) was positive for both methods (Table
II), meaning that both methods tended to overestimate the actual
minimum insert thickness that was measured from the explanted tibial inserts.
The <sc>sd</sc> of the mJSW measurement methods was similar. The
mean measurement error was significantly smaller for the model-based
measurement than for the conventional measurement for both the medial
(0.50 mm <italic>vs</italic> 0.94 mm, p < 0.01) and lateral condyle
(0.06 mm <italic>vs </italic>0.34 mm, p = 0.02); (paired <italic>t</italic>-tests).</p><fig id="f2" orientation="portrait" position="float"><label>Fig. 2</label><caption><p>Barplots of the estimated insert thicknesses
d<sub>C</sub> from the conventional mJSW method, d<sub>MB</sub> from
the model-based mJSW method, and actual minimum insert thickness
d<sub>0</sub> for each case. The cases are ordered as in Table I
and grouped by wear grade.</p></caption><graphic xlink:href="2000304-galleyfig2"/></fig><table-wrap id="t2" orientation="portrait" position="float"><label>Table II</label><caption><p>Statistics of the differences between
the mJSW measurements (conventional ∆C and model-based ∆MB) with respect
to the true minimum insert thickness</p></caption><table frame="hsides" rules="groups"><thead><tr><th rowspan="1" colspan="1"/><th colspan="3" rowspan="1"><bold>Medial condyle (N = 15)</bold></th><td rowspan="1" colspan="1"/><th colspan="3" rowspan="1"><bold>Lateral condyle (N = 15)</bold></th></tr><tr><th rowspan="1" colspan="1"/><th rowspan="1" colspan="1">∆<bold><sub>C</sub></bold></th><th rowspan="1" colspan="1">∆<bold><sub>MB</sub></bold></th><th rowspan="1" colspan="1"/><th rowspan="1" colspan="1"/><th rowspan="1" colspan="1">∆<bold><sub>C</sub></bold></th><th rowspan="1" colspan="1">∆<bold><sub>MB</sub></bold></th><th rowspan="1" colspan="1"/></tr></thead><tbody><tr><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">Mean (mm)</td><td align="left" rowspan="1" colspan="1">0.94</td><td align="left" rowspan="1" colspan="1">  0.50</td><td align="left" rowspan="1" colspan="1">p = 0.00<sup>*</sup></td><td rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1">  0.34</td><td align="left" rowspan="1" colspan="1">  0.06</td><td align="left" rowspan="1" colspan="1">p = 0.02<sup>*</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">Standard deviation (mm)</td><td align="left" rowspan="1" colspan="1">0.84</td><td align="left" rowspan="1" colspan="1">  0.79</td><td align="left" rowspan="1" colspan="1">p = 0.77<sup>†</sup></td><td rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1">  0.46</td><td align="left" rowspan="1" colspan="1">  0.46</td><td align="left" rowspan="1" colspan="1">p = 0.98<sup>†</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">Mean measurement error (mm)</td><td align="left" rowspan="1" colspan="1">1.02</td><td align="left" rowspan="1" colspan="1">  0.66</td><td align="left" rowspan="1" colspan="1">p = 0.00<sup>*</sup></td><td rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1">  0.44</td><td align="left" rowspan="1" colspan="1">  0.40</td><td align="left" rowspan="1" colspan="1">p = 0.31<sup>*</sup></td></tr><tr><td align="left" rowspan="1" colspan="1">N (err < 1 mm) (%)</td><td align="left" rowspan="1" colspan="1">9 (<italic>60</italic>)</td><td align="left" rowspan="1" colspan="1">11 (<italic>73</italic>)</td><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1">13 (<italic>87</italic>)</td><td align="left" rowspan="1" colspan="1">15 (<italic>100</italic>)</td><td rowspan="1" colspan="1"/></tr><tr><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/></tr></tbody></table><table-wrap-foot><fn><p>
* Paired <italic>t</italic>-test for equal means
† Levene’s test for homogeneity of variance
</p></fn></table-wrap-foot></table-wrap><p>The limits of agreement between the observers over the 15 cases
were calculated for both mJSW, measurement methods. For the model-based
mJSW, the values were 0.00 (<sc>sd</sc> 0.45) and 0.00 (<sc>sd</sc> 0.54)
(mean ± 1.96 × <sc>sd</sc>) for the medial and lateral condyles,
respectively. For the conventional mJSW, these values were -0.22
(<sc>sd</sc> 0.48) and -0.21 (<sc>sd</sc> 0.45). For both condyles
a systematic difference was found between the observers for the
conventional method (Student <italic>t</italic>-test, p < 0.01).
The Bland–Altman plots of the outcomes (Fig. 3) showed no other trends
for either mJSW measurement method. Two outliers (K2154 and K2156,
both condyles) were found in the distribution of the observer difference
for the model-based measurement. For the conventional measurement case,
a single outlier was found (K2154).</p><fig id="f3" orientation="portrait" position="float"><label>Figs. 3a - 3b</label><caption><p>Bland–Altman plots a) of the model-based
mJSW and b) of the conventional mJSW method</p></caption><graphic xlink:href="2000304-galleyfig3a"/><graphic xlink:href="2000304-galleyfig3b"/></fig><sec><title content-type="h3">Evaluation of the measurement points</title><p>The locations of the measurement point (p<sub>MB</sub>) was compared
with the minimum insert thickness location (p<sub>0</sub>) and the
wear scar area (Fig. 4) and the difference between the points in
terms of AP and ML distance was computed (Table III). The largest
distances were found in the AP direction, where the differences ranged
between -18 mm (anterior) and +6 mm (posterior). The distance was
less than 10 mm for 12 out of 15 cases medially and 13 out of 15
cases laterally. The median distance was 6 mm (1 to 18). For all
cases the locations were inside or at the edge of, the wear scar area. No significant correlation was found between
the distance and the measurement error of the model-based mJSW measurement (Spearman’s
rho = 0.07, p = 0.70).</p><fig id="f4" orientation="portrait" position="float"><label>Fig. 4</label><caption><p>Illustrations of the articular surfaces
of each explanted insert, showing the wear scar peripheries and
locations of the minimum insert thickness (p<sub>0</sub>) and the
femoral contact (p<sub>MB</sub>). These illustrations are plotted
as looking down on the superior surface of a right knee, with the
medial condyle always at the left side of the image. Illustrations
of inserts originating from left TKR are mirrored to fit this convention
and are indicated with an asterisk (*) after the case number.</p></caption><graphic xlink:href="2000304-galleyfig4"/></fig><table-wrap id="t3" orientation="portrait" position="float"><label>Table III</label><caption><p>The differences in position between
the femoral contact locations (p<sub>MB</sub>) and the minimum insert
thickness locations (p<sub>0</sub>) (as seen in Fig. 4). Values are
expressed in millimetres.</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1"><bold>Case</bold></th><th align="left" rowspan="1" colspan="1"><bold>Condition</bold></th><th colspan="2" align="left" rowspan="1"><bold>Medial compartment</bold></th><th colspan="2" align="left" rowspan="1"><bold>Lateral compartment </bold></th></tr></thead><tbody><tr><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td align="left" rowspan="1" colspan="1">AP difference</td><td align="left" rowspan="1" colspan="1">ML difference</td><td align="left" rowspan="1" colspan="1">AP difference</td><td align="left" rowspan="1" colspan="1">ML difference</td></tr><tr><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/></tr><tr><td align="left" rowspan="1" colspan="1">K2004</td><td align="left" rowspan="1" colspan="1">Mild</td><td align="left" rowspan="1" colspan="1">5.98</td><td align="left" rowspan="1" colspan="1">-1.84</td><td align="left" rowspan="1" colspan="1">0.63</td><td align="left" rowspan="1" colspan="1">1.06</td></tr><tr><td align="left" rowspan="1" colspan="1">K2133</td><td align="left" rowspan="1" colspan="1">Mild</td><td align="left" rowspan="1" colspan="1">5.98</td><td align="left" rowspan="1" colspan="1">-0.22</td><td align="left" rowspan="1" colspan="1">-2.06</td><td align="left" rowspan="1" colspan="1">0.81</td></tr><tr><td align="left" rowspan="1" colspan="1">K2145</td><td align="left" rowspan="1" colspan="1">Mild</td><td align="left" rowspan="1" colspan="1">-7.47</td><td align="left" rowspan="1" colspan="1">3.88</td><td align="left" rowspan="1" colspan="1">5.05</td><td align="left" rowspan="1" colspan="1">-0.68</td></tr><tr><td align="left" rowspan="1" colspan="1">K2154</td><td align="left" rowspan="1" colspan="1">Mild</td><td align="left" rowspan="1" colspan="1">0.52</td><td align="left" rowspan="1" colspan="1">0.76</td><td align="left" rowspan="1" colspan="1">-7.51</td><td align="left" rowspan="1" colspan="1">-0.96</td></tr><tr><td align="left" rowspan="1" colspan="1">K2171</td><td align="left" rowspan="1" colspan="1">Mild</td><td align="left" rowspan="1" colspan="1">2.02</td><td align="left" rowspan="1" colspan="1">-6.38</td><td align="left" rowspan="1" colspan="1">-1.29</td><td align="left" rowspan="1" colspan="1">2.26</td></tr><tr><td align="left" rowspan="1" colspan="1">K2178</td><td align="left" rowspan="1" colspan="1">Mild</td><td align="left" rowspan="1" colspan="1">1.67</td><td align="left" rowspan="1" colspan="1">-1.06</td><td align="left" rowspan="1" colspan="1">-0.46</td><td align="left" rowspan="1" colspan="1">1.20</td></tr><tr><td align="left" rowspan="1" colspan="1">K2035</td><td align="left" rowspan="1" colspan="1">Moderate </td><td align="left" rowspan="1" colspan="1">-1.95</td><td align="left" rowspan="1" colspan="1">-7.26</td><td align="left" rowspan="1" colspan="1">-0.14</td><td align="left" rowspan="1" colspan="1">-3.37</td></tr><tr><td align="left" rowspan="1" colspan="1">K2132</td><td align="left" rowspan="1" colspan="1">Moderate </td><td align="left" rowspan="1" colspan="1">-8.97</td><td align="left" rowspan="1" colspan="1">-8.80</td><td align="left" rowspan="1" colspan="1">-16.40</td><td align="left" rowspan="1" colspan="1">-2.59</td></tr><tr><td align="left" rowspan="1" colspan="1">K2137</td><td align="left" rowspan="1" colspan="1">Moderate </td><td align="left" rowspan="1" colspan="1">2.32</td><td align="left" rowspan="1" colspan="1">-6.57</td><td align="left" rowspan="1" colspan="1">-8.80</td><td align="left" rowspan="1" colspan="1">-3.39</td></tr><tr><td align="left" rowspan="1" colspan="1">K2144</td><td align="left" rowspan="1" colspan="1">Moderate </td><td align="left" rowspan="1" colspan="1">-9.04</td><td align="left" rowspan="1" colspan="1">-2.06</td><td align="left" rowspan="1" colspan="1">-9.10</td><td align="left" rowspan="1" colspan="1">-0.17</td></tr><tr><td align="left" rowspan="1" colspan="1">K2175</td><td align="left" rowspan="1" colspan="1">Moderate </td><td align="left" rowspan="1" colspan="1">-12.88</td><td align="left" rowspan="1" colspan="1">-1.16</td><td align="left" rowspan="1" colspan="1">-17.76</td><td align="left" rowspan="1" colspan="1">-2.18</td></tr><tr><td align="left" rowspan="1" colspan="1">K2046</td><td align="left" rowspan="1" colspan="1">Severe </td><td align="left" rowspan="1" colspan="1">-11.02</td><td align="left" rowspan="1" colspan="1">-3.69</td><td align="left" rowspan="1" colspan="1">-6.06</td><td align="left" rowspan="1" colspan="1">3.60</td></tr><tr><td align="left" rowspan="1" colspan="1">K2156</td><td align="left" rowspan="1" colspan="1">Severe </td><td align="left" rowspan="1" colspan="1">0.14</td><td align="left" rowspan="1" colspan="1">-1.07</td><td align="left" rowspan="1" colspan="1">-2.51</td><td align="left" rowspan="1" colspan="1">2.42</td></tr><tr><td align="left" rowspan="1" colspan="1">K2159<sup>*</sup></td><td align="left" rowspan="1" colspan="1">Severe </td><td align="left" rowspan="1" colspan="1">0.03</td><td align="left" rowspan="1" colspan="1">-0.53</td><td align="left" rowspan="1" colspan="1">1.27</td><td align="left" rowspan="1" colspan="1">1.99</td></tr><tr><td align="left" rowspan="1" colspan="1">K2161</td><td align="left" rowspan="1" colspan="1">Severe </td><td align="left" rowspan="1" colspan="1">4.89</td><td align="left" rowspan="1" colspan="1">-4.30</td><td align="left" rowspan="1" colspan="1">1.43</td><td align="left" rowspan="1" colspan="1">1.78</td></tr><tr><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/><td rowspan="1" colspan="1"/></tr></tbody></table><table-wrap-foot><fn><p>
AP, Anterioposterior direction; ML, mediolateral
direction
</p></fn></table-wrap-foot></table-wrap></sec></sec><sec><title>Discussion</title><p>The primary objective was to compare the accuracy and precision
of the model-based mJSW measurement and the conventional mJSW measurement
using minimum insert thickness measured from TKR retrievals as a
‘gold standard’. The accuracy (proximity to the truth) and precision
(measurement reproducibility) of both methods were determined by
applying the methods to pre-operative radiographs and comparing the outcomes with the minimum thickness of
the retrieved inserts. The results showed that the model-based measurement
method was more accurate than the conventional method for both condyles (0.50
mm <italic>vs</italic> 0.94 mm medially and 0.06 mm <italic>vs</italic> 0.34
mm laterally). The precision of the methods was similar (0.84 mm <italic>vs</italic> 0.79
mm medially and both 0.46 mm laterally). Both mJSW measurements
were more accurate and precise for the lateral condyle than for
the medial condyle. Since this occurred for both methods, this is
not a measurement error. Apparently a physical difference existed between
the femorotibial distance and the insert thickness, which may be
related to various clinical conditions such as varus malalignment.</p><p>Concerning the observer reproducibility for the model-based method
the mean difference was 0.0 mm and for the conventional method the
mean difference between the observers was 0.2 mm. The limits of
agreements of the mJSW measurement methods were similar. For the
cases K2154 and K2156 a large difference (> 0.5 mm) was found between
the model-based observers, mJSW measurements. For K2154, some bone
cement was still attached to the backside of the tibial baseplate
when it was scanned. This introduces a model inaccuracy and complicates
the matching procedure, as the respective contours of the tibial metal
baseplate should then not be used in the 2D/3D matching. One observer
deselected these particular contours, whereas the other observer
included this contour part, which may explain the measurement difference.
For K2156, one observer did not apply the 2D/3D matching process
for the tibial component correctly. This resulted in an out-of-plane
positioning error that affected the measurement outcome. Still,
the mean measurements for these cases were not remarkably far from
the actual minimum insert thickness. The outlier for the conventional
mJSW measurement (K2154) was related to a difference in setting the
height of the reference line at the tibial baseplate.</p><p>Four cases stand out (K2137, K2159, K2171 and K2178) as relatively
large overestimations (more than 1 mm) of the medial insert thickness
for both methods. For K2137 this seems to be related to the image
calibration: in the model-based optimisation the posterior edge
of the femoral component models is approximately 3 cm away from the
X-ray detector plate, which is physically unlikely. For K2159 there
is a large difference between the measurement location, p<sub>MB</sub>,
and the actual minimum insert thickness location, p<sub>0</sub>.
For the other cases no obvious explanation could be found, and it
may be possible that for these patients there was no actual contact
at the mJSW position p<sub>MB</sub> at the medial side.</p><p>Our secondary objective was to investigate whether the explanted
inserts truly show wear
scars at the points measured by the model-based mJSW technique.
The analysis showed that this was true for all inserts. It should be
noted that for some cases, such as K2156 and K2132, the wear scar
covers the majority of the inserts’ articular surface area, which
dilutes the information of this observation as any measurement is
bound to reside in the wear scar area. Still, this finding supports
the proposition that the mJSW measurement is suitable to detect
wear.</p><p>Concerning the difference between the minimum insert thickness
location (p<sub>0</sub>) and the femoral contact location (p<sub>MB</sub>),
the findings were volatile. The findings were similar for the medial
and lateral condyles: the Euclidean difference was less than 10
mm for 12 cases medially and 13 cases laterally. When this difference
was greater than 10 mm, the measurement point was always more anterior
than p<sub>0</sub>. This could be related to the patient positioning:
patients are standing with extended knees during the image acquisition,
whereas the femoral condyles reposition during dynamic activities.<sup><xref rid="r15" ref-type="bibr">15</xref></sup> In posterior cruciate
ligament-retaining TKR, knee flexion during activity can contribute
to posterior contact of the femoral condyles and posterior wear
scars.<sup><xref rid="r16" ref-type="bibr">16</xref></sup> This
is supported by the observation that three out of four cases with
severe wear had a relatively posterior location for p<sub>0</sub>.
The anteroposterior direction also corresponds to the film-focus direction
for a frontal plane radiograph, for which the 2D-3D model matching
algorithm is the least accurate. The difference in location can
therefore also be related to measurement error.</p><p>Collier et al<sup><xref rid="r7" ref-type="bibr">7</xref></sup> found
that conventional mJSW measurements had an accuracy within 1 mm
for 82% medially and 58% laterally. This is comparable to the findings with
the conventional method in the current study (60% medially and 87%
laterally within 1 mm), although the accuracy numbers for the condyles
are interchanged. Differences between these results could be caused
by the type of prosthesis that was evaluated. Whereas Collier et al<sup><xref rid="r7" ref-type="bibr">7</xref></sup> used a single, flat-surfaced
anatomic modular knee (Depuy, Warsaw, Indiana), the measurements in
the current study were applied to five different implant designs
to validate our measurement technique as a more generic application
to different implant models. This also included designs having a
metal rim capture mechanism on the tibial baseplate, which can distort
the projection image and for which an alternative approach of the
conventional mJSW method had to be used. Moreover, Collier, et al<sup><xref rid="r7" ref-type="bibr">7</xref></sup> achieved good measurement
accuracy only when TKR were well aligned relative to the projection
plane, necessitating that 28% to 39% of their radiographs be discarded
from the measurement analysis due to excessive anteroposterior tilt
of the tibial baseplate.<sup><xref rid="r3" ref-type="bibr">3</xref>,<xref rid="r7" ref-type="bibr">7</xref></sup> For the current
study, all radiographs were used regardless of baseplate tilt.</p><p>Moreover, in the prior validation study the model-based mJSW
measurement showed a <sc>sd</sc> of 0.2 mm in case of fixed-bearing
TKRs, against 0.79 mm medially and 0.46 mm laterally in the current
study.<sup><xref rid="r8" ref-type="bibr">8</xref></sup> An
explanation for this difference is that repeated measurements for
a single TKR were used in the validation study, whereas 15 different
TKRs were measured in our current study. Moreover, in the validation
study the inserts were replaced with a flat acrylic block.<sup><xref rid="r8" ref-type="bibr">8</xref></sup> This approach removed the
possibility that sagittal plane curvature of the articular surface
could lead to large variations in thickness with only slight deviations
in the anteroposterior position of the femoral condyle.</p><p>This study was set up in an attempt to capture a representative
range of wear severity in a limited number of implant designs and
to obtain a first impression of the accuracy that can be obtained
with the model-based mJSW method <italic>in vivo</italic>. In future
work the data need to be augmented to include a wider range of prosthesis
designs with varied insert curvature and to determine the precision
of the method when longitudinal data are analysed.</p><p>The model-based mJSW measurement requires accurate tibial and
femoral models. In this study, models were generated by reverse
engineering prosthesis components that were retrieved from the cohort
of included patients. This resulted in the best possible model accuracy
for the model-based method.<sup><xref rid="r11" ref-type="bibr">11</xref></sup> In
practice, it will not be possible to use such patient-specific models,
as longitudinal assessments of polyethylene wear are conducted without availability
of retrieved components. In that case scanned models (reverse engineered
models) are recommended that can be produced based on matching components (i.e.
of the same type and size) for which the costs of production are
relatively low.</p><p>Contour detection and optimisation can be time-consuming tasks
of the model-based mJSW measurement, which might limit the use in
clinical evaluation studies. A topic of further research is to reduce
the measurement time using further automation of the measurement
procedures. The measurement could also be improved by reducing the
out-of-plane error of the optimisation. For example, this could
be realised by restricting the freedom of the model pose using prior
knowledge on the allowed range of movement of the TKR.<sup><xref rid="r17" ref-type="bibr">17</xref></sup></p><p>In conclusion, the model-based mJSW measurement method delivers
a more accurate estimation of the <italic>in vivo</italic> insert
thickness from planar radiographs compared with the conventional
measurement. In addition, it provides information on the mJSW location
which is indicative for the site of the wear. Further research is
required to come to a standardised measurement protocol and to investigate
whether the model-based mJSW can hold its accuracy gain in longitudinal
data and for a broader range of prosthesis designs. </p><p><bold>Acknowledgement:</bold> The authors wish to thank H. van
der Linden, MD, (HvdL) for her contribution in the experiment.</p></sec> |
Patients’ perspectives on providing a stool sample to their GP: a qualitative study | <sec><title>Background</title><p>Stool specimen collection is challenging and informal feedback has indicated that participants find the process difficult. Increasing stool specimen returns would improve the investigation of outbreaks of diarrhoeal and food-borne disease.</p></sec><sec><title>Aim</title><p>To explore the barriers to stool sample collection and specimen return to ascertain which factors may help to improve the process.</p></sec><sec><title>Design and setting</title><p>Qualitative patient interview study in Gloucester, UK.</p></sec><sec><title>Method</title><p>A two-stage purposive sampling process was used to identify patients who had either previous experience or no experience of collecting a stool sample. The interview schedule, based on the theory of planned behaviour, was used to facilitate interviews with 26 patients. Interview transcripts were analysed using a modified framework analysis.</p></sec><sec><title>Results</title><p>Barriers to collection included embarrassment, fear of results, concerns around hygiene and contamination, discretion and privacy, and lack of information. Personal gain was identified as the main incentive to collecting and returning a stool sample. The need for an information leaflet on stool collection was emphasised by most patients.</p></sec><sec><title>Conclusions</title><p>GPs could make a number of small changes that could make a big difference for patients and potentially increase stool sample return. If they, rather than receptionists, distributed collection kits it may be easier for patients to ask any questions they had regarding collection. In addition, the provision of a stool-collection information leaflet could increase patients’ confidence regarding collecting the sample, and providing drop-off boxes for specimens could help prevent patients’ embarrassment regarding handing their stool over to a receptionist.</p></sec> | <contrib contrib-type="author"><name><surname>Lecky</surname><given-names>Donna M</given-names></name><degrees>PhD</degrees><role>Project manager</role></contrib><aff>Public Health England Primary Care Unit, Gloucester.</aff> | The British Journal of General Practice | <sec sec-type="intro"><title>INTRODUCTION</title><p>Collecting a stool specimen can be a definitive step in determining the diagnosis and appropriate treatment for suspected infectious diarrhoea and other gastrointestinal disease.<xref rid="b1" ref-type="bibr">1</xref> Stool culture results also form the basis of ongoing surveillance of infectious diarrhoea in the community.<xref rid="b2" ref-type="bibr">2</xref> Stool specimens may also be required for non-microbiological testing, for example faecal occult blood testing (FOBt), an early detection method for colorectal cancer.<xref rid="b3" ref-type="bibr">3</xref> Despite the fact that early FOBt screening has been shown to reduce mortality by 16%,<xref rid="b3" ref-type="bibr">3</xref> compliance is rarely >60%;<xref rid="b4" ref-type="bibr">4</xref> reasons given for the lack of patient compliance with stool collection for FOBt include embarrassment, concerns about screening results, and inconsistent or inadequate support from friends and family.<xref rid="b5" ref-type="bibr">5</xref></p><p>To the best of the authors' knowledge, no research has been published examining the patient perspective of collecting stool samples for microbiological testing. Due to the lack of current research in the area, this study aimed to explore, through patient interviews, the barriers to stool specimen collection and return, as well as factors that may help to improve the process. It was hoped that the results would inform the improvement of stool collection instructions for patients participating in a stool surveillance study and improve national guidance for GPs and patients on the investigation of suspected infectious intestinal disease.</p><p>Current GP guidance focuses on the treatment of diarrhoea<xref rid="b6" ref-type="bibr">6</xref> and not the collection of the stool sample itself. The theory of planned behaviour<xref rid="b7" ref-type="bibr">7</xref> has been used to develop the interview schedule to identify patients’ attitudes, subjective norms, and perceived behavioural control to submitting a stool sample in order to determine their intentions to act. The model suggests that individuals are more likely to intend to perform a behaviour (returning a stool sample) if they have a positive attitude towards it, perceive social pressure from others to perform the behaviour (subjective norms), and perceive that the performance of the behaviour is within their control (perceived behavioural control)<xref rid="b8" ref-type="bibr">8</xref> (<xref ref-type="fig" rid="fig1">Figure 1</xref>).</p><fig id="fig1" position="float"><label>Figure 1.</label><caption><p><bold><italic>Theory of planned behaviour intention to collect a stool specimen for microbiological testing.</italic></bold></p></caption><graphic xlink:href="bjgpNov-2014-64-628-e684-OA-1"/></fig></sec><sec sec-type="methods"><title>METHOD</title><sec><title>Participant selection and recruitment</title><p>A two-stage purposive sampling process was used to identify patients who had either previous experience (Group 1) or no previous experience (Group 2) of collecting a stool sample. Those included were patients aged >18 years, who had submitted a stool (Group 1) or blood sample (Group 2), as requested by a GP, to the microbiology department of Gloucestershire Royal Hospital in the 3 months leading up to the recruitment phase. Women who were pregnant or patients who were terminally ill were excluded.</p><p>Potential participants were stratified by ethnic origin, age, sex, and stool consistency (Group 1 only). White patients were selected randomly from each stratified list. All patients of non-white ethnicity were invited to take part due to their low numbers. The researchers aimed to recruit up to five participants from each stratum.</p><boxed-text position="float"><sec><title>How this fits in</title><p>Stool specimen collection is needed to inform the management of many gastrointestinal diseases and infections, but returns by patients are generally <60%. Through interviews based on the theory of planned behaviour with patients who had and had not submitted stool specimens, it was found that personal attitudes, subjective norms, and perceived behavioural controls all influenced specimen return. Patients perceived that handling stools was dirty and embarrassing, and it was found that a lack of information about why patients were collecting the stool, how to do it, receptionist involvement, privacy during returning specimens, and fear of results were all barriers to collection. Stool specimen returns may be increased through greater explanation about the reason for collection by the GP, providing the patient with plastic gloves (or telling them where to get them), and giving the patient Public Health England’s patient information leaflet on stool collection, which includes diagrams and opaque bags for return.</p></sec></boxed-text><p>Invitation letters containing study information from the local microbiologist and Public Health England Primary Care Unit (PCU) were sent to all selected patients. Letters describing the study were also sent to each participant’s GP practice. Willing participants provided written informed consent. Telephone calls were used to organise interview dates and venues. Patients were reimbursed for their time with a £10 voucher.</p></sec><sec><title>Interview sessions</title><p>Participants selected whether they preferred to be interviewed in their own home or a hospital office. Where possible, interviewers were matched in both ethnicity and sex to the participants.</p><p>Interviewers used a flexible interview schedule that had been developed by the investigators based on the theory of planned behaviour and comprised a mixture of closed and open-ended questions (<xref ref-type="app" rid="app1">Appendix 1</xref>). Visual aids in the form of a standard stool collection kit available in England and patient information leaflets were used to facilitate interviews. Each interview session lasted approximately 45 minutes.</p></sec><sec sec-type="methods"><title>Analysis</title><p>All interviews were recorded, with permission, and transcribed verbatim. Transcripts were analysed using a modified framework analysis. Transcripts were first read through for accuracy and to gain knowledge of the data. Two researchers independently coded categories and themes: the lead researcher coded all transcripts and a second researcher coded 10% of the transcripts to ensure coding consistency. The researchers then agreed categories and themes; discrepancies were resolved through discussion and referral to original transcripts until agreement was reached.</p><p>Themes were revised iteratively as the fieldwork and analysis progressed.<xref rid="b9" ref-type="bibr">9</xref> Use of NVivo software (version 10) facilitated the organisation of the data. The one sheet of paper (OSOP) method was used to clarify findings within, and between, themes.<xref rid="b10" ref-type="bibr">10</xref></p></sec></sec><sec sec-type="results"><title>RESULTS</title><p>Between March 2012 and June 2013, a total of 288 patients (Group 1, <italic>n</italic> = 118; Group 2, <italic>n</italic> = 170) were invited to participate. Thirty five (12.2%) agreed to do so; however, only 26 (9.0%) patients were interviewed due to patient drop out (<xref ref-type="fig" rid="fig2">Figure 2</xref>).</p><fig id="fig2" position="float"><label>Figure 2.</label><caption><p><bold><italic>Participant recruitment and characteristics.</italic></bold></p></caption><graphic xlink:href="bjgpNov-2014-64-628-e684-OA-2"/></fig><p>Stool collection methods previously used by Group 1 varied between participants but stool consistency had no effect on the collection method. Participants felt that the collection of a stool sample would be easier next time they were asked to do so. Forty per cent of Group 1 participants found it harder than they had expected due to lack of knowledge or instructions on how to collect the sample, the consistency of the stool, or the fact that it was messy. Group 2 participants’ main concern on stool collection was not knowing how to collect the specimen.</p><p>Overall six major themes emerged from the interviews:
<list list-type="bullet"><list-item><p>barriers to collection;</p></list-item><list-item><p>incentives to collection;</p></list-item><list-item><p>information and support;</p></list-item><list-item><p>collection management;</p></list-item><list-item><p>relationships; and</p></list-item><list-item><p>information and support.</p></list-item></list></p><p>These themes were further examined using the theory of planned behaviour model as outlined below.</p><sec><title>Personal attitudes: outcome beliefs</title><sec><title>Lack of information</title><p>Participants with previous experience of stool collection highlighted a lack of information from the GP on how to collect a stool sample:
<disp-quote><p>‘But I didn’t know what to do. I thought “how do you, how do you catch it here,” I thought, “without it ending up in the water?” So I thought if I pee or poo on some cardboard then take a little bit off and then that’s it in there?’</p><attrib>(Group 1, participant 5)</attrib></disp-quote>
<disp-quote><p>‘I think it might have been more helpful as to what they were, you know, what to include in, in the sample, then you know not just ordinary poo but the mucus and you know what, what they really wanted, so I think that would be more helpful.’</p><attrib>(Group 1, participant 1)</attrib></disp-quote></p></sec><sec sec-type="results"><title>Fear of results</title><p>Fear of the possibility of receiving bad results was also a frequent response:
<disp-quote><p>‘People, I mean I think people, um, don’t want to know the worst, and I’d rather live with what I don’t know than try and live with what I do know type of thing. I think I can be a bit like that sometimes.’</p><attrib>(Group 2, participant 4)</attrib></disp-quote>
<disp-quote><p>‘Sometimes not wanting to know the results. They know something is wrong but they don’t… sometimes some people can’t deal with it.’</p><attrib>(Group 2, participant 11)</attrib></disp-quote></p><p>Such a response was particularly the case when patients wrongly assumed the request for a stool sample was linked to colorectal cancer screening.</p></sec></sec><sec><title>Personal attitudes: rewards of action</title><sec><title>Personal gain</title><p>The main driver to returning the sample appeared to be personal benefit:
<disp-quote><p>‘I think if it’s to my benefit, I would do it.’</p><attrib>(Group 1, participant 3)</attrib></disp-quote>
<disp-quote><p>‘And somehow or other you have to create a shift in responsibility, that is, the patient takes responsibility for their own health and they’re having a stool sample tested because it’s clinically necessary.’</p><attrib>(Group 2, participant 8)</attrib></disp-quote></p><p>If the patient understood why they were being asked for the sample and when they should expect to get the results, they were happier to provide the sample:
<disp-quote><p><italic>‘They</italic> [GPs] <italic>have obviously got to emphasise what the results are going to show and whether that will have a positive or negative effect on your own personal health so therefore it has got to be stressed why they are doing it.’</italic></p><attrib>(Group 2, participant 10)</attrib></disp-quote></p></sec></sec><sec><title>Subjective norms: normative beliefs</title><sec><title>Embarrassment</title><p>The main barriers to stool collection included the embarrassment of other people knowing that they were carrying their stool in a container:
<disp-quote><p>It’s embarrassing, it’s just a subject that is, isn’t it? I think what I found embarrassing about it was that it was a clear jar so when you’re handing it over and it’s in clear polythene as well … I found that very embarrassing. You know, “here’s my sample”.’</p><attrib>(Group 1, participant 1)</attrib></disp-quote></p><p>In addition, participants were embarrassed about the actual process of collecting the sample as you are ‘taught not to handle poo’; they felt that this may be a main reason why some people lack the motivation to comply:
<disp-quote><p><italic>‘We have all been taught not to handle and deal with poo, and being embarrassed that you have had to do it yourself …</italic> [It] <italic>might be something that they don’t want to do.’</italic></p><attrib>(Group 2, participant 10)</attrib></disp-quote></p></sec></sec><sec><title>Subjective norms: motivation to comply</title><sec><title>Patient–GP relationships</title><p>Participants reported an extremely trusting relationship between themselves and their GP. All participants said they were happy for a GP to ask them for a stool sample without questioning why:
<disp-quote><p>‘You don’t question your doctor. You know it’s in your own interest really.’</p><attrib>(Group 1, participant 12)</attrib></disp-quote></p><p>Participants also viewed their GP as the main source of information on stool collection:
<disp-quote><p><italic>‘How did I find discussing it? Um, well we didn’t really. Um, we didn’t at all. I mean, he told me what it was for um, and then</italic> […] <italic>I went to reception and asked for a stool sample bottle and she gave it to me and that was it. There wasn’t really any discussion about it.’</italic></p><attrib>(Group 1, participant 1)</attrib></disp-quote>
<disp-quote><p>‘There wasn’t much discussion. It was just “give me a stool sample and go to the um, receptionist, err, to get it” and that was it.’</p><attrib>(Group 1, participant 4)</attrib></disp-quote></p></sec><sec><title>The family relationship</title><p>This plays an important part in, not only seeking medical advice, but also returning stool samples. Although, in many cases, participants were self-motivating in visiting the GP, most of them, particularly females, discussed the sample request openly with their family:
<disp-quote><p>‘Well I’d discuss it with my sister at the weekend because she and her husband have, um, both had to do samples recently. So um, yes, it’s not something we’d discuss but it’s something I might mention in passing ... we didn’t really go into details of how it works and what it is.’</p><attrib>(Group 2, participant 4)</attrib></disp-quote></p><p>Men more often said that they would only discuss it with their partner/wife:
<disp-quote><p>‘Well my wife would know because I would tell her but she wouldn’t have a problem as long as there was a reason for me having to do it.’</p><attrib>(Group 2, participant 10)</attrib></disp-quote></p><p>Male participants were the only ones who stated that they would not discuss this with their family.</p></sec><sec><title>Stool collection kits</title><p>Participants expected more from kits than what is currently provided by GP practices:
<disp-quote><p>‘I think that is a pathetic little kit. It’s insufficient, it’s … patients need as much information as possible and possibly you need to bore them with it, but a lot of what is said to a patient in a consulting room that’s forgotten when they leave. So all the instructions about how to go ahead and collect a stool sample needs to be in this.’</p><attrib>(Group 2, participant 8)</attrib></disp-quote></p><p>Expectations included instructions, a collection device such as a spoon or spatula, a collection pot, a coloured/paper bag in which to return the sample, and a pair of gloves to facilitate collection. As Group 1, participant 3, suggested:
<disp-quote><p>‘It could include a pair of surgical gloves, maybe help with that might encourage people to do it.’</p></disp-quote></p><p>Participants in Group 2 suggested that the lack of these materials may be a reason why some people do not return their sample.</p><p>Most participants from Group 1 first saw the stool collection kit when collecting it from the receptionist after the GP consultation. At that point, participants in Group 1 reported not feeling comfortable asking the receptionist questions but said it was too late to ask their GP.</p><p>Most participants reported that they would not be happy for the receptionist to ask them to collect a stool sample, mainly because they were not professionally trained. Those who were happy for the receptionist to ask for a sample felt it was because the GP had requested it.</p></sec></sec><sec><title>Perceived behavioural control: self-efficacy</title><sec><title>Information leaflets</title><p>Although only two participants received an information leaflet, the majority said they would have expected or liked to have been offered one. Participants wanted information on:
<list list-type="bullet"><list-item><p>why the sample was required;</p></list-item><list-item><p>how much sample was required;</p></list-item><list-item><p>how to collect the sample;</p></list-item><list-item><p>where they should return it; and</p></list-item><list-item><p>when they should expect to get the results.</p></list-item></list></p><p>All participants stated that any information leaflet should have images, large font and wording that would be easily understood by the general public. Participants also stated that the leaflets should:
<list list-type="bullet"><list-item><p>include a step-by-step guide using images or diagrams;</p></list-item><list-item><p>have clear, concise information in large font;</p></list-item><list-item><p>be in simple language that is easy to understand;</p></list-item><list-item><p>be in colour;</p></list-item><list-item><p>have a professional appearance;</p></list-item><list-item><p>be available in different languages;</p></list-item><list-item><p>be available on the internet to save the surgery or NHS printing costs;</p></list-item><list-item><p>provide information on how to collect the stool sample, why collecting the sample is important, when you should expect to receive the results, how to dispose of unwanted material, and emphasise the importance of washing hands; and</p></list-item><list-item><p>state where to hold the sample overnight (preferably not in the fridge).</p></list-item></list></p></sec></sec><sec><title>Perceived behavioural control: external factors</title><sec><title>Hygiene concerns</title><p>Getting their hands dirty, handling faeces, or disposing of the sample, and putting their hands near the toilet pan were common issues raised by participants:
<disp-quote><p>‘Well, I didn’t particularly want to get my hands dirty. Um, it’s something you can’t, you’re kind of brought up to, you know, that’s dirty and you don’t wanna touch it or ...’</p><attrib>(Group 1, participant 11)</attrib></disp-quote>
<disp-quote><p>‘I don’t like the idea of having to put your hand into the toilet as well when you’ve already been to the toilet.’</p><attrib>(Group 1, participant 17)</attrib></disp-quote></p><p>Six responders expressed concerns around potential contamination of the sample during collection or of the sample leaking and contaminating other things:
<disp-quote><p>‘… and as long as it wasn’t getting contaminated or anything like that then um, …. Equally it could … ah … the sort of thing, if it got damaged, it wouldn’t be very nice and also ah … the risk of, I don’t know, contamination maybe.’</p><attrib>(Group 1, participant 12)</attrib></disp-quote></p><p>Concern over how to dispose of collection devices was mentioned by participants who had no previous experience of stool collection:
<disp-quote><p>‘And then something to dispose the receptacle that you’ve caught it in, because you’re not going to want to put that in the bin and you’re not going to want to put it down the loo either, so what are you supposed to do with it?’</p><attrib>(Group 2, participant 8)</attrib></disp-quote></p></sec><sec><title>Discretion and privacy</title><p>Discretion and privacy were extremely important to patients regarding the request and return of a stool sample; many saw the receptionist as a barrier to this as they were not a medical professional:
<disp-quote><p>‘Um, if a receptionist asked me. Yeah I mean it’s not the nicest place the reception is it? You got a bit more of a public presence out there. Ahhh, and yeah you don’t wanna be discussing. So if ah … I mean if it was done discreetly — and I’m sure they’d try to — but ah … receptions are always a busy little area so it would not be an ideal place. It wouldn’t be my first choice anyway, if it was like a little room or a doctor’s own room then you got more privacy really.’</p><attrib>(Group 1, participant 12)</attrib></disp-quote>
<disp-quote><p>‘Well, as long as there was a certain amount of privacy involved, yes. You don’t want the whole waiting room listening!’</p><attrib>(Group 2, participant 8)</attrib></disp-quote></p><p>Responders indicated that, as the reception area lacked privacy, they would prefer to return the sample in a drop box or post it to the surgery, especially as the stool collection container was transparent and no opaque bag or cover was provided to return the sample. Ease of returning the sample was strongly linked with discretion and privacy:
<disp-quote><p>‘Oh that might be easier because then you can just go in, pop it in the box and then leave then without having to queue, you hand it over the reception desk, just drop it off it might be easier.’</p><attrib>(Group 1, participant 4)</attrib></disp-quote>
<disp-quote><p>‘It was actually a lot easier putting it into a box than giving it to a person.’</p><attrib>(Group 1, participant 11)</attrib></disp-quote></p></sec></sec></sec><sec sec-type="discussion"><title>DISCUSSION</title><sec sec-type="conclusions"><title>Summary</title><p>The present study used the theory of planned behaviour to develop an interview schedule to help identify patients’ attitudes towards collecting and returning a stool sample to their GP for microbiological examination. It is evident from this study that patients’ personal attitudes have a major influence on stool collection; patients do not view the request for a stool sample as routine and, therefore, fear the request and future results. In this instance they do not perceive any <italic>reward</italic> for their action.</p><p>The embarrassment of other people, including receptionists, knowing that patients had collected a stool sample and the taboo associated with the ‘dirtiness’ of human faeces were key subjective norms. Participants felt that this may be a key reason why some people lack the motivation to comply.</p><p>The greatest barrier to collection was perceived as being a lack of confidence or a lack of instructions on how to collect the sample, along with the undesired involvement of the receptionist in the process.</p></sec><sec><title>Strengths and limitations</title><p>To the best of the authors' knowledge, this is the first study examining the patient perspective of collecting and submitting a stool sample for routine microbiological examination. The findings are of particular importance because all participants were primary care patients and their responses reflect real issues about stool collection in this setting; previously, much research in this area has been with participants involved in colorectal cancer screening.<xref rid="b4" ref-type="bibr">4</xref>,<xref rid="b5" ref-type="bibr">5</xref></p><p>Although 27% of participants were non-white, the Asian community were under-represented and therefore the study could not identify cultural barriers to stool collection for this group.</p><p>Although the majority of participants in this study were aged 31–71 years, no differences between age groups were identified and so the authors believe that these findings will also be applicable to younger patients.</p></sec><sec><title>Comparison with existing literature</title><p>In line with the findings presented here, a survey of South Asian women in England found that the most important factors affecting FOBt response related to the difficulty of collecting the stool specimen;<xref rid="b11" ref-type="bibr">11</xref> other research has also suggested that perceived provider attitudes play an extremely important role in how comfortable patients feel in returning a stool sample.<xref rid="b12" ref-type="bibr">12</xref>–<xref rid="b14" ref-type="bibr">14</xref></p><p>Other literature has also suggested that patient health outcomes can be improved with good GP–patient communication.<xref rid="b15" ref-type="bibr">15</xref> In 2012, in almost one-quarter of calls to The Patients Association’s helpline, patients said their GP reacted ‘very poorly’ and refused to talk about their concerns.<xref rid="b16" ref-type="bibr">16</xref> An open dialogue between patient and doctor may go some way to removing the taboo associated with handling human faeces and the advice given could alleviate patient concern of sample contamination.<xref rid="b17" ref-type="bibr">17</xref> When discussing colorectal cancer screening Hynman <italic>et al,</italic><xref rid="b18" ref-type="bibr">18</xref> suggested that, to increase screening compliance, the potential illness should be explained to the patient to allay fears of hospitalisation and treatment, and that benefits should be explained to overcome reservations.</p><p>In the study reported here, it was found that most patients were asked to collect the stool collection kit from the receptionist. However, patients did not view the receptionist as a health professional and, therefore, did not feel comfortable asking questions about stool collection. Although the focus of this study was not on the patient–receptionist relationship, previous studies found that receptionists were perceived as rude, impersonal, insensitive or officious,<xref rid="b19" ref-type="bibr">19</xref> which may further explain the reasons behind patients’ perceived embarrassment in returning the sample to the receptionist.</p><p>The King’s Fund recently reported that practices performing well on delivering a good experience for their patients also perform well on measures of clinical quality.<xref rid="b20" ref-type="bibr">20</xref> In a recent English survey, 89% of GP responders (<italic>n</italic> = 477)<xref rid="b21" ref-type="bibr">21</xref> reported giving verbal advice to patients on how to collect a stool specimen although only 2% gave written instructions;<xref rid="b22" ref-type="bibr">22</xref> this is despite the fact that it has been shown that screening compliance is significantly improved when patients have an information leaflet.<xref rid="b23" ref-type="bibr">23</xref></p></sec><sec sec-type="discussion"><title>Implications for practice</title><p>The findings of this study suggest that patients trust the judgement of their GP; as such, there are a number of small changes that GPs could implement that could make a large difference to patients and potentially increase their stool sample returns.</p><sec><title>Improving personal attitudes</title><p>GPs, rather than receptionists, could give patients the stool collection kits; this would likely increase patient confidence in stool collection and allow patients to ask questions about the process. Explaining why the sample is required may remove the fear associated with being asked to submit a non-routine sample request, as well as highlighting the personal benefit to the patients of returning the sample.</p></sec><sec><title>Removing negative subjective norms</title><p>GPs discussing stool collection with their patients may also help to remove the taboo associated with stool collection. The provision of drop-off boxes for sample return may also help remove the embarrassment associated with returning the sample to the receptionist in front of other patients.</p></sec><sec><title>Increasing perceived behavioural control</title><p>The provision of gloves, or advice about where gloves could be obtained, may also help to remove the perceived ‘dirtiness’ that is associated with stool collection and, in turn, increase sample return. In addition, the provision of an opaque bag in which to return the sample, for example, would meet patients’ needs for privacy and discretion.</p><p>This research also found that participants wanted — and expected to receive — a stool collection information leaflet. The authors have used the patients’ suggestions to develop a new stool collection information leaflet for those participating in a stool surveillance study (<xref ref-type="app" rid="app2">Appendix 2</xref>); this will be modified for use with GPs and patients undergoing investigation of suspected infectious intestinal diseases. The modified leaflet will be made available alongside the management of diarrhoea guidance on Public Health England’s website in the next few months, and can also be requested directly from the lead author, but should also be promoted via GP computer systems, NHS information sources, laboratories, and through other professional bodies.</p></sec></sec></sec> |
Ethnicity and the diagnosis gap in liver disease: a population-based study | <sec><title>Background</title><p>Liver disease is a major cause of morbidity and mortality worldwide. Large numbers of liver function tests (LFTs) are performed in primary care, with abnormal liver biochemistry a common finding. Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver injury. Metabolic syndrome, common in people from South Asia, is an important risk factor for NAFLD.</p></sec><sec><title>Aim</title><p>It is hypothesised that a large gap exists between numbers of patients with abnormal LFTs and those with recorded liver diagnoses, and that NAFLD is more common among adults of South Asian ethnic groups.</p></sec><sec><title>Design and setting</title><p>A cross-sectional study of 690 683 adults in coterminous general practices in a region with high ethnic diversity.</p></sec><sec><title>Method</title><p>Data were extracted on LFTs, liver disease, and process of care measures from computerised primary care medical records.</p></sec><sec><title>Results</title><p>LFTs were performed on 218 032 patients, of whom 31 627 had elevated serum transaminases. The prevalence of abnormal LFTs was highest among individuals of Bangladeshi ethnicity. Of the patients with abnormal LFTs, 88.4% did not have a coded liver diagnosis. NAFLD was the most frequently recorded liver disease and was most common among Bangladeshi patients. In a multivariate analysis, independent risk factors for NAFLD included Bangladeshi ethnicity, diabetes, raised BMI, hypertension, and hypercholesterolaemia.</p></sec><sec><title>Conclusion</title><p>Abnormal LFTs are common in the population, but are underinvestigated and often remain undiagnosed. Bangladeshi ethnicity is an important independent risk factor for NAFLD.</p></sec> | <contrib contrib-type="author"><name><surname>Alazawi</surname><given-names>William</given-names></name><degrees>MA, PhD, MRCP</degrees><role>Senior lecturer and consultant in hepatology</role></contrib><aff>The Liver Unit, The Blizard Institute, Barts & The London School of Medicine, London, UK.</aff> | The British Journal of General Practice | <sec sec-type="intro"><title>INTRODUCTION</title><p>Hospital admissions and deaths from liver disease are rising across the Western world.<xref rid="b1" ref-type="bibr">1</xref>,<xref rid="b2" ref-type="bibr">2</xref> Most epidemiological studies focus on prevalence rates of individual diseases in selected hospital populations<xref rid="b3" ref-type="bibr">3</xref> as much early stage liver disease in primary care settings is asymptomatic. Alternative strategies include studying community cohorts, such as those found to have liver-related abnormalities on blood testing, or studying diagnostic prevalence in computerised primary care records.<xref rid="b4" ref-type="bibr">4</xref>–<xref rid="b7" ref-type="bibr">7</xref></p><p>Early identification of liver disease is important for timely intervention. Diagnoses are usually made after measurement of liver biochemical tests, chiefly alanine transaminase (ALT) and aspartate aminotransferase (AST). Increasing numbers of liver function tests (LFTs) are performed in primary care<xref rid="b5" ref-type="bibr">5</xref>,<xref rid="b8" ref-type="bibr">8</xref>,<xref rid="b9" ref-type="bibr">9</xref> for a variety of indications.<xref rid="b10" ref-type="bibr">10</xref>,<xref rid="b11" ref-type="bibr">11</xref> Despite high rates of testing, a large number of patients continue to present to secondary care in the later, decompensated, stages of disease, suggesting that there are important gaps between detecting abnormalities, making diagnoses, and implementing treatment.</p><p>Abnormal LFTs are not universal in chronic liver disease. Long periods in the natural history of conditions such as non-alcoholic fatty liver disease (NAFLD) or chronic hepatitis B virus (HBV) infection are characterised by transaminase values within the reference range, masking ongoing liver injury.</p><p>In the UK the most common cause of liver dysfunction is NAFLD, affecting 10–30% of the general population, rising to 80–90% in obese patients.<xref rid="b3" ref-type="bibr">3</xref>,<xref rid="b12" ref-type="bibr">12</xref>–<xref rid="b16" ref-type="bibr">16</xref> NAFLD is closely linked with other components of the metabolic syndrome: obesity, type 2 diabetes, hypercholesterolaemia, and hypertension.<xref rid="b17" ref-type="bibr">17</xref> The prevalence of NAFLD in different ethnic groups living in developed countries has received little attention. The prevalence of hepatic steatosis was most common in Hispanic patients in an American study,<xref rid="b13" ref-type="bibr">13</xref> and NAFLD may be more aggressive in those of Latino origin.<xref rid="b18" ref-type="bibr">18</xref> Little is known of prevalence in other populations, but given the high rates of metabolic syndrome in South Asian populations,<xref rid="b19" ref-type="bibr">19</xref> the authors hypothesise that NAFLD is more prevalent in these ethnic groups.</p><p>A regional database was used to identify patients with abnormal LFTs to estimate the burden of undiagnosed liver disease in primary care. Rates of recorded NAFLD were studied in patients from different ethnic groups to determine whether South Asian ethnicities are at increased risk.</p></sec><sec sec-type="methods"><title>METHOD</title><p>The study was conducted in three east London boroughs (Tower Hamlets, Newham, and City & Hackney), where more than 50% of the population are from ethnic minority groups. Data were anonymised and extracted from the electronic record for all patients aged ≥18 years registered with the 150 out of 154 GP practices that use the EMIS Web clinical system (total 817 700). Data were managed according to UK NHS information governance requirements.</p><boxed-text position="float"><sec><title>How this fits in</title><p>Abnormal liver function tests (LFTs) are a common finding in primary care, and in this study it is shown that many patients with such abnormalities have no recorded liver diagnoses. Ethnic group and components of the metabolic syndrome are among key independent risk factors for abnormal LFTs, and a large number of these patients are likely to have undiagnosed non-alcoholic fatty liver disease (NAFLD). NAFLD is the most common liver diagnosis in the general population and being of Bangladeshi, but not any other South Asian ethnicity, is identified as a novel independent risk factor for this condition.</p></sec></boxed-text><p>Demographic variables included age, sex, ethnic group, and social deprivation. Self-reported ethnic group was collapsed into categories: white, Bangladeshi, Indian, Pakistani, African, Caribbean, and other (including mixed) and not stated (ethnicity not determined because of non-response or coding error). Social deprivation was classified according to the Townsend score.</p><p>Liver diagnoses included NAFLD, alcoholic liver disease, hepatitis A, B, C, D, and E, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, haemochromatosis, Wilson’s disease, alpha-1 antitrypsin deficiency, portal vein thrombosis, Budd Chiari syndrome, glycogen storage disorder, cholestasis of pregnancy, liver disease in pregnancy, and HELLP syndrome. Glandular fever and pre-eclampsia were included as causes of abnormal transaminases.</p><p>Comorbid diabetes, hypertension, and cardiovascular disease were recorded. Clinical care measures included viral serology completion, tobacco and alcohol consumption, and latest recorded values for body mass index (BMI), serum ALT, AST, and lipids. Use of drugs known to commonly cause abnormal LFTs was recorded: amiodarone, azathioprine, carbamazepine, methotrexate, phenytoin, antituberculous medications (isoniazid, pyrazinamide, rifampicin, ethambutol), and statins.</p><p>BMI values were collapsed into four categories of underweight (<18.5 kg/m<sup>2</sup>), normal weight (18.5–24.9 kg/m<sup>2</sup>), overweight (25.0–29.9 kg/m<sup>2</sup>), and obese (≥30.0 kg/m<sup>2</sup>). For Bangladeshi, Indian, and Pakistani patients, the category cut-offs were set at underweight (<18.5 kg/m<sup>2</sup>), normal weight (18.5–22.9 kg/m<sup>2</sup>), overweight (23.0–27.4 kg/m<sup>2</sup>), and obese (≥27.5 kg/m<sup>2</sup>).<xref rid="b20" ref-type="bibr">20</xref> Alcohol use was categorised into within or greater than recommended limits (males ≤21 units/week, females ≤14 units/week), and ‘not recorded’. Statistical analyses were conducted using Stata (version 12).</p></sec><sec sec-type="results"><title>RESULTS</title><sec><title>Abnormal liver function in primary care</title><p>The study population comprised 690 683 adults (<xref ref-type="table" rid="table1">Table 1</xref>). Ethnicity was recorded for 93.1% of the adult population and for subsequent analyses, the focus was on the six most populous ethnic groups: white, Bangladeshi, Pakistani, Indian, African, and Caribbean. BMI was recorded for 89.2% of all adults, and units of alcohol/week for 91.0%.</p><table-wrap id="table1" position="float"><label>Table 1.</label><caption><p>Demographic and clinical characteristics of the study population</p></caption><table frame="hsides" rules="groups"><thead><tr><th valign="bottom" align="left" rowspan="1" colspan="1"/><th valign="bottom" align="center" rowspan="1" colspan="1"><bold>Bangladeshi</bold></th><th valign="bottom" align="center" rowspan="1" colspan="1"><bold>Indian</bold></th><th valign="bottom" align="center" rowspan="1" colspan="1"><bold>Pakistani</bold></th><th valign="bottom" align="center" rowspan="1" colspan="1"><bold>White</bold></th><th valign="bottom" align="center" rowspan="1" colspan="1"><bold>African</bold></th><th valign="bottom" align="center" rowspan="1" colspan="1"><bold>Caribbean</bold></th><th valign="bottom" align="center" rowspan="1" colspan="1"><bold>Total population</bold></th></tr></thead><tbody><tr><td valign="top" align="left" rowspan="1" colspan="1"><italic>n</italic></td><td valign="top" align="center" rowspan="1" colspan="1">93 315</td><td valign="top" align="center" rowspan="1" colspan="1">53 342</td><td valign="top" align="center" rowspan="1" colspan="1">31 794</td><td valign="top" align="center" rowspan="1" colspan="1">278 944</td><td valign="top" align="center" rowspan="1" colspan="1">47 152</td><td valign="top" align="center" rowspan="1" colspan="1">23 388</td><td valign="top" align="center" rowspan="1" colspan="1">690 683</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Mean age, years (SD)</td><td valign="top" align="center" rowspan="1" colspan="1">36.5 (14.0)</td><td valign="top" align="center" rowspan="1" colspan="1">38.6 (14.8)</td><td valign="top" align="center" rowspan="1" colspan="1">37.2 (14.1)</td><td valign="top" align="center" rowspan="1" colspan="1">41 (16.3)</td><td valign="top" align="center" rowspan="1" colspan="1">40.8 (13.6)</td><td valign="top" align="center" rowspan="1" colspan="1">49.7 (17.9)</td><td valign="top" align="center" rowspan="1" colspan="1">39.5 (15.4)</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Mean Townsend deprivation score (SD)</td><td valign="top" align="center" rowspan="1" colspan="1">6.3 (1.7)</td><td valign="top" align="center" rowspan="1" colspan="1">4.7 (1.8)</td><td valign="top" align="center" rowspan="1" colspan="1">4.8 (1.7)</td><td valign="top" align="center" rowspan="1" colspan="1">5.4 (1.9)</td><td valign="top" align="center" rowspan="1" colspan="1">5.9 (1.8)</td><td valign="top" align="center" rowspan="1" colspan="1">5.6 (1.8)</td><td valign="top" align="center" rowspan="1" colspan="1">5.5 (1.9)</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Female, <italic>n</italic> (%)</td><td valign="top" align="center" rowspan="1" colspan="1">42 221 (45.2)</td><td valign="top" align="center" rowspan="1" colspan="1">23 340 (43.8)</td><td valign="top" align="center" rowspan="1" colspan="1">12 353 (38.9)</td><td valign="top" align="center" rowspan="1" colspan="1">146 113 (52.4)</td><td valign="top" align="center" rowspan="1" colspan="1">24 835 (52.7)</td><td valign="top" align="center" rowspan="1" colspan="1">13 227 (56.6)</td><td valign="top" align="center" rowspan="1" colspan="1">340 455 (49.3)</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Male, <italic>n</italic> (%)</td><td valign="top" align="center" rowspan="1" colspan="1">51 094 (54.8)</td><td valign="top" align="center" rowspan="1" colspan="1">30 002 (56.2)</td><td valign="top" align="center" rowspan="1" colspan="1">19 441 (61.1)</td><td valign="top" align="center" rowspan="1" colspan="1">132 831 (47.6)</td><td valign="top" align="center" rowspan="1" colspan="1">22 317 (47.3)</td><td valign="top" align="center" rowspan="1" colspan="1">10 161 (43.4)</td><td valign="top" align="center" rowspan="1" colspan="1">350 228 (50.7)</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Diabetes, <italic>n</italic> (%)</td><td valign="top" align="center" rowspan="1" colspan="1">11 554 (12.4)</td><td valign="top" align="center" rowspan="1" colspan="1">5028 (9.4)</td><td valign="top" align="center" rowspan="1" colspan="1">2991 (9.4)</td><td valign="top" align="center" rowspan="1" colspan="1">12 666 (4.5)</td><td valign="top" align="center" rowspan="1" colspan="1">3575 (7.6)</td><td valign="top" align="center" rowspan="1" colspan="1">3618 (15.5)</td><td valign="top" align="center" rowspan="1" colspan="1">47 232 (6.8)</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Hypertension, <italic>n</italic> (%)</td><td valign="top" align="center" rowspan="1" colspan="1">9599 (10.3)</td><td valign="top" align="center" rowspan="1" colspan="1">6055 (11.4)</td><td valign="top" align="center" rowspan="1" colspan="1">2981 (9.4)</td><td valign="top" align="center" rowspan="1" colspan="1">29 910 (10.7)</td><td valign="top" align="center" rowspan="1" colspan="1">8078 (17.1)</td><td valign="top" align="center" rowspan="1" colspan="1">6540 (28.0)</td><td valign="top" align="center" rowspan="1" colspan="1">76 704 (11.1)</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><bold>BMI</bold></td><td valign="top" align="center" rowspan="1" colspan="1"/><td valign="top" align="center" rowspan="1" colspan="1"/><td valign="top" align="center" rowspan="1" colspan="1"/><td valign="top" align="center" rowspan="1" colspan="1"/><td valign="top" align="center" rowspan="1" colspan="1"/><td valign="top" align="center" rowspan="1" colspan="1"/><td valign="top" align="center" rowspan="1" colspan="1"/></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">  Underweight, <italic>n</italic> (%)</td><td valign="top" align="center" rowspan="1" colspan="1">1482 (1.6)</td><td valign="top" align="center" rowspan="1" colspan="1">6225 (11.7)</td><td valign="top" align="center" rowspan="1" colspan="1">445 (1.4)</td><td valign="top" align="center" rowspan="1" colspan="1">3933 (1.4)</td><td valign="top" align="center" rowspan="1" colspan="1">247 (0.5)</td><td valign="top" align="center" rowspan="1" colspan="1">149 (0.6)</td><td valign="top" align="center" rowspan="1" colspan="1">9458 (1.4)</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">  Normal, <italic>n</italic> (%)</td><td valign="top" align="center" rowspan="1" colspan="1">10 352 (11.1)</td><td valign="top" align="center" rowspan="1" colspan="1">10 811 (20.3)</td><td valign="top" align="center" rowspan="1" colspan="1">3121 (9.8)</td><td valign="top" align="center" rowspan="1" colspan="1">74 890 (26.8)</td><td valign="top" align="center" rowspan="1" colspan="1">6154 (13.1)</td><td valign="top" align="center" rowspan="1" colspan="1">3344 (14.3)</td><td valign="top" align="center" rowspan="1" colspan="1">133 287 (19.3)</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">  Overweight, <italic>n</italic> (%)</td><td valign="top" align="center" rowspan="1" colspan="1">21 098 (22.6)</td><td valign="top" align="center" rowspan="1" colspan="1">10 269 (19.3)</td><td valign="top" align="center" rowspan="1" colspan="1">5636 (17.7)</td><td valign="top" align="center" rowspan="1" colspan="1">45 331 (16.3)</td><td valign="top" align="center" rowspan="1" colspan="1">9404 (19.9)</td><td valign="top" align="center" rowspan="1" colspan="1">5112 (21.9)</td><td valign="top" align="center" rowspan="1" colspan="1">120 060 (17.4)</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">  Obese, <italic>n</italic> (%)</td><td valign="top" align="center" rowspan="1" colspan="1">17 929 (19.2)</td><td valign="top" align="center" rowspan="1" colspan="1">25 139 (47.1)</td><td valign="top" align="center" rowspan="1" colspan="1">7441 (23.4)</td><td valign="top" align="center" rowspan="1" colspan="1">36 211 (13.0)</td><td valign="top" align="center" rowspan="1" colspan="1">10 367 (22.0)</td><td valign="top" align="center" rowspan="1" colspan="1">6361 (27.2)</td><td valign="top" align="center" rowspan="1" colspan="1">106 800 (15.5)</td></tr></tbody></table><table-wrap-foot><fn id="tfn1"><p>BMI = body mass index. SD = standard deviation.</p></fn></table-wrap-foot></table-wrap><p>LFTs were performed for 31.6% of this population in the previous 2 years (<italic>n =</italic> 218 032 patients; <xref ref-type="fig" rid="fig1">Figure 1</xref>). The tested population was older than the general adult population (49.1 versus 39.4 years). Testing varied by ethnicity: 46.5% of adults of Caribbean ethnicity had liver testing, compared with 40.6% of Bangladeshi, 37.8% of Pakistani, 36.3% of Indian, 36.0% of African, and 28.6% of white adults. Of the tested population, 19.1% had diabetes (compared with 6.8% of the total adult population, <italic>P</italic><0.001).</p><fig id="fig1" position="float"><label>Figure 1.</label><caption><p><bold><italic>Venn diagram showing patients with abnormal liver tests and most commonly recorded liver diagnoses. (A+B+C+D) = All adults tested (n = 218 032); Set A = Tested and normal LFTs (n = 196 360). (B+C) = Tested and abnormal LFTs (n = 31 672); Set B = Tested, abnormal LFTs but no diagnosis (n = 27 985); Set C = Tested, abnormal LFTs and liver diagnosis recorded (n = 3687). (C+D+E) = Liver diagnosis recorded; Set D = Tested, normal LFTs and liver diagnosis recorded (n = 4384); Set E = Not tested, liver diagnosis recorded (n =3965). This group of sets is further broken down into the proportions of patients with the common liver diagnoses. ALD = alcoholic liver disease. HBV = hepatitis B virus. HCV = hepatitis C virus.</italic></bold></p></caption><graphic xlink:href="bjgpNov-2014-64-628-e694-OA-1"/></fig><p>Among the tested population, 14.5% had an ALT or AST elevated above the upper limit of the normal range on at least one occasion during the 2-year period. The key demographic and clinical data for the populations with normal and abnormal LFTs are shown in <xref ref-type="table" rid="table2">Table 2</xref>. Within the total tested population, the mean ages of patients with normal and abnormal LFTs were comparable and most patients with abnormal LFTs were male. The prevalence of abnormal LFTs was highest among Bangladeshi (18.4%) and Pakistani (17.6%) adults, and lowest among adults of Caribbean ethnicity (10.2%), while the prevalence rates among patients of Indian (14.8%), white (13.5%) and African (11.8%) ethnicities were comparable. The mean ALT in the group of patients with raised transaminases was 67i U/ml (SD = 62.2, median 52, interquartile range [IQR] 24) and the mean AST was 65 iU/ml (SD 72.5, median 46, IQR 24).</p><table-wrap id="table2" position="float"><label>Table 2.</label><caption><p>Demographic and clinical characteristics of tested patients<xref ref-type="table-fn" rid="tfn2"><sup>a</sup></xref> with normal and abnormal liver function tests (LFTs)</p></caption><table frame="hsides" rules="groups"><thead><tr><th valign="bottom" align="left" rowspan="1" colspan="1"><bold>Variable</bold></th><th valign="bottom" align="center" rowspan="1" colspan="1"><bold>Normal LFTs (<italic>N</italic> = 186 360) <italic>n</italic></bold></th><th valign="bottom" align="center" rowspan="1" colspan="1"><bold>Abnormal LFTs (<italic>N</italic> = 31 672) <italic>n</italic> (%)</bold></th></tr></thead><tbody><tr><td valign="top" align="left" rowspan="1" colspan="1">Female</td><td valign="top" align="center" rowspan="1" colspan="1">108 540</td><td valign="top" align="center" rowspan="1" colspan="1">9596 (8.1)</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Male</td><td valign="top" align="center" rowspan="1" colspan="1">77 820</td><td valign="top" align="center" rowspan="1" colspan="1">22 076 (22.1)</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Bangladeshi</td><td valign="top" align="center" rowspan="1" colspan="1">30 954</td><td valign="top" align="center" rowspan="1" colspan="1">6962 (18.4)</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Indian</td><td valign="top" align="center" rowspan="1" colspan="1">16 507</td><td valign="top" align="center" rowspan="1" colspan="1">2876 (14.8)</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Pakistani</td><td valign="top" align="center" rowspan="1" colspan="1">9917</td><td valign="top" align="center" rowspan="1" colspan="1">2111 (17.6)</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">White</td><td valign="top" align="center" rowspan="1" colspan="1">69 028</td><td valign="top" align="center" rowspan="1" colspan="1">10 791 (13.5)</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">African</td><td valign="top" align="center" rowspan="1" colspan="1">14 975</td><td valign="top" align="center" rowspan="1" colspan="1">2004 (11.8)</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Caribbean</td><td valign="top" align="center" rowspan="1" colspan="1">9772</td><td valign="top" align="center" rowspan="1" colspan="1">1112 (10.2)</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Mean age, years (SD)</td><td valign="top" align="center" rowspan="1" colspan="1">49.6 (17.4)</td><td valign="top" align="center" rowspan="1" colspan="1">47.0 (14.3)</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Diabetes</td><td valign="top" align="center" rowspan="1" colspan="1">33 673</td><td valign="top" align="center" rowspan="1" colspan="1">22 804 (40.4)</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Hypertension</td><td valign="top" align="center" rowspan="1" colspan="1">52 480</td><td valign="top" align="center" rowspan="1" colspan="1">9226 (29.1)</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Cardiovascular disease</td><td valign="top" align="center" rowspan="1" colspan="1">11 968</td><td valign="top" align="center" rowspan="1" colspan="1">6248 (34.2)</td></tr></tbody></table><table-wrap-foot><fn id="tfn2"><label>a</label><p>n = 218 032. SD = standard deviation.</p></fn></table-wrap-foot></table-wrap><p>The odds of a finding of abnormal LFTs in the tested population were calculated for a range of potential risk factors in a multivariate analysis (<xref ref-type="table" rid="table3">Table 3</xref>). The odds of having abnormal LFTs in the tested population were similar in the Bangladeshi and white ethnic groups, and significantly reduced in patients of Indian, Pakistani, African, or Caribbean ethnicity. Key independent risk factors for abnormal LFTs include male sex, alcohol consumption in excess of recommended limits, and components of the metabolic syndrome (diagnosed diabetes, hypertension, raised BMI, and hypercholesterolaemia). There was a gradient of risk of abnormal LFTs with increasing BMI.</p><table-wrap id="table3" position="float"><label>Table 3.</label><caption><p>Multivariate regression examining the odds of having abnormal liver function tests in the previous 2 years<xref ref-type="table-fn" rid="tfn3"><sup>a</sup></xref></p></caption><table frame="hsides" rules="groups"><thead><tr><th valign="bottom" align="left" rowspan="1" colspan="1"><bold>Explanatory variable</bold></th><th valign="bottom" align="center" rowspan="1" colspan="1"><bold>Odds ratio</bold></th><th valign="bottom" align="center" rowspan="1" colspan="1"><bold>95% CI</bold></th><th valign="bottom" align="center" rowspan="1" colspan="1"><bold><italic>P</italic>-value</bold></th></tr></thead><tbody><tr><td valign="top" align="left" rowspan="1" colspan="1"><bold>Ethnicity</bold></td><td valign="top" align="center" rowspan="1" colspan="1"/><td valign="top" align="center" rowspan="1" colspan="1"/><td valign="top" align="center" rowspan="1" colspan="1"/></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">  White (ref)</td><td valign="top" align="center" rowspan="1" colspan="1">1</td><td valign="top" align="center" rowspan="1" colspan="1">–</td><td valign="top" align="center" rowspan="1" colspan="1"/></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">  Bangladeshi</td><td valign="top" align="center" rowspan="1" colspan="1">1.09</td><td valign="top" align="center" rowspan="1" colspan="1">1.03 to 1.14</td><td valign="top" align="center" rowspan="1" colspan="1">0.001</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">  Indian</td><td valign="top" align="center" rowspan="1" colspan="1">0.84</td><td valign="top" align="center" rowspan="1" colspan="1">0.78 to 0.91</td><td valign="top" align="center" rowspan="1" colspan="1"><0.001</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">  Pakistani</td><td valign="top" align="center" rowspan="1" colspan="1">0.92</td><td valign="top" align="center" rowspan="1" colspan="1">0.85 to 1.00</td><td valign="top" align="center" rowspan="1" colspan="1">0.055</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">  African</td><td valign="top" align="center" rowspan="1" colspan="1">0.77</td><td valign="top" align="center" rowspan="1" colspan="1">0.71 to 0.82</td><td valign="top" align="center" rowspan="1" colspan="1"><0.001</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">  Caribbean</td><td valign="top" align="center" rowspan="1" colspan="1">0.82</td><td valign="top" align="center" rowspan="1" colspan="1">0.75 to 0.89</td><td valign="top" align="center" rowspan="1" colspan="1"><0.001</td></tr><tr><td colspan="4" valign="top" align="center" rowspan="1">
<hr/></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Age (continuous)</td><td valign="top" align="center" rowspan="1" colspan="1">0.98</td><td valign="top" align="center" rowspan="1" colspan="1">0.97 to 0.98</td><td valign="top" align="center" rowspan="1" colspan="1"><0.001</td></tr><tr><td colspan="4" valign="top" align="center" rowspan="1">
<hr/></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><bold>Sex</bold></td><td valign="top" align="center" rowspan="1" colspan="1"/><td valign="top" align="center" rowspan="1" colspan="1"/><td valign="top" align="center" rowspan="1" colspan="1"/></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">  Female (ref)</td><td valign="top" align="center" rowspan="1" colspan="1">1</td><td valign="top" align="center" rowspan="1" colspan="1">–</td><td valign="top" align="center" rowspan="1" colspan="1"/></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">  Male</td><td valign="top" align="center" rowspan="1" colspan="1">2.94</td><td valign="top" align="center" rowspan="1" colspan="1">2.80 to 3.10</td><td valign="top" align="center" rowspan="1" colspan="1"><0.001</td></tr><tr><td colspan="4" valign="top" align="center" rowspan="1">
<hr/></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Diagnosed diabetes</td><td valign="top" align="center" rowspan="1" colspan="1">1.58</td><td valign="top" align="center" rowspan="1" colspan="1">1.51 to 1.65</td><td valign="top" align="center" rowspan="1" colspan="1"><0.001</td></tr><tr><td colspan="4" valign="top" align="center" rowspan="1">
<hr/></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Diagnosed hypertension</td><td valign="top" align="center" rowspan="1" colspan="1">1.16</td><td valign="top" align="center" rowspan="1" colspan="1">1.11 to 1.21</td><td valign="top" align="center" rowspan="1" colspan="1"><0.001</td></tr><tr><td colspan="4" valign="top" align="center" rowspan="1">
<hr/></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Diagnosed CVD</td><td valign="top" align="center" rowspan="1" colspan="1">0.90</td><td valign="top" align="center" rowspan="1" colspan="1">0.84 to 0.95</td><td valign="top" align="center" rowspan="1" colspan="1">0.001</td></tr><tr><td colspan="4" valign="top" align="center" rowspan="1">
<hr/></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><bold>BMI category</bold></td><td valign="top" align="center" rowspan="1" colspan="1"/><td valign="top" align="center" rowspan="1" colspan="1"/><td valign="top" align="center" rowspan="1" colspan="1"/></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">  Normal (ref)</td><td valign="top" align="center" rowspan="1" colspan="1">1</td><td valign="top" align="center" rowspan="1" colspan="1">–</td><td valign="top" align="center" rowspan="1" colspan="1"/></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">  Underweight</td><td valign="top" align="center" rowspan="1" colspan="1">0.84</td><td valign="top" align="center" rowspan="1" colspan="1">0.70 to 0.99</td><td valign="top" align="center" rowspan="1" colspan="1">0.042</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">  Overweight</td><td valign="top" align="center" rowspan="1" colspan="1">1.63</td><td valign="top" align="center" rowspan="1" colspan="1">1.53 to 1.74</td><td valign="top" align="center" rowspan="1" colspan="1"><0.001</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">  Obese</td><td valign="top" align="center" rowspan="1" colspan="1">2.44</td><td valign="top" align="center" rowspan="1" colspan="1">2.27 to 2.62</td><td valign="top" align="center" rowspan="1" colspan="1"><0.001</td></tr><tr><td colspan="4" valign="top" align="center" rowspan="1">
<hr/></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><bold>Alcohol consumption</bold></td><td valign="top" align="center" rowspan="1" colspan="1"/><td valign="top" align="center" rowspan="1" colspan="1"/><td valign="top" align="center" rowspan="1" colspan="1"/></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">  Safe (ref)</td><td valign="top" align="center" rowspan="1" colspan="1">1</td><td valign="top" align="center" rowspan="1" colspan="1">–</td><td valign="top" align="center" rowspan="1" colspan="1"/></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">  Unsafe</td><td valign="top" align="center" rowspan="1" colspan="1">1.92</td><td valign="top" align="center" rowspan="1" colspan="1">1.80 to 2.05</td><td valign="top" align="center" rowspan="1" colspan="1"><0.001</td></tr></tbody></table><table-wrap-foot><fn id="tfn3"><label>a</label><p>n <italic>= 125 429 out of 218 032 cases with full data sets. Adjusted for Townsend deprivation score, locality and clustered by practice (139 clusters). CVD = cardiovascular disease. ref = reference category.</italic></p></fn></table-wrap-foot></table-wrap></sec><sec><title>Most liver abnormalities are undiagnosed</title><p>Of the 31 672 patients with abnormal transaminases, only 3687 (11.6%) had a liver-related diagnosis in the clinical record, the most common of which were NAFLD, viral infection, and alcoholic liver disease (<xref ref-type="app" rid="app1">Appendix 1</xref>).</p><p>There is no Read Code for drug-induced liver injury, but 900 patients were identified who were taking medication associated with abnormal LFTs (amiodarone, azathioprine, carbamazepine, methotrexate, phenytoin, and antituberculous medications). In the adult population, 87 532 patients (12.7%) were prescribed a statin. Of 27 985 patients who did not have a diagnosis, 11 111 were taking a statin.</p><p>Only 494 patients with abnormal LFTs had a recorded diagnosis of alcoholic liver disease (ALD). Alcohol usage was recorded in 28 802 (91.0%) adults with abnormal LFTs, of whom 3866 (13.4%) were drinking in excess of recommended limits. Therefore, in 3372 patients without a recorded diagnosis the abnormal LFTs were at least associated with alcohol.</p><p>Of patients with abnormal LFTs, 6026 (19.0%) were recorded as drinking within recommended limits and had undergone testing for viral hepatitis and did not have a positive result. This group represents a population that may have high rates of NAFLD and/or other chronic liver diseases.</p></sec><sec><title>Prevalence of recorded diagnosis of liver disease</title><p>It was reasoned that a large number of patients with liver disease may not have abnormal LFTs. Patients with recorded liver-related diagnoses in the whole adult population were investigated therefore, irrespective of biochemical testing.</p><p>A liver-related diagnosis was recorded in 12 239 (1.7%) of the total adult population. The most commonly recorded liver diagnosis was NAFLD in 42.9% (<italic>n</italic> = 5250), followed by HBV (23.8%, <italic>n</italic> = 2910), hepatitis C virus (HCV) infection (15.7%, <italic>n</italic> = 1922), and ALD (9.9%, <italic>n</italic> = 1215). Only 436 (3.6%) had more than one liver diagnosis. A diagnosis of ALD was most prevalent among people of white (0.3%), HBV in people of African (1.49%) and HCV in people of Pakistani (0.68%) ethnicity (<xref ref-type="fig" rid="fig2">Figure 2</xref>).</p><fig id="fig2" position="float"><label>Figure 2.</label><caption><p><bold><italic>Prevalence of NAFLD, ALD, HBV and HCV in different ethnic groups within the general population.</italic></bold></p></caption><graphic xlink:href="bjgpNov-2014-64-628-e694-OA-2"/></fig><p>Not all patients with liver-related diagnoses had abnormal LFTs in the 2-year period of the study. Liver function tests had been performed in 82.5% of patients with NAFLD, of whom 55.7% had normal results.</p></sec><sec><title>Risk factors for NAFLD</title><p>As NAFLD was the most prevalent liver disease, the risk factors for NAFLD in the general population were determined. Diagnosed NAFLD was significantly more prevalent among people of Bangladeshi ethnicity (1.8% of the adult population) than any other ethnic group, including other South Asian groups (<xref ref-type="app" rid="app2">Appendices 2</xref> and <xref ref-type="app" rid="app3">3</xref>). As expected, the prevalence of NAFLD was significantly lower in the African and Caribbean ethnic groups.</p><p>The odds of a diagnosis of NAFLD in the general adult population were calculated, by ethnicity, adjusting for a range of previously established risk factors (<xref ref-type="table" rid="table4">Table 4</xref>). The highest risk for NAFLD was in patients with raised BMI (with a gradient of risk of NAFLD with increasing BMI) and in patients with comorbid diabetes or hypertension. Other risk factors include hypertension but not sex, smoking, age, social deprivation, or a diagnosis of cardiovascular disease. Bangladeshi, but not Indian or Pakistani, ethnicity remained an independent risk factor for NAFLD in the adjusted analysis.</p><table-wrap id="table4" position="float"><label>Table 4.</label><caption><p>Multivariate regression examining the odds of having diagnosed NAFLD<xref ref-type="table-fn" rid="tfn4"><sup>a</sup></xref></p></caption><table frame="hsides" rules="groups"><thead><tr><th valign="bottom" align="left" rowspan="1" colspan="1"><bold>Explanatory variable</bold></th><th valign="bottom" align="center" rowspan="1" colspan="1"><bold>Odds ratio</bold></th><th valign="bottom" align="center" rowspan="1" colspan="1"><bold>95% CI</bold></th><th valign="bottom" align="center" rowspan="1" colspan="1"><bold><italic>P</italic>-value</bold></th></tr></thead><tbody><tr><td valign="top" align="left" rowspan="1" colspan="1"><bold>Ethnicity</bold></td><td valign="top" align="left" rowspan="1" colspan="1"/><td valign="top" align="left" rowspan="1" colspan="1"/><td valign="top" align="left" rowspan="1" colspan="1"/></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">  White (ref)</td><td valign="top" align="center" rowspan="1" colspan="1">1</td><td valign="top" align="center" rowspan="1" colspan="1"/><td valign="top" align="center" rowspan="1" colspan="1"/></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">  Bangladeshi</td><td valign="top" align="center" rowspan="1" colspan="1">1.86</td><td valign="top" align="center" rowspan="1" colspan="1">1.56 to 2.23</td><td valign="top" align="center" rowspan="1" colspan="1"><0.001</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">  Indian</td><td valign="top" align="center" rowspan="1" colspan="1">0.94</td><td valign="top" align="center" rowspan="1" colspan="1">0.71 to 1.24</td><td valign="top" align="center" rowspan="1" colspan="1">0.657</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">  Pakistani</td><td valign="top" align="center" rowspan="1" colspan="1">1.31</td><td valign="top" align="center" rowspan="1" colspan="1">0.99 to 1.74</td><td valign="top" align="center" rowspan="1" colspan="1">0.062</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">  African</td><td valign="top" align="center" rowspan="1" colspan="1">0.58</td><td valign="top" align="center" rowspan="1" colspan="1">0.47 to 0.72</td><td valign="top" align="center" rowspan="1" colspan="1"><0.001</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">  Caribbean</td><td valign="top" align="center" rowspan="1" colspan="1">0.45</td><td valign="top" align="center" rowspan="1" colspan="1">0.35 to 0.58</td><td valign="top" align="center" rowspan="1" colspan="1"><0.001</td></tr><tr><td colspan="4" valign="top" align="center" rowspan="1">
<hr/></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Age (continuous)</td><td valign="top" align="center" rowspan="1" colspan="1">1.01</td><td valign="top" align="center" rowspan="1" colspan="1">1.01 to 1.02</td><td valign="top" align="center" rowspan="1" colspan="1"><0.001</td></tr><tr><td colspan="4" valign="top" align="center" rowspan="1">
<hr/></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><bold>Sex</bold></td><td valign="top" align="center" rowspan="1" colspan="1"/><td valign="top" align="center" rowspan="1" colspan="1"/><td valign="top" align="center" rowspan="1" colspan="1"/></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">  Female (ref)</td><td valign="top" align="center" rowspan="1" colspan="1">1</td><td valign="top" align="center" rowspan="1" colspan="1"/><td valign="top" align="center" rowspan="1" colspan="1"/></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">  Male</td><td valign="top" align="center" rowspan="1" colspan="1">1.02</td><td valign="top" align="center" rowspan="1" colspan="1">0.95 to 1.09</td><td valign="top" align="center" rowspan="1" colspan="1">0.574</td></tr><tr><td colspan="4" valign="top" align="center" rowspan="1">
<hr/></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Diagnosed diabetes</td><td valign="top" align="center" rowspan="1" colspan="1">2.74</td><td valign="top" align="center" rowspan="1" colspan="1">2.51 to 2.99</td><td valign="top" align="center" rowspan="1" colspan="1"><0.001</td></tr><tr><td colspan="4" valign="top" align="center" rowspan="1">
<hr/></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Diagnosed hypertension</td><td valign="top" align="center" rowspan="1" colspan="1">1.34</td><td valign="top" align="center" rowspan="1" colspan="1">1.15 to 1.57</td><td valign="top" align="center" rowspan="1" colspan="1"><0.001</td></tr><tr><td colspan="4" valign="top" align="center" rowspan="1">
<hr/></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">Diagnosed CVD</td><td valign="top" align="center" rowspan="1" colspan="1">0.84</td><td valign="top" align="center" rowspan="1" colspan="1">0.75 to 0.95</td><td valign="top" align="center" rowspan="1" colspan="1">0.007</td></tr><tr><td colspan="4" valign="top" align="center" rowspan="1">
<hr/></td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1"><bold>BMI category</bold></td><td valign="top" align="center" rowspan="1" colspan="1"/><td valign="top" align="center" rowspan="1" colspan="1"/><td valign="top" align="center" rowspan="1" colspan="1"/></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">  Normal (ref)</td><td valign="top" align="center" rowspan="1" colspan="1">1</td><td valign="top" align="center" rowspan="1" colspan="1"/><td valign="top" align="center" rowspan="1" colspan="1"/></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">  Underweight</td><td valign="top" align="center" rowspan="1" colspan="1">0.55</td><td valign="top" align="center" rowspan="1" colspan="1">0.25 to 1.23</td><td valign="top" align="center" rowspan="1" colspan="1">0.148</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">  Overweight</td><td valign="top" align="center" rowspan="1" colspan="1">4.52</td><td valign="top" align="center" rowspan="1" colspan="1">3.72 to 5.51</td><td valign="top" align="center" rowspan="1" colspan="1"><0.001</td></tr><tr><td valign="top" align="left" rowspan="1" colspan="1">  Obese</td><td valign="top" align="center" rowspan="1" colspan="1">9.59</td><td valign="top" align="center" rowspan="1" colspan="1">7.77 to 11.8</td><td valign="top" align="center" rowspan="1" colspan="1"><0.001</td></tr></tbody></table><table-wrap-foot><fn id="tfn4"><label>a</label><p>n = 268 657 out of 690 683 cases with full data sets. Adjusted for Townsend deprivation score, alcohol consumption, locality and clustered by practice (139 clusters). CVD = cardiovascular disease. ref = reference category.</p></fn></table-wrap-foot></table-wrap></sec></sec><sec sec-type="discussion"><title>DISCUSSION</title><sec sec-type="conclusions"><title>Summary</title><p>Abnormal liver tests are a common finding in primary care,<xref rid="b21" ref-type="bibr">21</xref> yet this study shows that most patients with abnormal LFTs who are likely to have significant liver disease are undiagnosed.</p><p>Abnormal LFTs exist in 14.5% of the tested population and the marked male predilection may be related to alcohol consumption<xref rid="b22" ref-type="bibr">22</xref> and HCV infection.<xref rid="b23" ref-type="bibr">23</xref> Ethnicity is an independent risk factor for abnormal LFTs, with Bangladeshi and white patients at higher risk than other groups. Risk factors identified in the current study include raised BMI and a recorded diagnosis of diabetes, both of which are strongly associated with NAFLD.</p><p>More than half of patients with abnormal LFTs had no recorded aetiology or evidence of serological investigation, despite the fact that detection of even mild derangements in LFTs is a significant risk factor for liver disease, all-cause and liver-related mortality, and despite the higher risk of viral liver disease in a multiethnic population.<xref rid="b6" ref-type="bibr">6</xref> Excess alcohol consumption was common among patients with abnormal LFTs (13.4%), which is lower than rates reported in other series, although it is important to remember that a significant proportion of patients in the present cohort were from religious and social backgrounds in which alcohol is rarely consumed or where consumption is stigmatised.</p><p>A large proportion of patients with recorded liver diagnoses did not have abnormal LFTs, and this highlights the limitation of transaminase testing as a screening tool for liver disease.<xref rid="b24" ref-type="bibr">24</xref> Even patients with ALT 20–40 iU/ml are at increased risk of liver-related mortality and this is independent of the liver diagnosis.<xref rid="b25" ref-type="bibr">25</xref> Furthermore, other key abnormalities in liver biochemistry were not recorded — gamma-glutamyltransferase or alkaline phosphatase — which may be elevated in the context of normal transaminases. Therefore, it is likely that although this study has identified patients with diagnosed liver disease and a probable cohort with undiagnosed significant liver disease, these data still underestimate the true burden of liver disease in the population.</p></sec><sec><title>Strengths and limitations</title><p>This study is the largest community-based study of the association of ethnicity with LFTs and NAFLD to date, and was conducted in one of the most ethnically diverse regions of the UK. It benefits from high levels of data completeness for key characteristics: ethnicity, BMI, and alcohol consumption. Unlike other cohorts, the present data are derived from contiguous (unselected) GP practices across the region, and therefore include all adult patients. This includes practices that specialise in the care of homeless persons and injecting drug populations. Despite this, it is possible that data are lacking from unregistered patients who may be among the most at-risk sectors of society.</p><p>Although ALT was shown in the BALLETS study to be the best liver biochemistry test for the exclusion of significant hepatocellular disease,<xref rid="b26" ref-type="bibr">26</xref> by focusing on transaminases and not alkaline phosphatases, patients with biliary disease or hepatobiliary tumours may have been overlooked, and therefore the true burden of liver disease in primary care may well be even higher than suggested here.</p><p>The EMIS database is principally a clinical tool and reflects current practice in primary care. There may be bias in data entry, therefore, towards mandatory or positive data. A priori clinical suspicion usually exists, which is not captured in the electronic record, before LFTs are requested. The present estimates are comparable with published data regarding the distribution of liver disease by ethnicity;<xref rid="b2" ref-type="bibr">2</xref>,<xref rid="b27" ref-type="bibr">27</xref> HBV is most common among Africans, HCV among Pakistanis, and ALD among white people. However given the ethnic mix in the study population, the relatively low rates of viral testing in patients with abnormal LFTs but without a liver diagnosis suggest a diagnostic gap in the estimates of HBV and HCV infection rates.</p><p>It is not possible to evaluate the accuracy of a coded diagnosis of NAFLD, although it is likely that most were made after attendance at specialist liver clinics. Similarly, among the undiagnosed patients with abnormal LFTs, it was not possible to determine the reasons for the absence of a diagnostic code. In some, the abnormality may have resolved after a period of watchful waiting, or resolution of a concurrent illness.</p></sec><sec><title>Comparison with existing literature</title><p>This study found that NAFLD was the most commonly recorded cause of abnormal LFTs in the population in keeping with data from others,<xref rid="b28" ref-type="bibr">28</xref> although the prevalence rate in the present study is lower than estimates from series where the diagnosis was made radiologically in the general population or histologically in selected series of patients. This is partially explained by the high proportion of patients in the present cohort in whom NAFLD is the probable cause of abnormal LFTs but who do not have a liver diagnosis. It is also likely that a large proportion of patients with NAFLD have simply not been diagnosed yet.</p><p>The prevalence of recorded NAFLD varied considerably by ethnic group. To the authors' knowledge, this is the first study to identify Bangladeshi ethnicity as an independent risk factor for NAFLD, albeit in the context of low overall rates of liver diagnoses. Among Bangladeshis, there are high rates of type 2 diabetes and cardiovascular disease that may have a genetic basis. Increased prevalence may be related to intergenerational influences, early years and immigration impacts on lifestyle and health beliefs.<xref rid="b29" ref-type="bibr">29</xref>,<xref rid="b30" ref-type="bibr">30</xref></p></sec><sec sec-type="discussion"><title>Implications for research and practice</title><p>Further work is required to understand the effect of ethnicity on the natural history of NAFLD and why Bangladeshis are at increased risk, and, in particular, the relative contributions of genetics, diet, social deprivation, and cultural health behaviours.</p><p>The high proportion of patients with abnormal LFTs without a diagnosis is a challenge to primary care clinicians. Among this group many will have a liver disease that is amenable to further management, which may prevent complications. Where a patient has had abnormal LFTs, normalisation does not necessarily mean that liver injury was transient, as shown by the high proportion of patients with NAFLD and normal LFTs in the previous 2 years.</p><p>The authors recommend that when abnormal liver tests are identified, every reasonable effort should be made to make a diagnosis and to record this. There is a need to evaluate the cost-effectiveness of increased investigations in primary care and onward referral to liver specialists, which may be in conflict with current financial pressures to reduce laboratory tests and traditional outpatient attendance. Therefore, the authors support the development of evidence-based guidelines for the investigation, referral, and management of patients with abnormal LFTs in the community, to ensure early identification of treatable disease.</p></sec></sec> |
Recent Research on Febrile Seizures: A Review | <p id="P1">Febrile seizures are common and mostly benign. They are the most common cause of seizures in children less than five years of age. There are two categories of febrile seizures, simple and complex. Both the International League against Epilepsy and the National Institute of Health has published definitions on the classification of febrile seizures. Simple febrile seizures are mostly benign, but a prolonged (complex) febrile seizure can have long term consequences. Most children who have a febrile seizure have normal health and development after the event, but there is recent evidence that suggests a small subset of children that present with seizures and fever may have recurrent seizure or develop epilepsy. This review will give an overview of the definition of febrile seizures, epidemiology, evaluation, treatment, outcomes and recent research.</p> | <contrib contrib-type="author"><name><surname>Syndi Seinfeld</surname><given-names>DO</given-names></name><xref rid="FN1" ref-type="author-notes">*</xref></contrib><contrib contrib-type="author"><name><surname>Pellock</surname><given-names>John M.</given-names></name></contrib><aff id="A1">Department of Neurology, Virginia Commonwealth University, Richmond, USA</aff> | Journal of neurology & neurophysiology | <sec sec-type="intro" id="S1"><title>Introduction</title><p id="P2">Febrile seizures (FS) are the single most common seizure type and occur in 2 to 5% of children younger than age 5 years with a peak incidence in the second year of life [<xref rid="R1" ref-type="bibr">1</xref>]. Historically FS were studied using large epidemiologic studies. Initial studies did not exclude seizures associated with underlying neurological disturbance [<xref rid="R2" ref-type="bibr">2</xref>], and prognosis was pessimistic because of the inclusion criteria [<xref rid="R3" ref-type="bibr">3</xref>]. It is currently accepted that most children who have a FS often have normal health and development after the event. FS are considered benign, but there is recent evidence that suggests a small subset of children that present with seizures and fever may have recurrent FS or develop epilepsy.</p><p id="P3">The incidence and prevalence of FS is similar across the numerous FS studies. There is variation of incidence of FS based on geographic location, with higher prevalence found in Japan and Guam [<xref rid="R4" ref-type="bibr">4</xref>–<xref rid="R6" ref-type="bibr">6</xref>]. FS are not considered a form of epilepsy, but a FS can be the first presentation of subsequent epilepsy. At this time it is not possible to predict which child will develop an a febrile seizure after presenting with FS [<xref rid="R7" ref-type="bibr">7</xref>]. This review will give an overview of the definition of FS, epidemiology, evaluation, treatment, outcomes and recent research.</p></sec><sec id="S2"><title>Definition</title><p id="P4">The International League against Epilepsy (ILAE) defines a FS as a seizure occurring in childhood after one month of age, associated with a febrile illness that is not caused by an infection of the central nervous system. A child with the diagnosis of FS cannot have a history of neonatal seizures, a previous unprovoked seizure or meet criteria for other acute symptomatic seizures [<xref rid="R8" ref-type="bibr">8</xref>]. The lower age limit of the ILAE definition is younger than the limit proposed previously by the National Institutes of Health (NIH). The NIH Consensus Conference definition of FS is an event usually occurring between 3 months and 5 years of age, associated with fever, but without evidence of intracranial infection or defined cause [<xref rid="R9" ref-type="bibr">9</xref>].</p><p id="P5">FS can be separated into two categories, simple and complex. A simple febrile seizure is isolated, brief and generalized [<xref rid="R10" ref-type="bibr">10</xref>]. Complex FS is one with focal onset, one that occurs more than once during a febrile illness, or one that lasts more than 10 to 15 minutes [<xref rid="R11" ref-type="bibr">11</xref>]. Developmental delay and younger age are associated with prolonged FS [<xref rid="R12" ref-type="bibr">12</xref>]. Subsequent febrile seizures can be prolonged if the initial febrile seizure was prolonged [<xref rid="R13" ref-type="bibr">13</xref>]. Febrile status epilepticus (FSE) is a subgroup of complex FS.</p></sec><sec id="S3"><title>Epidemiology</title><p id="P6">FS have a peak incidence at 18 months of age and are most common between 6 months and 5 years [<xref rid="R10" ref-type="bibr">10</xref>]. Most FS are simple with approximately 20–30% being complex [<xref rid="R13" ref-type="bibr">13</xref>]. The distribution of a first FS duration can be described using a two population model, one with short seizure duration and the other with long seizure duration, with the cut-off at approximately 10 minutes [<xref rid="R12" ref-type="bibr">12</xref>]. Approximately 5% of FS will last ≥30 minutes [<xref rid="R14" ref-type="bibr">14</xref>]. No correlation has been identified between duration of the first FS and duration of the second FS [<xref rid="R12" ref-type="bibr">12</xref>]. Although, it has been observed that a recurrent febrile seizure is more likely to be prolonged if the initial FS was prolonged [<xref rid="R13" ref-type="bibr">13</xref>].</p><p id="P7">By definition, a febrile illness is required for a child to have a FS. Children with FS have higher temperatures with illness compared to febrile controls [<xref rid="R15" ref-type="bibr">15</xref>]. The rapid onset of fever was previously thought to be precipitating factor of FS, but this is no longer thought to be true [<xref rid="R16" ref-type="bibr">16</xref>]. Gender predominance of FS has also been studied. There are studies that conclude a higher incidence of FS in males [<xref rid="R17" ref-type="bibr">17</xref>] and others showed no significant difference based on gender [<xref rid="R18" ref-type="bibr">18</xref>].</p><p id="P8">FS occur in the setting of a febrile illness, which could cause seasonal variation. In Japan a study of FS showed two peaks of incidence, November to January and June to August, which correspond to peaks of viral upper respiratory infections and gastrointestinal infections respectively [<xref rid="R19" ref-type="bibr">19</xref>]. A study performed in Italy, which looked at 188 first FS, found that there is a significant increase in FS from 6 PM to 11:59 PM and a seasonal peak in January [<xref rid="R20" ref-type="bibr">20</xref>]. There have been multiple studies have supported the conclusion that FS have a peak in the winter and end of the summer [<xref rid="R21" ref-type="bibr">21</xref>–<xref rid="R23" ref-type="bibr">23</xref>]. Influenza A has been found to have a significant relationship with recurrence of FS [<xref rid="R24" ref-type="bibr">24</xref>]. Although it has been suggested that FS are more likely to occur with respiratory illnesses compared to viral gastrointestinal illness [<xref rid="R15" ref-type="bibr">15</xref>,<xref rid="R25" ref-type="bibr">25</xref>], any febrile illness can cause a FS. For example, in malaria endemic regions there is an increased incidence of FS when there is increased malaria transmission [<xref rid="R26" ref-type="bibr">26</xref>].</p></sec><sec id="S4"><title>Provoking Factors</title><sec id="S5"><title>Genetics</title><p id="P9">FS can be seen in multiple family members and there is evidence of genetic and environmental causes. There is a variable inheritance pattern, with no single accepted mechanism. A positive family history of FS can be found in 25–40% of cases when a child present with a FS [<xref rid="R27" ref-type="bibr">27</xref>–<xref rid="R29" ref-type="bibr">29</xref>]. The number of FS a child has affects the risk of a sibling experiencing a FS [<xref rid="R30" ref-type="bibr">30</xref>]. Significantly higher concordance rates are seen for FS in monozygotic twins as compared to dizygotic twins in multiple twin registries [<xref rid="R31" ref-type="bibr">31</xref>].</p><p id="P10">The phenotype of FS plus may account for children without a specific epilepsy syndrome who have FS and then developed generalized epilepsy [<xref rid="R32" ref-type="bibr">32</xref>]. These patients or family members have a have a history of FS, often complex and frequently occur beyond 5 years of age. Epilepsy with variable seizure types develop later in childhood or adulthood. A variety of mutations including SCN1A, SCN1B, and GABGR2 have been demonstrated in these families [<xref rid="R33" ref-type="bibr">33</xref>]. The proposed genetic syndrome that is called generalized epilepsy with febrile seizures plus (GEFS+) is a spectrum of clinical epilepsy phenotypes, with the most severe phenotype of myoclonic-astatic epilepsy [<xref rid="R32" ref-type="bibr">32</xref>].</p></sec><sec id="S6"><title>Intrauterine risk factor</title><p id="P11">A population-based, prospective questionnaire study from early fetal life onward evaluated the occurrence of FS in 3,372 subjects at age 12 and 24 months [<xref rid="R34" ref-type="bibr">34</xref>]. Children in the lowest percentile of transverse cerebellar diameter in the second trimester were at increased risk of developing FS, compared with children in the highest percentile. In the third trimester, children in the lowest percentile of all general growth characteristics (femur length, abdominal circumference, and estimated fetal weight) were at increased risk of developing FS. Children in the lowest percentile of biparietal diameter in the third trimester also were at increased risk of FS. The study concluded fetal growth retardation is associated with increased risk of FS and that adverse environmental and genetic factors during pregnancy may be important in the development of FS. Furthermore, the Danish birth cohorts (Aarhus Birth Cohort, Aalborg-Odense cohort and the Danish National Birth Cohort) have demonstrated that low birth weight and short gestational age are significant risk factors for FS [<xref rid="R35" ref-type="bibr">35</xref>].</p></sec><sec id="S7"><title>Vaccinations</title><p id="P12">Vaccinations are important to pediatric health and are recommended by the American Academy of Pediatrics (AAP) for members of the youngest age group at risk for experiencing the disease for which efficacy and safety have been demonstrated [<xref rid="R36" ref-type="bibr">36</xref>]. The World Health Organization (WHO), in 2002, released a publication on immunization safety. WHO recommends that children be vaccinated. In general, FS that occur after vaccination have not been found to be different from FS from other causes [<xref rid="R37" ref-type="bibr">37</xref>]. Historically, there were a group of children that were thought to have severe FS and encephalopathy from vaccine induced FS. The children presented with a first FS after vaccination and developed encephalopathy and recurrent seizures, but it is known that this subgroup have a genetic sodium channel mutation that causes a susceptibility to severe seizures and encephalopathy. The FS is commonly the first manifestation of the sodium channel mutation known as Dravet syndrome, but any febrile illness could cause the first seizure in a genetically susceptible individual. Vaccinations can trigger the onset of seizures in one third of patients with Dravet syndrome [<xref rid="R37" ref-type="bibr">37</xref>]. FS will not cause an epileptic encephalopathy in a child without a mutation conferring genetic susceptibility. Neither the AAP nor WHO recommend stopping or changing the immunization schedule after a FS, even in children with underlying genetic mutations.</p><p id="P13">The public fear of vaccines causing FS has led to numerous studies. Children less than 2 years of age have an increased risk of FS after the first dose of a measles containing vaccine when it is administered with a varicella vaccine [<xref rid="R38" ref-type="bibr">38</xref>]. The measles containing vaccines have not been found to be associated with an increased risk of FS in children over 4, regardless of whether varicella is given at the same time [<xref rid="R39" ref-type="bibr">39</xref>]. The previously used whole-cell diphtheria/tetanus/pertussis and measles-containing vaccines have an established association with FS, but the less reactogenic diphtheria, tetanus, and a cellular pertussis (DTaP) vaccine has been developed and is currently used and does not increase the risk of FS [<xref rid="R37" ref-type="bibr">37</xref>]. There is no evidence that children should not be vaccinated.</p></sec><sec id="S8"><title>Metabolic abnormalities and deficiencies</title><p id="P14">Whereas some have reported a statistical association between iron deficiency anemia and simple febrile seizures [<xref rid="R40" ref-type="bibr">40</xref>,<xref rid="R41" ref-type="bibr">41</xref>], other cross sectional studies have not found a significant association [<xref rid="R42" ref-type="bibr">42</xref>]. A study of febrile seizures in Indian children found lower zinc levels in patients with FS compared to age matched febrile children without seizures [<xref rid="R43" ref-type="bibr">43</xref>]. Other studies have proposed that there is a link between FS and a systemic respiratory alkalosis, irrespective of the severity of the underlying infection [<xref rid="R44" ref-type="bibr">44</xref>]. These associations require large population studies to determine if they can be predictive or if they can ultimately be factors associated with prevention.</p></sec></sec><sec id="S9"><title>Evaluation</title><p id="P15">The AAP has guidelines for evaluation of first simple febrile seizure, and state that clinicians should work to identify the source of the fever when a child presents within 12 hours of a simple febrile seizure [<xref rid="R45" ref-type="bibr">45</xref>]. There have been recent US studies to suggest that the risk of bacterial meningitis presenting as a first simple FS is very low and that the above guidelines are not being strictly followed [<xref rid="R46" ref-type="bibr">46</xref>,<xref rid="R47" ref-type="bibr">47</xref>]. Alternatively, in countries with high prevalence of diseases, such as malaria, these guidelines for evaluation cannot be as easily followed. Children with malaria who develop fever frequently have complex FS, and it is often difficult to differentiate children with intracranial infection from those without [<xref rid="R48" ref-type="bibr">48</xref>].</p><sec id="S10"><title>Laboratory studies</title><p id="P16">It is not recommended to perform routine serum labs when a child has a simple febrile seizure [<xref rid="R49" ref-type="bibr">49</xref>]. The labs that are performed should be based on the clinical presentation of the febrile illness.</p><p id="P17">The Consequences of Prolonged Febrile Seizure Study (FEBSTAT), which enrolled 199 children from 1 month to 5 years of age with FSE, evaluated children for the presence of HHV-6A, HHV-6B, or HHV-7 DNA and RNA in serum [<xref rid="R50" ref-type="bibr">50</xref>,<xref rid="R51" ref-type="bibr">51</xref>]. Children that had central nervous system infection were excluded from this study, thus no subject had encephalitis. The study concluded HHV-6B infection is commonly associated with FSE, and HHV-7 infection is less frequently associated with FSE. Together the HHV infections accounted infection in one third of FSE subjects in the study [<xref rid="R50" ref-type="bibr">50</xref>]. The study did not detect HHV-6B or HHV-7 DNA in the CSF of the 23 subjects who presented in FSE with documented HHV-6B or HHV-7 viremia.</p></sec><sec id="S11"><title>Lumbar puncture</title><p id="P18">The AAP guidelines strongly recommend a lumbar puncture in any child who presents with a seizure, a fever and has meningeal signs and symptoms. It is also recommended in any child whose history or examination suggests the presence of meningitis or intracranial infection [<xref rid="R45" ref-type="bibr">45</xref>]. As part of the FEBSTAT study, cerebrospinal fluid (CSF) samples were reviewed for the 136 of the 199 children who had a nontraumatic lumbar puncture performed. The study confirmed that FSE rarely causes CSF pleocytosis [<xref rid="R52" ref-type="bibr">52</xref>]. The CSF glucose and protein levels of the sample were unremarkable, and the temperature, age, seizure focality, and seizure duration did not affect results. This supports that CSF pleocytosis should not be attributed to FSE.</p></sec><sec id="S12"><title>EEG</title><p id="P19">There is no evidence that an EEG can be used to predict if a child will develop epilepsy after a simple febrile seizure. A clinician should consider performing EEG if more than one complex feature is present [<xref rid="R53" ref-type="bibr">53</xref>], but a routine practice of obtaining an early EEG in neurologically normal children with complex febrile seizures was not initially justified [<xref rid="R54" ref-type="bibr">54</xref>]. EEG does have a role if a child remains encephalopathic after a FS.</p><p id="P20">The FEBSTAT study performed baseline EEGs within 72 hours of the episode of FSE. Review of the baseline EEGs showed that there is focal EEG slowing or attenuation in a substantial proportion of children [<xref rid="R55" ref-type="bibr">55</xref>]. The slowing and attenuation are highly associated with MRI evidence of acute hippocampal injury [<xref rid="R55" ref-type="bibr">55</xref>]. These findings may be a sensitive and readily obtainable marker of acute injury associated with FSE.</p></sec><sec id="S13"><title>Imaging</title><p id="P21">Neuroimaging is not recommended after a simple febrile seizure [<xref rid="R49" ref-type="bibr">49</xref>]. If a patient presents with focal complex FS and/or FSE, one should consider performing brain MRI to evaluate for a structural abnormality as an explanation for the seizure [<xref rid="R51" ref-type="bibr">51</xref>]. A head MRI is more sensitive for abnormalities that can cause seizures, but when unavailable computed tomography can be performed.</p><p id="P22">The FEBSTAT study included baseline imaging on recruited subjects [<xref rid="R51" ref-type="bibr">51</xref>]. One hundred ninety one of the children had baseline MRI of the brain with emphasis on the hippocampus. A total of 22 children had definitely abnormal or equivocal increased T2 signal in the hippocampus following FSE [<xref rid="R56" ref-type="bibr">56</xref>]. None of the children in the control group had this abnormality, which was statistically significant [<xref rid="R56" ref-type="bibr">56</xref>]. The imaging also showed that developmental abnormalities of the hippocampus were more common in the FSE group than in controls, with hippocampal malrotation being the most common. This study has demonstrated that children with FSE are at risk for acute hippocampal injury and that a substantial number also have abnormalities in hippocampal development. These cohorts of FSE subjects are still being followed to determine the long-term outcomes in these children.</p></sec></sec><sec id="S14"><title>Treatment</title><sec id="S15"><title>Acute</title><sec id="S16"><title>Simple FS</title><p id="P23">There is no evidence that treatment of simple febrile seizures can prevent later development of epilepsy [<xref rid="R57" ref-type="bibr">57</xref>] and thus is not recommended (<xref rid="F1" ref-type="fig">Figure 1</xref>).</p></sec><sec id="S17"><title>Complex FS</title><p id="P24">Prolonged febrile seizures, lasting more than 10 minutes, are unlikely to stop spontaneously [<xref rid="R51" ref-type="bibr">51</xref>]. A child that is actively having a seizure should receive acute abortive treatment after 5 minutes of the seizure starting. Pediatric status epileptics (SE) have traditionally been defined as a seizure that lasts for more than 30 minutes. This operational definition has been revised. More recent articles and studies have advocated that the duration of pediatric SE should be operationally shortened to seizures lasting more than 5–10 minutes [<xref rid="R58" ref-type="bibr">58</xref>,<xref rid="R59" ref-type="bibr">59</xref>]. Prolonged seizures are accompanied by an increased risk of complications, and treatment should be initiated before it reaches 10 minutes in duration. Prolonged FS should be treated acutely using the same algorithm as prolonged seizures caused by other etiologies. A prolonged FS should be treated acutely by emergency medical services (EMS) or the emergency department. Initial use of a benzodiazepine is recommended, and if continued seizure then a full SE protocol should be initiated [<xref rid="R10" ref-type="bibr">10</xref>]. FSE is a neurological emergency and the most common cause of SE in children younger than two years of age [<xref rid="R60" ref-type="bibr">60</xref>].</p></sec></sec><sec id="S18"><title>Chronic</title><p id="P25">Using around-the-clock prophylactic administration of antipyretics has not been shown to affect the incidence of recurrence of FS, and is not recommended [<xref rid="R61" ref-type="bibr">61</xref>]. It is not recommended to treat children with FS using daily anti-epileptic medications because of high likelihood of adverse effects [<xref rid="R62" ref-type="bibr">62</xref>].</p></sec></sec><sec id="S19"><title>Recurrence</title><p id="P26">The risk of recurrence is influenced by both the age of the child and the type of FS. About one-third of children with a first FS will have a recurrence. Risk factors for recurrence include family history of FS, less than 18 months of age, temperature lower than 40.0°C at first convulsion and less than 1 hour between onset of febrile illness and first convulsion [<xref rid="R10" ref-type="bibr">10</xref>,<xref rid="R62" ref-type="bibr">62</xref>,<xref rid="R63" ref-type="bibr">63</xref>]. The numbers of risk factors are directly proportional to the risk of recurrence. A child with two or more risk factors has a more than 30% recurrence risk at 2 years of age, and that risk doubles with three risk factors [<xref rid="R61" ref-type="bibr">61</xref>] (<xref rid="T1" ref-type="table">Table 1</xref>).</p></sec><sec id="S20"><title>Differential Diagnosis</title><p id="P27">There are seizures in the setting of fever that are not considered FS. For example, falciparum malaria is a cause of acute symptomatic seizures in children admitted to hospitals in sub-Saharan Africa, and these seizures are associated with neurological disabilities and epilepsy [<xref rid="R64" ref-type="bibr">64</xref>]. These children may have fever, but the seizures are a different category compared to FS. Dravet syndrome, a severe epilepsy syndrome, can present with early febrile seizures, but the seizures progress and become a febrile and intractable [<xref rid="R65" ref-type="bibr">65</xref>]. These children initially present as FS, but are not true FS. Mitochondrial disease can present similarly to Dravet syndrome, with early febrile seizures and progression to developmental regression, ataxia, behavior change and a febrile seizures [<xref rid="R65" ref-type="bibr">65</xref>]. When a child has an underlying metabolic or mitochondrial disease they may have a seizure associated with a febrile illness, but they would not be classified as FS. It may be difficult to determine if there is an underlying disorder with an initial FS, but the progressive course would indicate different etiology of seizure.</p></sec><sec id="S21"><title>Outcome</title><p id="P28">There is evidence that FS are associated with an increased risk of subsequent epilepsy, and that epilepsy develops in 2 to 4% of children with a history of FS [<xref rid="R18" ref-type="bibr">18</xref>]. Although it is accepted that a single brief simple FS is benign with no clinical consequences, the risk of developing epilepsy can be as great as 57% in children with focal, prolonged, and recurrent FS [<xref rid="R28" ref-type="bibr">28</xref>].</p><p id="P29">A prospective cohort study performed using children presenting with first FS observed that developmental delay is associated with prolonged FS [<xref rid="R12" ref-type="bibr">12</xref>]. There are greater frequencies of delays in reaching developmental motor milestones at baseline in children with long versus short FS. Thus, children with prolonged FS are more likely to be neurologically abnormal than children with single, short, nonfocal FS [<xref rid="R13" ref-type="bibr">13</xref>].</p><p id="P30">Additionally, having had more than one complex feature of a febrile seizure further increased the risk of developing subsequent unprovoked seizures [<xref rid="R66" ref-type="bibr">66</xref>]. Risk factors for developing epilepsy after febrile seizure include neurodevelopmental abnormality, complex FS, family history of epilepsy and duration of fever [<xref rid="R10" ref-type="bibr">10</xref>] (<xref rid="T2" ref-type="table">Table 2</xref>). There is no evidence that preventing FS prevents the development of epilepsy.</p></sec><sec id="S22"><title>Current Research</title><p id="P31">The National Institute of Neurological Disorders and Stroke (NINDS), which is part of the National Institutes of Health (NIH), sponsors febrile seizure research in the United States, but FS are the focus of numerous current studies worldwide. There are basic science studies that are using a rat model to evaluate neuronal injury from FS, and looking at future development of epilepsy following FS [<xref rid="R67" ref-type="bibr">67</xref>]. The North London Status Epileptics in Childhood Surveillance Study (NLSTEPSS) in the United Kingdom is evaluating incidence, morbidity and treatment children with SE, including FSE [<xref rid="R68" ref-type="bibr">68</xref>]. The FEBSTAT study is evaluating the long-term consequences of FSE using MRI, EEG, developmental/neuropsychological testing, virology, genetics, psychiatric interview and parental interviews [<xref rid="R51" ref-type="bibr">51</xref>]. The genetics of FS, the sub-types and sub-syndromes are also being studied using twin pairs [<xref rid="R69" ref-type="bibr">69</xref>].</p></sec><sec id="S23"><title>Summary</title><p id="P32">FS are common and mostly benign. Unfortunately, FSE is frequently not recognized and prolonged seizures frequently need medication to terminate the seizure [<xref rid="R51" ref-type="bibr">51</xref>]. Fortunately, studies have found that a simple FS is mostly benign, but prolonged febrile seizures do have long term consequences. They should be treated acutely if they continue for past 5 minutes. It is important to educate parents about the risks associated with febrile seizures and the at home treatments for prolonged febrile seizures (ex. rectal diazepam). Education about seizure safety and precautions should be explained to the family, and are important to prevent consequences of prolonged seizures.</p></sec> |
A Perspective on the Potential of Human iPS Cell-Based Therapies for Muscular Dystrophies: Advancements so far and Hurdles to Overcome | Could not extract abstract | <contrib contrib-type="author"><name><surname>Darabi</surname><given-names>Radbod</given-names></name></contrib><contrib contrib-type="author"><name><surname>Perlingeiro</surname><given-names>Rita C.R.</given-names></name><xref rid="FN1" ref-type="author-notes">*</xref></contrib><aff id="A1">Lillehei Heart Institute, Department of Medicine, University of Minnesota, 312 Church Street SE, Minneapolis, MN 55455, USA</aff> | Journal of stem cell research & therapy | <p id="P1">Pluripotent stem cells [<xref rid="R1" ref-type="bibr">1</xref>,<xref rid="R2" ref-type="bibr">2</xref>] are well-known for their unique self-renewal and differentiation capabilities, which make these cells very favorable for cell-based therapeutic applications in degenerative disorders such as muscular dystrophies. Major concerns associated with embryonic stem (ES) cells, such as immunological compatibility and ethical considerations had limited their clinical applications [<xref rid="R3" ref-type="bibr">3</xref>]. However, the technology of reprogramming somatic cells into induced pluripotent stem (iPS) cells provides a new breath of hope for a potential therapeutic application in incurable diseases [<xref rid="R4" ref-type="bibr">4</xref>–<xref rid="R6" ref-type="bibr">6</xref>]. iPS cells have been generated initially from mouse and later from human somatic cells by introduction of the four reprogramming transcription factors using retroviral vectors. More recently, this has been accomplished by using non-integrating vectors [<xref rid="R7" ref-type="bibr">7</xref>,<xref rid="R8" ref-type="bibr">8</xref>], which is much safer as the associated risk of insertional mutagenesis is significantly reduced. Consequently, by proper differentiation of iPS cells into tissue specific progenitors, one can foresee the therapeutic application of iPS cells in a relatively near future, upon extensive testing for the safety of these cell preparations is achieved.</p><p id="P2">Muscular dystrophies (MDs) are an heterogeneous group of degenerative disorders caused by various gene defects that lead to progressive muscle damage, defective regeneration and fibrosis [<xref rid="R9" ref-type="bibr">9</xref>]. Among these, Duchene Muscular Dystrophy (DMD) is the most common type, and is caused by mutations in the dystrophin gene [<xref rid="R10" ref-type="bibr">10</xref>]. DMD is characterized by progressive muscle weakness and atrophy, leading patients to be confined to a wheelchair before their teenage years and eventual death due to respiratory and/or cardiac failure. To date, there is no definitive treatment for DMD or any other type of MD. Reprogramming technology potentially allows for a cell replacement therapy using patient-derived iPS cells, which combined with gene correction, and subsequent controlled differentiation into myogenic progenitors, could be used clinically in the autologous transplantation setting. In the current perspective, we review recent studies in this field, and point out the advantages and the shortcomings of each approach.</p><sec id="S1"><title>Gene Correction of Human DMD iPS Cells using a Human Artificial Chromosome (HAC)</title><p id="P3">This is the first report describing genetic correction in human DMD iPS cells [<xref rid="R11" ref-type="bibr">11</xref>]. The authors utilized a HAC containing the complete human dystrophin sequence (DYS-HAC). The use of HAC for gene therapy has certain advantages including the ability to carry large inserts such as the dystrophin gene, stable episomal maintenance, and importantly, minimum risk of insertional mutagenesis. The DMD mutation corrected in this study was a large deletion of axons 4-43, a mutation not suitable for conventional gene correction methods. The DYS-HAC was transferred via microcell-mediated chromosome transfer (MMCT) into patient fibroblasts and then corrected fibroblasts were used to generate iPS cells. Using FISH analysis, the authors showed stable maintenance of the DYS-HAC in long-term cultures of corrected iPS cells. In teratoma assays, they detected the presence of DYS-HAC in 90% of the cells as well as dystrophin expression in muscle-like tissues within the tumor. For potential clinical application, it will be necessary for corrected DMD cells to be differentiated <italic>in vitro</italic> into myogenic progenitors, and used for <italic>in vivo</italic> regeneration in an animal model for DMD.</p></sec><sec id="S2"><title>Derivation of Myogenic Progenitors from Human iPS Cells using Pax7</title><p id="P4">In this report from our group [<xref rid="R12" ref-type="bibr">12</xref>], we have applied conditional expression of Pax7, a critical molecular regulator of the skeletal myogenic program, to efficiently promote myogenesis from human ES/iPS cells, a method that worked well in the mouse system [<xref rid="R13" ref-type="bibr">13</xref>–<xref rid="R15" ref-type="bibr">15</xref>]. A tet-on inducible Pax7 lentiviral vector was introduced into the iPS cells and upon differentiation of these cells into early mesodermal progenitors, Pax7 was induced by adding doxycycline to the cultures, which promoted the generation of proliferating myogenic precursors. Following FACS purification, Pax7<sup>+</sup> (GFP<sup>+</sup>) cells expanded exponentially <italic>in vitro</italic>, and expressed CD56, M-cadherin and α7 integrin, surface markers associated with early myogenic cells. Following transplantation into an immunodeficient mouse model of DMD, human iPS-derived skeletal myogenic progenitors engrafted into diseased muscle, restored dystrophin expression, improved contractility and seeded the satellite cell compartment. Moreover, long-term engraftment was detected in recipient mice 46 weeks post-transplant. By using non-integrating methods for Pax7 induction, in combination with gene correction approaches, such as DYS-HAC or nuclease mediated gene correction strategies [<xref rid="R16" ref-type="bibr">16</xref>–<xref rid="R19" ref-type="bibr">19</xref>], one can envision a potential strategy for gene/cell-based therapy in MDs.</p></sec><sec id="S3"><title>Differentiation of Human iPS Cells into Myogenic Cells using MyoD</title><p id="P5">Recently, two research groups have used MyoD for myogenic induction of differentiating human iPS cells. In the first study, Tedesco and colleagues [<xref rid="R20" ref-type="bibr">20</xref>] have described a novel approach to differentiate human iPS cells into mesoangioblast-like cells, a cell population they have demonstrated to have skeletal muscle regeneration potential [<xref rid="R21" ref-type="bibr">21</xref>,<xref rid="R22" ref-type="bibr">22</xref>]. Due to the limitation in obtaining sufficient numbers of mesoangioblasts from the vessels of patient biopsies, a protocol to generate these cells from patient-specific iPS was sought. In this investigation, the authors succeeded in doing so using iPS cells from Limb-Girdle MD type 2D (LGMD2D) patients, which are deficient in α-sarcoglycan. A tamoxifen inducible MyoD lentiviral vector was used to promote the <italic>in vitro</italic> myogenic differentiation of these cells into mature myotubes. Using a lentiviral vector encoding human α-sarcoglycan under a muscle specific promoter, corrected human iPS cell-derived mesoangioblast-like cells were shown to engraft and express the missing protein in a mouse model of LGMD2D (Sgca-null/scid/beige) following intramuscular and intra-arterial delivery. One great advantage of this method is the ability of engraftment following systemic delivery.</p><p id="P6">In the second study, Gougenege and colleagues [<xref rid="R23" ref-type="bibr">23</xref>] utilized a two-step protocol, in which human ES and DMD iPS cells were initially differentiated into CD73<sup>+</sup> mesenchymal-like cells, and then infected with an adenoviral vector expressing MyoD under a ubiquitous promoter (CAG) to generate myogenic cells. Following transplantation into damaged muscles of an immunodeficient mouse model of DMD (<italic>Rag/mdx</italic>), iPS-derived myogenic cells engrafted and differentiated into mature myofibers expressing human spectrin. Although myogenic potential of human ES-cell derived CD73<sup>+</sup> mesenchymal cells had been reported previously by Barberi and colleagues [<xref rid="R24" ref-type="bibr">24</xref>], this approach has not been successfully replicated by other laboratories. By tweaking the protocol and by over-expressing MyoD, these authors have improved differentiation efficiency and reduced culture duration. Another advantage of this study is the use of non-integrating adenoviruses for MyoD induction, reducing the risk of mutagenesis.</p><p id="P7">A possible limitation associated with the use of MyoD is its direct effect to induce cell cycle arrest [<xref rid="R25" ref-type="bibr">25</xref>], which may limit myogenic cell expansion to the levels needed for cell therapy as well as their <italic>in vivo</italic> self-renewal. Recently, Iacovino and colleagues [<xref rid="R26" ref-type="bibr">26</xref>] have used Myf5 to induce myogenesis in human ES cells. By inserting the myogenic regulator gene Myf5 into an inducible cassette exchange locus, human H9 ES cells with regulated Myf5 expression were generated. These were differentiated via EBs into mesenchymal cells which underwent efficient myogenesis after Myf5 induction. In this regard, Myf5 might be a better alternative to MyoD induction to avoid cell cycle arrest.</p></sec><sec id="S4"><title>Derivation of Myogenic Mesenchymal Cells from Human ES/iPS Cells</title><p id="P8">Recently Awaya et al. [<xref rid="R27" ref-type="bibr">27</xref>] reported a method for the differentiation of myogenic mesenchymal cells from human ES/iPS cells, which is somewhat similar to the strategy reported for the murine counterparts by the same group [<xref rid="R28" ref-type="bibr">28</xref>, <xref rid="R29" ref-type="bibr">29</xref>]. Human ES/iPS cells were grown initially as embryoid bodies and then as monolayers in the presence of muscle induction medium. In this condition, clusters of Pax3<sup>+</sup> or Pax7<sup>+</sup> cells randomly emerged after 3 weeks, which were subsequently enriched for myogenic cells by sub-culturing on collagen-I coated plates. The peak of expression for myogenic genes was reached after 50 days in culture, at which point cells were able to terminally differentiate into multinucleated myotubes. Transplantation of these cells into immunodeficient non-dystrophic mice resulted in engraftment, as shown by the detection of human laminin-α2. Myogenic cells were able to respond to muscle re-injury and seed the satellite cell compartment to some degree. In both cases, additional transplantation studies in mouse models of MD are warranted. An advantage of this method is the fact that it does not require genetic modification to induce myogenesis. Limitations include the low efficiency of myogenic induction and the adhesion-based purification, which requires long-term <italic>in vitro</italic> culture.</p></sec><sec id="S5"><title>Future Directions</title><p id="P9">iPS technology provides a new doorway for cell-based therapies. However, there are major safety concerns associated with iPS cells, which need to be overcome before they can be seriously considered for clinical applications [<xref rid="R30" ref-type="bibr">30</xref>,<xref rid="R31" ref-type="bibr">31</xref>]. In the case of skeletal muscle regeneration, obstacles include the development of i) an efficient and safe (integration-free) myogenic induction/purification protocol, ii) safe gene correction strategies, and iii) efficient cell delivery approaches.</p><p id="P10">Because <italic>in vitro</italic> differentiation of pluripotent stem cells into the skeletal muscle lineage is very inefficient, most studies rely on the over-expression of genes that are critical on the regulation of the myogenic program (Pax7, Myf5 and MyoD). These approaches, though effective for differentiation, carry the potential of insertional mutagenesis since these genes are commonly delivered using lentiviral vectors.</p><p id="P11">Another point to be considered is the need for cell purification of the target cells to be transplanted since the presence of residual undifferentiated cells in these cell preparations can lead to undesired tumor formation [<xref rid="R31" ref-type="bibr">31</xref>]. Therefore, approaches need to be optimized to guarantee an efficient and safe method for the myogenic induction of human iPS cells in order to be considered for future applications.</p><p id="P12">Likewise, a site specific gene correction approach would be desirable for MD patient-specific iPS cells, as this technology would avoid the risks associated with random integration. Strategies involving nuclease mediated homologous recombination gene correction [<xref rid="R17" ref-type="bibr">17</xref>–<xref rid="R19" ref-type="bibr">19</xref>,<xref rid="R32" ref-type="bibr">32</xref>–<xref rid="R34" ref-type="bibr">34</xref>], are ideal to safely genetically edit and correct mutations in a site specific manner.</p><p id="P13">Finally, since the skeletal muscle is the largest organ of the human body, and in most of the MDs, multiple muscle groups are involved, local intramuscular cell injection is not a practical and feasible option [<xref rid="R35" ref-type="bibr">35</xref>–<xref rid="R37" ref-type="bibr">37</xref>]. Derivation of the appropriate myogenic population from human iPS cells endowed with efficient and selective skeletal muscle homing ability following systemic delivery is another major hurdle to overcome.</p><p id="P14">As a proof of principle, the studies reviewed above have highlighted the therapeutic potential of human iPS cells in muscular dystrophies. Overcoming the safety hurdles associated with myogenic induction and gene correction of human iPS cells will allow researchers to find practical methods for the generation of safe and clinical grade cell preparations for future therapeutic application.</p></sec> |
Thermally assisted electric field control of magnetism in flexible multiferroic heterostructures | <p>Thermal and electrical control of magnetic anisotropy were investigated in flexible Fe<sub>81</sub>Ga<sub>19</sub> (FeGa)/Polyvinylidene fluoride (PVDF) multiferroic heterostructures. Due to the large anisotropic thermal deformation of PVDF (<italic>α</italic><sub>1</sub> = −13 × 10<sup>−6</sup> K<sup>−1</sup> and <italic>α</italic><sub>2</sub> = −145 × 10<sup>−6</sup> K<sup>−1</sup>), the in-plane uniaxial magnetic anisotropy (UMA) of FeGa can be reoriented 90° by changing the temperature across 295 K where the films are magnetically isotropic. Thus, the magnetization of FeGa can be reversed by the thermal cycling between 280 and 320 K under a constant magnetic field lower than coercivity. Moreover, under the assistance of thermal deformation with slightly heating the samples to the critical temperature, the electric field of ± 267 kV cm<sup>−1</sup> can well align the UMA along the two orthogonal directions. The new route of combining thermal and electrical control of magnetic properties realized in PVDF-based flexible multiferroic materials shows good prospects in application of flexible thermal spintronic devices and flexible microwave magnetic materials.</p> | <contrib contrib-type="author"><name><surname>Liu</surname><given-names>Yiwei</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Zhan</surname><given-names>Qingfeng</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Dai</surname><given-names>Guohong</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Zhang</surname><given-names>Xiaoshan</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Wang</surname><given-names>Baomin</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Liu</surname><given-names>Gang</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Zuo</surname><given-names>Zhenghu</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Rong</surname><given-names>Xin</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Yang</surname><given-names>Huali</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Zhu</surname><given-names>Xiaojian</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Xie</surname><given-names>Yali</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Chen</surname><given-names>Bin</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Li</surname><given-names>Run-Wei</given-names></name><xref ref-type="corresp" rid="c2">b</xref><xref ref-type="aff" rid="a1">1</xref></contrib><aff id="a1"><label>1</label><institution>Key Laboratory of Magnetic Materials and Devices & Zhejiang Province Key Laboratory of Magnetic Materials and Application Technology, Ningbo Institute of Materials Technology and Engineering (NIMTE)</institution>, Chinese Academy of Sciences (CAS), Ningbo 315201, People's Republic of China</aff> | Scientific Reports | <p>The possibility of controlling magnetism with an electric field, which can be realized by using a kind of multiferroic materials showing coupled ferroelectricity and ferromagnetism, opens a new way for appealing novel magnetization control schemes in future low power consumed spintronic devices<xref ref-type="bibr" rid="b1">1</xref><xref ref-type="bibr" rid="b2">2</xref><xref ref-type="bibr" rid="b3">3</xref>. However, most of the single-phase multiferroic materials (such as BiMnO<sub>3</sub>, TbMnO<sub>3</sub>) exhibit a low Curie temperature and/or a weak intrinsic magnetoelectric (ME) coupling especially above room temperature<xref ref-type="bibr" rid="b4">4</xref><xref ref-type="bibr" rid="b5">5</xref>.</p><p>BiFeO<sub>3</sub> uniquely shows the room temperature ME coupling, but it is G-type antiferromagnetic or very weak ferromagnetic<xref ref-type="bibr" rid="b6">6</xref><xref ref-type="bibr" rid="b7">7</xref>. An attractive alternative way is to use ferromagnetic/ferroelectric (FM/FE) heterostructures, in which electric control of magnetism can be realized through either strain-mediated ME coupling across the interface, exchange bias, or charge-driven ME coupling. Among the three mechanisms, the strain-mediated ME coupling has proven to be more promising<xref ref-type="bibr" rid="b8">8</xref><xref ref-type="bibr" rid="b9">9</xref><xref ref-type="bibr" rid="b10">10</xref>. In strain-mediated FM/FE heterostructures, a uniaxial strain, which is produced through the converse piezoelectric effect when an electric field applied on FE layer, is transferred to FM layer, due to the inverse magnetostrictive effect, resulting in the change of magnetic properties including magnetic anisotropy, coercive fields, magnetic domains, giant magnetoresistance, etc<xref ref-type="bibr" rid="b11">11</xref><xref ref-type="bibr" rid="b12">12</xref><xref ref-type="bibr" rid="b13">13</xref><xref ref-type="bibr" rid="b14">14</xref>. However, for FM/FE thin-film heterostructures deposited on rigid substrates, the remarkable clamping effect of substrates limit the tunability of magnetic properties by electric field<xref ref-type="bibr" rid="b15">15</xref><xref ref-type="bibr" rid="b16">16</xref>. Flexible or freestanding FM/FE heterostructures can reduce or eliminate the substrate clamping effect, thus enhance the ME coupling and the tunability of magnetic properties<xref ref-type="bibr" rid="b17">17</xref><xref ref-type="bibr" rid="b18">18</xref><xref ref-type="bibr" rid="b19">19</xref>.</p><p>Up to now, most of the previously investigated FM/FE multiferroic composites were fabricated by using oxide ferroelectric materials, such as BaTiO<sub>3</sub>, Pb(Zr, Ti)O<sub>3</sub>, PbZn<sub>1/3</sub>Nb<sub>2/3</sub>O<sub>3</sub>–PbTiO<sub>3</sub>, and PbMg<sub>1/3</sub>Nb<sub>2/3</sub>O<sub>3</sub>–PbTiO<sub>3</sub><xref ref-type="bibr" rid="b20">20</xref><xref ref-type="bibr" rid="b21">21</xref><xref ref-type="bibr" rid="b22">22</xref><xref ref-type="bibr" rid="b23">23</xref>. However, these oxide ferroelectric materials are extremely fragile and not suitable for preparing flexible or freestanding FM/FE heterostructures<xref ref-type="bibr" rid="b24">24</xref>. Organic ferroelectric materials, in particular polyvinylidene fluoride (PVDF) and its copolymers, exhibit excellent mechanical flexibility, high piezoelectric voltage constants, and low production cost, which are good candidates for developing flexible multiferroic composites used in magnetic control of polarization<xref ref-type="bibr" rid="b25">25</xref><xref ref-type="bibr" rid="b26">26</xref><xref ref-type="bibr" rid="b27">27</xref><xref ref-type="bibr" rid="b28">28</xref>. For example, flexible SmFe/PVDF heterostructure with a large voltage output of 230 μV at 2.3 kOe has been reported to show potential application in weak magnetic-field detection<xref ref-type="bibr" rid="b29">29</xref>. In contrast, due to the small piezoelectric coefficients (<italic>d</italic><sub>31</sub> = 21.4 pC N<sup>−1</sup>, <italic>d</italic><sub>32</sub> = 2.3 pC N<sup>−1</sup>) and the low Young's modulus (1.5 GPa) of PVDF<xref ref-type="bibr" rid="b29">29</xref><xref ref-type="bibr" rid="b30">30</xref>, it seems not possible to achieve an effective electric control of magnetism in PVDF based FM/FE heterostructures. On the other hand, PVDF membrane exhibits a large and anisotropic thermal deformation with thermal expansion coefficients <italic>α</italic><sub>1</sub> = −13 × 10<sup>−6</sup> K<sup>−1</sup> along the stretch direction during preparation and <italic>α</italic><sub>2</sub> = −145 × 10<sup>−6</sup> K<sup>−1</sup> perpendicular to that direction<xref ref-type="bibr" rid="b30">30</xref>. The characteristics of large thermal deformation have already been used as thermal actuators in micro-electro-mechanical-systems<xref ref-type="bibr" rid="b31">31</xref><xref ref-type="bibr" rid="b32">32</xref>. In this work, we demonstrate that a significant electric field control of magnetic anisotropy can be realized under assistance of thermal deformation in Fe<sub>81</sub>Ga<sub>19</sub>/PVDF heterostructures. The Fe<sub>81</sub>Ga<sub>19</sub> (FeGa) alloy selected here as the FM layer is a typical magnetostrictive material exhibiting a moderate magnetostriction of 350 ppm under a very low magnetic field and excellent mechanical properties<xref ref-type="bibr" rid="b33">33</xref>. The multiferroic heterostructure of FeGa/PVDF exhibit a good interfacial coupling, which ensures an efficient transfer of mechanical strain between the two phases. Due to the large anisotropic thermal deformation of PVDF, the uniaxial magnetic anisotropy (UMA) can be reoriented in the plane of FeGa films by varying the temperature. Under a constant magnetic field, the magnetization of FeGa can be reversed by thermal cycling across the critical temperature of 295 K where FeGa films are magnetically isotropic. In addition, the combined electric field and thermal deformation can lead to the reorientation of UMA in FeGa/PVDF films through a strain-mediated converse ME coupling.</p><sec disp-level="1" sec-type="results"><title>Results</title><p>FeGa films with 60 nm in thickness were deposited by magnetron-sputtering at an ambient temperature of 291 K on 30-µm-thick PVDF membrane coated with 50-nm-thick Al layers on both sides. A 5 nm Au layer was deposited to protect against oxidation. The sample structure is schematically shown in <xref ref-type="fig" rid="f1">Figure 1a</xref>. We define in the plane of PVDF membrane that <italic>d</italic><sub>31</sub> and <italic>α</italic><sub>1</sub> are along the <italic>x</italic> direction, and <italic>d</italic><sub>32</sub> and <italic>α</italic><sub>2</sub> are along the <italic>y</italic> direction. The X-Ray diffraction measurement confirms that the PVDF membrane is <italic>β</italic>-phase which possesses the ferroelectricity (<xref ref-type="supplementary-material" rid="s1">Figure S1</xref>)<xref ref-type="bibr" rid="b34">34</xref>. The magnetic hysteresis loops of FeGa/PVDF films were measured at different temperatures by a superconducting quantum interference device-vibrating sample magnetometer (SQUID-VSM) and at different applied electric fields by a magneto-optical Kerr effect (MOKE) setup. During the magnetic measurements, an in-plane magnetic field <italic>H</italic> was applied at an angle of <italic>θ</italic> with respect to the <italic>x</italic> direction (<xref ref-type="fig" rid="f1">Figure 1a</xref>).</p><p>The hysteresis loop of the as-prepared FeGa/PVDF film measured along the <italic>x</italic> direction at 291 K exhibits a relative slanted shape with a <italic>M</italic><sub>r</sub>/<italic>M</italic><sub>s</sub> ratio of 0.65 (<xref ref-type="fig" rid="f1">Figure 1b</xref>), while the corresponding hysteresis loop measured along the <italic>y</italic> direction is square with a <italic>M</italic><sub>r</sub>/<italic>M</italic><sub>s</sub> ratio of 0.91 (<xref ref-type="fig" rid="f1">Figure 1c</xref>), where <italic>M</italic><sub>r</sub> and <italic>M</italic><sub>s</sub> are the remanent and saturation magnetization, respectively. These magnetic features indicate that the FeGa/PVDF film shows a UMA along the <italic>y</italic> direction. When the temperature increases to 295 K, the <italic>M</italic><sub>r</sub>/<italic>M</italic><sub>s</sub> ratios for <italic>H</italic> along the <italic>x</italic> and <italic>y</italic> directions are nearly identical, which indicates that the FeGa/PVDF film is magnetically isotropic at this critical temperature. At 300 K, the <italic>M</italic><sub>r</sub>/<italic>M</italic><sub>s</sub> ratio of 0.93 along the <italic>x</italic> direction becomes remarkably greater than that of 0.43 along the <italic>y</italic> direction, which indicates that the easy magnetization direction of FeGa/PVDF film is reoriented from the <italic>y</italic> direction at 291 K to the <italic>x</italic> direction at 300 K. The additional angular dependent magnetic measurements confirm that the FeGa/PVDF film at the critical temperature of 295 K is magnetically isotropic with <italic>M</italic><sub>r</sub>/<italic>M</italic><sub>s</sub> of 0.82 at arbitrary orientation of magnetic field (<xref ref-type="fig" rid="f1">Figure 1d</xref>). The symmetries of the angular dependence of <italic>M</italic><sub>r</sub>/<italic>M</italic><sub>s</sub> ratios obtained at 291 and 300 K confirm that the UMA in FeGa film below and above the critical temperature are oriented along the <italic>y</italic> and <italic>x</italic> directions, respectively (<xref ref-type="fig" rid="f1">Figure 1d</xref>). We ascribe the origin of UMA and the change with temperature to the anisotropic thermal deformation of PVDF. Although we did not set a substrate temperature during the deposition of FeGa layer, the heating effect of sputtered atoms gives rise to a slight thermal contraction of PVDF. After cooling down to the ambient temperature of 291 K, the difference in the thermal expansion of PVDF between the <italic>x</italic> and <italic>y</italic> directions results in an uniaxial tensile strain <italic>ε</italic><sub>T</sub> = <italic>k<sub>c</sub></italic> (α<sub>1</sub>-<italic>α</italic><sub>2</sub>)Δ<italic>T</italic> along the <italic>y</italic> direction, which can be transferred to FeGa layer across the interface. The interfacial coupling factor <italic>k<sub>c</sub></italic> (0 ≤ <italic>k<sub>c</sub></italic> ≤ 1) results from the clamping effect of metallic layers on PVDF, which depends on the Young's moduli, the thicknesses of each layer (including ferromagnetic FeGa layer, ferroelectric PVDF membrane, and two Al electrodes), and the interfacial bondings between the neighboring layers<xref ref-type="bibr" rid="b35">35</xref>. The Young's moduli of metallic layers (<italic>E<sub>f</sub></italic> = 60 GPa for FeGa and <italic>E<sub>a</sub></italic> = 66 GPa for Al) are about 40 times larger than that of PVDF (<italic>E<sub>p</sub></italic> = 1.5 GPa)<xref ref-type="bibr" rid="b29">29</xref><xref ref-type="bibr" rid="b36">36</xref>. The metallic layers with a comparable thickness would significantly clamp the deformation of PVDF with changing temperature. However, in our samples, the thickness of PVDF (<italic>t<sub>p</sub></italic> = 30 µm) is two orders in magnitude larger than the total thickness of both FeGa (<italic>t<sub>f</sub></italic> = 60 nm) and Al (<italic>t<sub>a</sub></italic> = 100 nm)<xref ref-type="bibr" rid="b37">37</xref>, which leads to a theoretical value of <italic>k<sub>c</sub></italic> about 81.5% by using the relation of <inline-formula id="m1"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e609" xlink:href="srep06925-m1.jpg"/></inline-formula> (<xref ref-type="supplementary-material" rid="s1">Supporting information, Figure S2</xref>)<xref ref-type="bibr" rid="b38">38</xref>. Good and tight chemical bondings between the neighboring layers can ensure that the experimental value of <italic>k<sub>c</sub></italic> is close to the theoretical value. Consequently, due to the inverse magnetostriction effect of FeGa film, an UMA <italic>K</italic><sub>T</sub> = 3/2<italic>λ</italic><sub>s</sub><italic>σ</italic><sub>T</sub> along the <italic>y</italic> direction can be induced when the as-prepared FeGa/PVDF film is cooled down, where <italic>λ</italic><sub>s</sub> is the magnetostriction constant of FeGa film. The stress applied on FeGa film is <italic>σ</italic><sub>T</sub> = <italic>ε</italic><sub>T</sub><italic>E<sub>f</sub></italic>/(1-<italic>ν</italic><sup>2</sup>)<xref ref-type="bibr" rid="b36">36</xref>, where <italic>ν</italic> is the Poisson ratio of FeGa film. With increasing temperature from below to above the critical temperature, the thermal contraction along the <italic>y</italic> direction would reverse the strain imposed on FeGa film from tensile to compressive, leading to the reorientation of UMA from the <italic>y</italic> to <italic>x</italic> directions. Similarly, decreasing the temperature across the critical temperature may also result in the inverse reorientation of UMA from the <italic>x</italic> to <italic>y</italic> directions. The further increasing or decreasing temperature away from the critical point would enhance the strength of UMA.</p><p>The thermally induced reorientation of UMA in FeGa/PVDF films can be employed to reverse the magnetization under a constant magnetic field. Before thermal cycling, the FeGa film is fully magnetized along the <italic>x</italic> direction by a saturation magnetic field of 200 Oe at 320 K. <xref ref-type="fig" rid="f2">Figure 2a</xref> shows the temperature dependence of magnetization by cooling and warming the FeGa/PVDF film between 320 and 280 K with applying different magnetic fields from 0 to -25 Oe along the <italic>x</italic> direction. The magnetic fields are selected to be smaller than the measured coercive fields which are 27 Oe at 280 K and 44 Oe at 320 K (<xref ref-type="supplementary-material" rid="s1">Supporting information, Figure S3</xref>). With cooling the sample without magnetic field from 320 to 280 K, the magnetization of FeGa film <italic>decays</italic> about 68 percent from 0.19 to 0.06 memu. In the following warming process from 280 to 320 K, the magnetization returns to the original value. It should be noted that the spontaneous magnetization of FeGa is oppositely <italic>increased</italic> about 2.8 percent for the temperature varied from 320 to 280 K (<xref ref-type="supplementary-material" rid="s1">Supporting information, Figure S4</xref>), which is far less than the strain effect caused by the thermal deformation of PVDF. A negative magnetic field applied during thermal cycling results in a significant difference of magnetization between before and after thermal cycling. The increase in the strength of magnetic field may enlarge this difference and leads to the magnetization after thermal cycling changing from 0.05 to −0.16 memu for the magnetic field increasing from −10 to −25 Oe. As shown in <xref ref-type="fig" rid="f2">Figure 2b</xref>, the thermal cycling with a negative magnetic field of −20 Oe can switch the magnetization of FeGa films from 0.19 to −0.16 memu. The following thermal cycle with a positive magnetic field of 20 Oe may reverse the magnetization back to 0.19 memu (<xref ref-type="fig" rid="f2">Figure 2c</xref>). In <xref ref-type="fig" rid="f2">Figure 2d</xref>, we demonstrate that the thermal cycling associated with an alternatively positive and negative magnetic field can switch the magnetization of FeGa/PVDF film between the positive and negative directions.</p><p>The magnetization reversed by the thermal cycling under the assistance of magnetic field can be understood with considering the change of domain orientations in FeGa/PVDF with temperature, as schematically shown in the insets of <xref ref-type="fig" rid="f2">Figure 2a to 2c</xref>. At 320 K, the orientations of magnetic domains in FeGa layer are squeezed into a narrow distribution around the positive direction of UMA along the <italic>x</italic> direction by a saturation magnetic field. With zero-field cooling the sample, the strength of UMA is reduced due to the tensile strain along the <italic>y</italic> direction produced by the thermal expansion of PVDF. Consequently, the distribution of domain orientations becomes much broader. At the critical point of 295 K where FeGa/PVDF film is magnetically isotropic, the domain orientations of FeGa distribute arbitrarily in the film plane with components along the positive <italic>x</italic> direction. The further decrease of temperature results in the enhancement of the reoriented UMA along the <italic>y</italic> direction and squeezes the FeGa domain orientations along both the positive and negative <italic>y</italic> directions. Therefore, the magnetization of FeGa film measured along the <italic>x</italic> direction decreases in the zero-field cooling process. When warming the sample back to 320 K, the reorientation of UMA can drive the FeGa domain orientations back to the original state of narrow distribution along the <italic>x</italic> direction. In case of applying a negative magnetic field during thermal cycling, the magnetic field rotates FeGa domain orientations towards the negative <italic>x</italic> direction (<xref ref-type="fig" rid="f2">Figure 2b</xref>). The domain orientations with the negative <italic>x</italic> component, which amount depends on the strength of applied field, would be squeezed into a narrow distribution along the negative <italic>x</italic> direction after a thermal cycle of cooling and warming processes across the critical temperature of magnetic isotropy. It is noted that since the applied magnetic field is lower than the coercivity, not all of the magnetization can be switched from positive to negative after thermal cycling under a negative magnetic field. In contrast, a following thermal cycle under an identical positive field can reverse all the magnetization with negative component to positive (<xref ref-type="fig" rid="f2">Figure 2c</xref>).</p><p>The electric field control of magnetic properties in FeGa/PVDF film was measured by a MOKE magnetometer with an electric field applied through the thickness of PVDF. At the ambient temperature of 291 K, the Kerr hysteresis loops measured at different orientations of magnetic field indicate that the as-prepared FeGa/PVDF sample possesses a UMA along the <italic>y</italic> direction (<xref ref-type="fig" rid="f3">Figure 3a</xref>). When applying an electric field on PVDF, the remanent magnetization of FeGa measured along the <italic>x</italic> and <italic>y</italic> directions are increased and decreased linearly with increasing the electric field from −267 to 267 kV cm<sup>−1</sup> due to the strain-mediated converse ME coupling, respectively (<xref ref-type="supplementary-material" rid="s1">Supporting information, Figure S5</xref>). Except a slight change in <italic>M</italic><sub>r</sub>/<italic>M</italic><sub>s</sub>, the hysteresis loops measured at <italic>E</italic> = −267 kV cm<sup>−1</sup> are similar to that obtained without external electric field, which suggests that the UMA of FeGa layer remains aligned along the <italic>y</italic> direction under a negative electric field, but the strength is slightly enhanced (<xref ref-type="fig" rid="f3">Figure 3b</xref>). However, when <italic>E</italic> = 267 kV cm<sup>−1</sup>, the hysteresis loops measured along both the <italic>x</italic> and <italic>y</italic> directions are almost identical, which indicates a magnetic isotropy in FeGa/PVDF film (<xref ref-type="fig" rid="f3">Figure 3c</xref>). The angular dependent magnetic measurements confirm the changes of UMA in FeGa/PVDF at various electric fields (<xref ref-type="fig" rid="f3">Figure 3g</xref>). It is clearly seen that at <italic>T</italic> = 291 K the strain-mediated converse ME coupling cannot lead to a reorientation of UMA in FeGa layer by applying an electric field on PVDF membrane due to the rather small piezoelectric coefficients of PVDF and the UMA in FeGa layer.</p><p>In the present PVDF based heterostructures, we demonstrate that the thermal deformation can be used to eliminate the UMA in FeGa layer and assist electric field to realize the reorientation of UMA in FeGa/PVDF film. When we increase the temperature to 295 K, the hysteresis loops measured with a magnetic field applied along the <italic>x</italic> and <italic>y</italic> directions at <italic>E</italic> = 0 show that the FeGa/PVDF film is almost magnetically isotropic (<xref ref-type="fig" rid="f3">Figure 3d</xref>), which indicates that the UMA is remarkably reduced by slightly increasing the temperature through the effect of anisotropic thermal contraction. When applying an electric field of −267 kV cm<sup>−1</sup> on PVDF, the <italic>M</italic><sub>r</sub>/<italic>M</italic><sub>s</sub> ratio measured along the <italic>y</italic> direction becomes larger than that obtained along the <italic>x</italic> direction (<xref ref-type="fig" rid="f3">Figure 3e</xref>). On the contrary, for <italic>E</italic> = 267 kV/cm, the <italic>M</italic><sub>r</sub>/<italic>M</italic><sub>s</sub> ratio along the <italic>y</italic> direction is smaller than that along the <italic>x</italic> direction (<xref ref-type="fig" rid="f3">Figure 3f</xref>). Obviously, after warming to 295 K, depending on the electric field applied on PVDF, the UMA of FeGa layer can be adjusted along the <italic>x</italic> or <italic>y</italic> directions. The in-plane reorientation of UMA driven by electric field can be clearly seen in the angular dependence of normalized remanent magnetization measured at <italic>T</italic> = 295 K with different electric fields applied on PVDF (<xref ref-type="fig" rid="f3">Figure 3h</xref>).</p><p><xref ref-type="fig" rid="f4">Figure 4a</xref> shows the conventional strain-mediated mechanism for electric control of magnetism in FM/FE multiferroic heterostructures. Due to the converse piezoelectric effect, an electric field applied on FE phase produces a strain which can be transferred to FM phase. Then, the magnetic properties of FM can be modified by this strain through the inverse magnetostrictive effect. For FM/FE heterostructures using PVDF as the ferroelectric layer, thanks to the characteristics of large anisotropic thermal deformation of PVDF, a variation of temperature may additionally generate a mechanical strain, assisting electric field to change the magnetic properties of FM phase, which provides us a new route for multi-field control of magnetism in FM/FE heterostructures (<xref ref-type="fig" rid="f4">Figure 4b</xref>).</p></sec><sec disp-level="1" sec-type="discussion"><title>Discussion</title><p>In order to quantitatively understand the thermally assisted electric field control of magnetic anisotropy in FeGa/PVDF film, the total free energy density <italic>F</italic> of FeGa film can be written as: <italic>F</italic> = (<italic>K</italic><sub>E</sub>+<italic>K</italic><sub>T</sub>)cos<sup>2</sup><italic>φ</italic>-<italic>MH</italic>cos(<italic>θ-φ</italic>), where <italic>K</italic><sub>E</sub> and <italic>K</italic><sub>T</sub> are the UMAs induced by the electric field through the converse piezoelectric effect and by the variation of temperature through the anisotropic thermal deformation effect, respectively, and <italic>φ</italic> is the angle between the magnetization and the <italic>x</italic> direction. Due to the dimensions of FeGa films, the in-plane demagnetization energy is too weak to be taken into account<xref ref-type="bibr" rid="b39">39</xref>, which only provides a negligible constant term into the free energy. <italic>K</italic><sub>E</sub> depends on the piezoelectric coefficients of PVDF and the applied electric field. Thus, <italic>K</italic><sub>E</sub> = 3<italic>ε</italic><sub>E</sub><italic>λ</italic><sub>s</sub><italic>E<sub>f</sub></italic>/2(1-<italic>ν</italic><sup>2</sup>), where the electric-field-induced strain <italic>ε</italic><sub>E</sub> is evaluated to be <italic>k<sub>c</sub></italic> (<italic>d</italic><sub>31</sub>-<italic>d</italic><sub>32</sub>)<italic>E</italic>. λ<sub>s</sub> and <italic>E<sub>f</sub></italic> are estimated to be about 100 ppm and 60 GPa for polycrystalline FeGa film, respectively<xref ref-type="bibr" rid="b36">36</xref>. <italic>ν</italic> is chosen to be a typical value of 0.3 for metals<xref ref-type="bibr" rid="b40">40</xref>. We assume <italic>K</italic><sub>T</sub> = 0, i.e., magnetic isotropy for PVDF at 295 K. According to these parameters, the energy landscapes of FeGa/PVDF film under different applied electric fields at 291 and 295 K under zero magnetic field can be numerically calculated, as respectively shown in <xref ref-type="fig" rid="f4">Figure 4c and 4d</xref>. At 291 K, the FeGa film shows the UMA along the <italic>y</italic> direction, which is originated from the strain caused by the anisotropic thermal deformation of PVDF. A uniaxial strain produced by an electric field of 267 kV cm<sup>−1</sup> applied on PVDF via the converse piezoelectric effect can greatly reduce the strength of UMA, but not reverse the orientation of UMA in FeGa/PVDF. As a contrast, a negative electric field of −267 kV cm<sup>−1</sup> may enhance the UMA along the <italic>y</italic> direction. The slight increase of temperature to 295 K generates a compressive strain along the <italic>y</italic> direction due to the anisotropic thermal contraction, and reduces the energy barrier of UMA. Consequently, in this case, a positive electric field applied on PVDF is able to reorient the UMA of FeGa along the <italic>x</italic> direction, while a negative electric field enhances the UMA along the <italic>y</italic> direction, which are consistent with the experimental observations shown in <xref ref-type="fig" rid="f3">Figure 3</xref>.</p><p>In conclusion, we have successfully fabricated flexible multiferroic FeGa/PVDF heterostructures. Due to the large anisotropic thermal deformation of the PVDF membrane, the in-plane UMA of FeGa layer can be reoriented 90° by changing temperature across 295 K where FeGa films are magnetically isotropic. Utilizing this special characteristic, the magnetization of FeGa can be reversed by thermal cycling between 280 and 320 K under a constant magnetic field lower than the coercivity. Because of the rather small piezoelectric coefficients, a sufficient electric field applied on PVDF cannot lead to a reorientation of UMA in FeGa at the ambient temperature of 291 K. Under the assistance of thermal deformation by slightly heating the sample to 295 K, the electric field of ± 267 kV cm<sup>−1</sup> is able to align the UMA along the two orthogonal directions through a strain-mediated converse ME coupling. Our experimental results suggest that the thermal control of magnetic properties based on the anisotropic thermal deformation of PVDF have promising applications in thermally tunable or thermally assisted electric field tunable flexible spintronic devices and flexible microwave magnetic materials.</p></sec><sec disp-level="1" sec-type="methods"><title>Methods</title><sec disp-level="2"><title>Specimen Fabrication</title><p>The commercial PVDF membranes with 30 µm in thickness from were sputtered with 50-nm-thick Al layers on both sides. The 60 nm FeGa layers were deposited onto PVDF at an ambient temperature of 291 K by using a magnetron sputtering system with a base pressure better than 5×10<sup>−5</sup> Pa. During sputtering, the argon flow was kept at 50 sccm and the pressure was set at 1.0 Pa. The growth rate of FeGa film was controlled at 10.0 nm min<sup>−1</sup>. A deposition rate of 2.0 nm min<sup>−1</sup> was used for growing a 5 nm Au protection layer.</p></sec><sec disp-level="2"><title>Characterization</title><p>The crystalline structure of PVDF was checked by an x-ray diffraction meter (D8 Advance, Bruker) with Cu Kα radiation. The thicknesses of FeGa and Au layers were calibrated by X-ray reflectivity. A standardized ferroelectric test system (Precision Premier II, Radiant Technologies) was used to measure the electric hysteresis loops of PVDF. A superconducting quantum interference device-vibrating sample magnetometer (Quantum Design) was employed to measure the magnetic hysteresis loops of FeGa/PVDF in the temperature range from 280 to 320 K. The angular dependence of magnetic hysteresis loops of FeGa layer was measured at different polarization states of PVDF by magneto-optical Kerr effect (MOKE) from 291 to 300 K. A hot plate mounted on the back of samples was used to heat the samples during MOKE measurements. The Al layers on both sides of PVDF were connected to a voltage source (Keithley 237 High-Voltage Source-Measure Unit) with thin Pt wires. During the MOKE measurements, the voltage source provides an electric field to polarize the ferroelectric PVDF membrane thought the thickness.</p></sec></sec><sec disp-level="1"><title>Author Contributions</title><p>Y.L., Q.Z. and R.L. conceived the experiments. Y.L., G.D. and X.Z. carried out experiments. Y.L. and Q.Z. did modeling. Y.L., Q.Z. and R.L. wrote the manuscript. B.W., G.L., Z.Z., X.R., H.Y., X.Z., Y.X. and B.C. discussed the data and the results, and commented the manuscript.</p></sec><sec sec-type="supplementary-material" id="s1"><title>Supplementary Material</title><supplementary-material id="d33e24" content-type="local-data"><caption><title>Supplementary Information</title><p>Supplementary information</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="srep06925-s1.doc"/></supplementary-material></sec> |
Symmetrically periodic segregation in a vertically vibrated binary granular bed | <p>Periodic segregation behaviors in fine mixtures of copper and alumina particles, including both percolation and eruption stages, are experimentally investigated by varying the ambient air pressure and vibrational acceleration. For the cases with moderate air pressure, the heaping profile of the granular bed keeps symmetrical in the whole periodic segregation. The symmetrical shape of the upper surface of the granular bed in the eruption stage, which resembles a miniature volcanic eruption, could be described by the Mogi model that illuminates the genuine volcanic eruption in the geography. When the air pressure increases, an asymmetrical heaping profile is observed in the eruption stage of periodic segregation. With using the image processing technique, we estimate a relative height difference between the copper and the alumina particles as the order parameter to quantitatively characterize the evolution of periodic segregation. Both eruption and percolation time, extracted from the order parameter, are plotted as a function of the vibration strength. Finally, we briefly discuss the air effect on the granular segregation behaviors.</p> | <contrib contrib-type="author"><name><surname>Wen</surname><given-names>Pingping</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Zheng</surname><given-names>Ning</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Li</surname><given-names>Liangsheng</given-names></name><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Shi</surname><given-names>Qingfan</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><aff id="a1"><label>1</label><institution>School of Physics, Beijing Institute of Technology</institution>, Beijing 100081, <country>China</country></aff><aff id="a2"><label>2</label><institution>Key Laboratory of Cluster Science of Ministry of Education</institution>, Beijing 100081, <country>China</country></aff><aff id="a3"><label>3</label><institution>Science and Technology on Electromagnetic Scattering Laboratory</institution>, Beijing 100854, <country>China</country></aff> | Scientific Reports | <p>Granular segregation induced by external excitation is of importance in industrial and scientific society, widely applied in the agriculture, food and pharmaceutical processing, and ore dressing<xref ref-type="bibr" rid="b1">1</xref><xref ref-type="bibr" rid="b2">2</xref><xref ref-type="bibr" rid="b3">3</xref><xref ref-type="bibr" rid="b4">4</xref>. In last two decades, shaken, sliding or rotary segregation of binary particles exhibits various patterns, such as the well-known Brazil nut effect (BNE)<xref ref-type="bibr" rid="b3">3</xref><xref ref-type="bibr" rid="b4">4</xref>, reverse Brazil nut effect (RBNE)<xref ref-type="bibr" rid="b5">5</xref><xref ref-type="bibr" rid="b6">6</xref>, sandwich pattern (SP)<xref ref-type="bibr" rid="b7">7</xref>, axial or radial streaks<xref ref-type="bibr" rid="b8">8</xref><xref ref-type="bibr" rid="b9">9</xref><xref ref-type="bibr" rid="b10">10</xref>, and sliding spontaneous stratification<xref ref-type="bibr" rid="b11">11</xref><xref ref-type="bibr" rid="b12">12</xref> etc. Different mechanisms including void filling (also called percolation, small particles tend to fall downward through the gaps in between the large particles)<xref ref-type="bibr" rid="b5">5</xref><xref ref-type="bibr" rid="b13">13</xref><xref ref-type="bibr" rid="b14">14</xref>, density-driven<xref ref-type="bibr" rid="b6">6</xref>, inertia<xref ref-type="bibr" rid="b7">7</xref>, buoyancy<xref ref-type="bibr" rid="b15">15</xref>, convection<xref ref-type="bibr" rid="b16">16</xref>, geometric shape of particles<xref ref-type="bibr" rid="b17">17</xref>, the influence of different frictions<xref ref-type="bibr" rid="b18">18</xref> and interstitial media<xref ref-type="bibr" rid="b19">19</xref>, were presented to account for these seemingly simple segregation behaviors. Afterwards it is found that in many situations one or several of these mechanisms simultaneously take effect on the segregation, each being dominant in different parameter regimes and thus leading to this complexity<xref ref-type="bibr" rid="b20">20</xref><xref ref-type="bibr" rid="b21">21</xref>. However, most experiments and theories only concentrate on the time-independent segregation, in which once spatial pattern is formed, the configuration of the whole granular bed remains a steady state.</p><p>Compared with segregation with stable spatial patterns, few well-segregated configurations are time-dependent. Burtally et al observed that the upper surface and the separation boundary periodically swing together back and forth between two alternative configurations where both surfaces were tilted at an angle close to the dynamical repose angle<xref ref-type="bibr" rid="b22">22</xref>. Strictly speaking, the boundaries were time-dependent, but the basic configurations of the granular bed at all times were unchanged. Until recently, we have observed that in some regimes of specific shaken parameters, the configuration of a copper and alumina mixture evolves with time development, showing the time-dependence characteristic<xref ref-type="bibr" rid="b23">23</xref>. Traditionally, segregation experiments are performed at atmospheric pressure. However, researches have demonstrated that interstitial air plays an important role on segregation of granular materials<xref ref-type="bibr" rid="b24">24</xref><xref ref-type="bibr" rid="b25">25</xref><xref ref-type="bibr" rid="b26">26</xref>, which suggests that the manipulation of the air pressure may introduce a wealth of new time-dependent segregation phenomena, providing more information for the deep understanding of the time-dependent separation behaviors.</p><p>In this study, we observe a time-dependent periodic segregation (PS) behavior in a vertically vibrated binary granular mixture. When tuning the pressure of interstitial air, the PS exhibits two different forms: symmetrically periodic segregation (SPS) at the region of low air pressure and asymmetrically periodic segregation (APS) at the region of high air pressure. Here we systematically investigate the SPS behavior. We first use a sequence of successive snapshots to illustrate two alternating stages in the SPS pattern: the percolation stage where the alumna particles pass through the copper layer and assemble at the bottom; the eruption stage where the deposited alumina layer heaps and then surges onto the top from the middle of the upper copper layer, which looks like a miniature volcanic eruption. To quantitatively characterize the periodic behaviors, we introduce an order parameter defined as a relative height difference between copper and alumina particles by the image processing technique. Not only can the order parameter be used to extract the percolation time, eruption time and whole period of the SPS quantitatively and accurately, but to characterize every status in time-dependent segregation. With the aid of the order parameter, we plot the periodic time at the percolation and the eruption stage as a function of vibrational acceleration Γ. We also concern about the eruption stage in SPS since it appears analogous to a volcanic eruption in nature. Thus we attempt to apply Mogi model which was a classical model in the earthquake field for the deformational evolution of the earth surface<xref ref-type="bibr" rid="b27">27</xref>, to fit the arching profile of upper surface of the granular bed. It is surprisingly found that the model is able to describe the deformational evolution of the upper surface of the granular volcano. Finally, we briefly discuss the APS pattern and the air effect on the granular segregation.</p><sec disp-level="1" sec-type="results"><title>Results</title><sec disp-level="2"><title>Experimental setup</title><p>A vertical vibration system is used to investigate the periodic segregation behaviors of the fine mixture. The fundamental diagram of the experimental establishment is illustrated in <xref ref-type="fig" rid="f1">Fig. 1</xref>. A top-opened rectangular glass container with a size 40 mm (length) × 10 mm (width) × 50 mm (height) is mounted on the platform of an electromechanical shaker that is used to generate vertical sinusoidal motion, with the ratio of the horizontal vibration amplitude to the vertical vibration amplitude less than 3%. An accelerometer continuously monitors the vertical vibration, which is conveniently characterized by two control parameters <italic>f</italic> and Γ = 4Aπ<sup>2</sup><italic>f</italic><sup>2</sup>/g, where <italic>f</italic> is the vibrational frequency, and Γ is a dimensionless acceleration, namely the ratio of the maximum acceleration of the shaker to the gravitational acceleration <italic>g</italic>, <italic>A</italic> is the vibration amplitude. In this experiment, we choose the frequency <italic>f</italic> at 42 Hz and the acceleration Γ within the ranges of 5.4–6.8. A plexiglass cylinder, of a diameter 120 mm and a height 240 mm, bolted to the shaker acts as a vacuum chamber which can be evacuated to a lowest pressure around 30 Pa through a mechanical pump. By rotating a fine tuning valve on the chamber, the background air pressure inside the chamber can be quantitatively adjusted.</p><p>The binary mixture in the glass container consists of copper particles of density 8.38 g/cm<sup>3</sup> and of diameter 320 ± 30 μm and alumina particles of density 1.65 g/cm<sup>3</sup> and of diameter 170 ± 10 μm. Unless specified otherwise, each volume of the granular components, in the proportion 50%:50%, used in the binary mixture is 3 ml for all experimental trials. Before the experiment, both types of particles are mixed intimately and handled by an antielectrostatic process. To eliminate the build-up of static charges, we replace the mixture by freshly prepared particles every 20 min, and maintain the humility of the laboratory at the range of 45%–55%. After all experimental conditions are well established, we use a digital camcorder to record the spatial configuration of the granular bed, and also apply a high-speed camera (Phantom V7.3) to capture the details of the dynamical deformation process of the upper surface of the granular bed.</p></sec><sec disp-level="2"><title>Phase diagram</title><p>Vibrating patterns of the binary mixture at various air pressures and dimensionless accelerations are recorded. To avoid the air flow caused by the pump evacuation, the mechanical pump is halted before turning on the shaker. The initial status of the mixture is always mixed state, and all stable configurations are identified as a pattern that may be maintained at least ten min at given conditions.</p><p><xref ref-type="fig" rid="f2">Fig. 2</xref> represents the regions of different patterns in the Γ<italic>-P</italic> plane. At low pressure region, the mixture retains a mixed state (MS) and no segregation behaviors are observed. Any tendency to separate particles is thwarted by strong global convection. As the air pressure is steadily increased, a segregation behavior emerges. The mixture first evolves into a spatial configuration of one single copper rich layer sandwiched between two alumina rich layers in seconds. Subsequently, the alumina particles on the top percolate through the copper layer and accumulate on the bottom, and thus the two alumina layers merge into one. The final configuration which large particles move to the top of a collection of small particles is the well-known BNE. The boundary of the two components is not necessarily horizontal for BNE; two types of heaping boundaries are observed. When the air pressure is relatively low, a symmetrical heap occasionally occurs in the middle of the container. Remarkably, the symmetrical heap is unstable and soon vanishes, and then reappears after a while, forming an oscillation. At the region with high air pressure and large value of Γ, an asymmetrical heap always arises and it is a stable configuration (see the inset in <xref ref-type="fig" rid="f2">Fig. 2</xref>). Whatever the boundary looks like, the spatial configuration of BNE is time-independent. When Γ continues to increase, the mixture enters into a time-dependent segregation status, namely the PS region. In this region, either the symmetrical or the asymmetrical heap keeps growing until the heap breaks through the suppression of the upper copper layer, surging onto the top surface. According to the difference from the spatial conformation of the eruption stage, we divide the PS into two categories, the symmetrical PS pattern (SPS) and the asymmetrical PS pattern (APS). The corresponding insets in <xref ref-type="fig" rid="f2">Fig. 2</xref> illustrate the inversion moments of alumina particles for two patterns. The APS takes place in the high pressure regime. In contrast, the SPS is a new type of periodic segregation which always occurs at low air pressure, as shown in <xref ref-type="fig" rid="f2">Fig. 2</xref>. Next we focus on the SPS pattern as a detailed investigation of the PS behaviors.</p></sec><sec disp-level="2"><title>Symmetrically periodic segregation</title><p><xref ref-type="fig" rid="f3">Fig. 3</xref> illustrates a complete cycle of the segregation using a series of consecutive snapshots at <italic>P</italic> = 7 kPa and Γ = 6.4. Initially the mixture is vibrated for a while, forming a RBNE configuration, as shown in <xref ref-type="fig" rid="f3">Fig. 3(a)</xref>. However, the RBNE is unstable. As more time elapses, the alumina particles percolate through the copper layer and constitute a new layer below the copper one; the RBNE evolves to be a sandwich pattern [<xref ref-type="fig" rid="f3">Fig. 3(b)</xref>]. With the amount of alumina particles on the top layer steadily decreasing, the alumina particles finally deposit at the bottom of the container, developing into a BNE configuration [see <xref ref-type="fig" rid="f3">Fig. 3(c)</xref>]. Here it is natural that the duration from <xref ref-type="fig" rid="f3">Fig. 3(a)</xref> to <xref ref-type="fig" rid="f3">Fig. 3(c)</xref> is defined as the percolation stage. It should be pointed out that BNE is not final state for the system. The horizontal alumina layer becomes unstable; namely the alumina particles begin to gather in the middle of the granular bed, forming a symmetrical heap, as shown in <xref ref-type="fig" rid="f3">Fig. 3(d)</xref>. The symmetrical alumina heap gradually grows and eventually breaks through the upper copper layer, surging onto the top. The duration from <xref ref-type="fig" rid="f3">Fig. 3(c)</xref> to <xref ref-type="fig" rid="f3">Fig. 3(i)</xref> is referred to as the eruption stage; including the heaping process or called the pre-eruption process [see <xref ref-type="fig" rid="f3">Fig. 3(c)</xref>–<xref ref-type="fig" rid="f3">Fig. 3 (f)</xref>] and the inversion process [refer to <xref ref-type="fig" rid="f3">Fig. 3(f)</xref>–<xref ref-type="fig" rid="f3">Fig. 3(i)</xref>], which resembles a miniature volcano being in violent eruption. In general, the granular bed in SPS undergoes a stable cycle of RBNE-SP-BNE-RBNE, which consists of the percolation and the eruption stage.</p></sec><sec disp-level="2"><title>Relative height difference <italic>ϕ</italic></title><p>To date, the segregation behaviors in most literatures were described with qualitative methods, for instance, BNE or RBNE pattern was judged only by the photo images. The approach for quantitatively determining the segregation degree is still scarce. Even if a quantitative definition was presented to differentiate the pure segregation from light mixed state, the requirement of a flat boundary of the separated layers restricted its application<xref ref-type="bibr" rid="b6">6</xref>. Particularly, if the segregation behavior is time-dependent, such an approach in Ref. <xref ref-type="bibr" rid="b6">6</xref> for various segregation patterns becomes inaccurate and inconvenient. In order to quantitatively characterize the time-dependent segregation patterns, we introduce a relative height difference <italic>ϕ</italic> between copper and alumina particles by using the image processing technique <disp-formula id="m1"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e300" xlink:href="srep06914-m1.jpg"/></disp-formula>Where <inline-formula id="m6"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e303" xlink:href="srep06914-m6.jpg"/></inline-formula>and <inline-formula id="m7"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e307" xlink:href="srep06914-m7.jpg"/></inline-formula> are height coordinates of pixels of copper and alumina particles, and <italic>N<sub>Cu</sub></italic> and <italic>N<sub>Al</sub></italic> are the numbers of pixels of copper and alumina particles in a snapshot. The container is reasonably considered quasi-two dimension<xref ref-type="bibr" rid="b30">30</xref>, and we have observed almost identical configuration of the granular bed at the opposite large face of the container. In this sense, the relative height difference ϕ can be applied to estimate the normalized relative position of the centroids of two components in the granular bed. We can utilize this relative height difference ϕ as an order parameter to distinguish different segregation states. <xref ref-type="fig" rid="f4">Fig. 4</xref> shows a time evolution of the order parameter in five SPS periods at <italic>P</italic> = 7 kPa and Γ = 6.4. The excellent repeatability of the order parameter indicates the periodicity of the SPS behavior. Furthermore, the time at the percolation and the eruption stage can be extracted from <xref ref-type="fig" rid="f4">Fig. 4</xref>. Combining with the visual observation, we build the corresponding relations between the order parameter and the real physical process, see <xref ref-type="fig" rid="f4">Fig. 4</xref>. The order parameter ϕ at point <italic>a</italic> gains a maximum negative value, which means two species of particles have reached a high degree of separation. The centroid of the copper particles is lower than that of the alumina particles, and the case implies a RBNE status [see <xref ref-type="fig" rid="f3">Fig. 3(a)</xref>]. Similarly, the maximum positive value of the order parameter ϕ at point <italic>c</italic> corresponds to a BNE state. The duration from the RBNE to the BNE, namely from ϕ(a) to ϕ(c) is the percolation stage. The sharp decrease of the order parameter from point <italic>c</italic> to point <italic>i</italic> suggests a dramatic transformation of the spatial configuration of the granular bed, and corresponds to the eruption stage. The corresponding relation demonstrates that the relative height difference <italic>ϕ</italic> can precisely distinguish different segregation states in the time-dependent segregation behavior, and quantitatively and conveniently extract the percolation and the eruption time, which are used to investigate the relations with the external driving parameters, see <xref ref-type="fig" rid="f5">Fig. 5</xref>.</p></sec><sec disp-level="2"><title>Periodic time</title><p>In this section, we measure the percolation time <italic>T<sub>p</sub></italic>, the eruption time <italic>T<sub>e</sub></italic>, and whole cycle time <italic>T<sub>c</sub> = T<sub>p</sub> + T<sub>e</sub></italic> as a function of the vibration acceleration Γ in SPS, respectively. The measurements apparently show a non-monotonic dependence of the percolation time on the vibration acceleration, whereas the eruption time exhibits a monotonic relation. The monotonic decrease of the eruption time indicates that it is easier for the alumina layer to break the suppression of the copper layer with stronger vibrations. At a higher Γ, once most alumina particles have accumulated below the copper layer, the symmetrical heap immediately grows. While at a lower Γ, the heap of the alumina layer does not start until the copper layer becomes more pure. It is worth noting that for all Γ, it only takes several seconds from the onset of the heap to the end of the eruption.</p><p>Furthermore, when Γ> 6.2, the eruption time approaches a stable value which is small compared with the percolation time. Thus, the whole cycle time is dominated by the percolation time, basiclly following the curve shape of percolation time except Γ< 6.2. The non-monotonic behavior implies that underlying physics still keeps open.</p></sec><sec disp-level="2"><title>Mogi model</title><p>In a mono-disperse granular system, the center-heaping formations derive from the instabilities of the flat surface, and the underlying mechanism attributes to downward convection<xref ref-type="bibr" rid="b28">28</xref><xref ref-type="bibr" rid="b29">29</xref>. However, for a bi-disperse granular bed, the deformation of the arched profile of upper surface in eruption stage more likely originates from the heaping push of the bottom layer. It can be clearly seen that the arch of the upper surface is much gentler than that of the interface between the two layers [see <xref ref-type="fig" rid="f3">fig. 3(e)</xref>]. Thus single-layer heaping model may not be applicable for this bottom-heap-pushed deformation of the upper surface. Visually, the symmetrical eruption stage is similar to the process of a volcanic eruption in nature, and hence we attempt to introduce the Mogi model<xref ref-type="bibr" rid="b27">27</xref> to fit the profile of the upper surface. To obtain the profile evolution throughout the eruption stage, we use a high-speed camera at a sample rate 400 fps. We average the 400 pictures to acquire the topography of the upper surface in one second, and to eliminate the profile fluctuation of the upper surface. The topography of the upper surface is averaged as <disp-formula id="m2"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e397" xlink:href="srep06914-m2.jpg"/></disp-formula>Here <italic>x</italic> is the horizontal coordinate of the upper surface and <italic>H(x, t)</italic> is the height of the surface from the bottom of the container [see the illustration in <xref ref-type="fig" rid="f6">Fig. 6(a)</xref>]. We then apply the Mogi model to fit the averaged profile as follows<xref ref-type="bibr" rid="b27">27</xref>, <disp-formula id="m3"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e412" xlink:href="srep06914-m3.jpg"/></disp-formula>Where <italic>H<sub>0</sub></italic> is the height of the granular bed at rest, <italic>x<sub>0</sub></italic> is the horizontal projection of the upper surface center, <italic>V</italic> and <italic>d</italic> are fitting parameters. When <italic>x = x<sub>0</sub></italic>, <italic>H(x, t)</italic> is the apex of the profile. Then, <disp-formula id="m4"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e440" xlink:href="srep06914-m4.jpg"/></disp-formula>Therefore, the upper surface profile can be fitted by the equation below, <disp-formula id="m5"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e443" xlink:href="srep06914-m5.jpg"/></disp-formula>In our experiment, <italic>H<sub>0</sub></italic>, <italic>ΔH<sub>max</sub></italic> and <italic>x<sub>0</sub></italic> are measurable parameters, and thus <italic>V</italic> is the only fitting parameter. <xref ref-type="fig" rid="f6">Fig. 6(b)</xref> shows the comparison between the fitting result from Eq. (5) and the averaged experimental profile in four seconds at f = 42 Hz, Γ = 6.2 and <italic>P</italic> = 7 kPa. The result shows that the Mogi curves are in good agreement with the topographic growth of the upper surface. <xref ref-type="fig" rid="f6">Fig. 6(I)–(IV)</xref> illustrate the profile evolution of the upper surface with four consecutive figures in a more intuitive way.</p><p>The inset in <xref ref-type="fig" rid="f6">Fig. 6(b)</xref> shows the fitting parameter monotonically decreases with time. It is noteworthy that the fitting parameter has actual physical meaning in the volcanic eruption; that is to say, <italic>V</italic> is the spherical volume of the lava as a heat point<xref ref-type="bibr" rid="b27">27</xref>. However, the heat point in the Mogi model cannot be directly measured and examined in our experiments.</p></sec><sec disp-level="2"><title>Asymmetrically periodic segregation</title><p>In the APS region, the mixture undergoes a similar time-dependent segregation procedure with that in the SPS region. <xref ref-type="fig" rid="f7">Fig. 7</xref> shows the order parameter of the APS in five periods for <italic>P</italic> = 50 kPa and Γ = 6.2. The processes marked I and II represent the percolation and the eruption stage, respectively. The percolation stage of the APS has insignificant difference compared with the SPS. We have investigated the relations between the percolation time and the numbers of two species of particles in atmosphere, where the amount of either alumina or copper particles increases, the percolation time monotonically increases<xref ref-type="bibr" rid="b23">23</xref>. Nevertheless, the eruption stage of the APS is distinctly different from the SPS. The lower layer of the alumina particles forms a tilting heap at one side of the container, and then the alumina particles surge onto the top surface along either wall of the container, covering the copper layer. Because of the asymmetric profile of the heap, the Mogi model is incapable of fitting the profile evolution of the upper surface during the eruption stage in the APS.</p></sec></sec><sec disp-level="1" sec-type="discussion"><title>Discussion</title><p>In this experiment, the air effect on the segregation patterns is conclusive. First, all segregation behaviors vanish at sufficiently low pressure, which is consistent with other observations in the powder system<xref ref-type="bibr" rid="b22">22</xref><xref ref-type="bibr" rid="b23">23</xref><xref ref-type="bibr" rid="b24">24</xref><xref ref-type="bibr" rid="b25">25</xref><xref ref-type="bibr" rid="b26">26</xref>. Second, by modulating the air pressure inside the chamber, segregation behaviors can switch between the time-independent segregation pattern (BNE) and the time-dependent segregation pattern (PS). In addition, numerous experimental and theoretical researches have demonstrated that ambient air is of significant importance on the heap formations<xref ref-type="bibr" rid="b28">28</xref><xref ref-type="bibr" rid="b29">29</xref><xref ref-type="bibr" rid="b30">30</xref><xref ref-type="bibr" rid="b31">31</xref><xref ref-type="bibr" rid="b32">32</xref><xref ref-type="bibr" rid="b33">33</xref>. The air pressure can influence the symmetry of the heap, which distinguishes between the APS and the SPS pattern in this experiment. Due to the experimental difficulty in measuring the distribution of the air pressure and the air flow inside the granular bed, the quantitative investigation of the air effect on granular segregation has been seldom involved. In our experiment, we preliminarily study a critical value of the air pressure, <italic>P<sub>c</sub></italic> as a function of vibration acceleration Γ over which an eruption can occur [see the left boundary of the APS region in <xref ref-type="fig" rid="f2">Fig. 2</xref>].</p><p>In summary, we observe a time-dependent periodic segregation behavior, which presents in two different patterns, namely the SPS and the APS, in a vertically vibrated binary granular mixture. A complete cycle of the SPS is described by the successive snapshots. We also plot a Γ-<italic>P</italic> phase diagram that delineates different segregation behaviors. In the phase diagram, it is clearly shown that the APS and the SPS are reversible by tuning the pressure of interstitial air. Additionally, a well-defined order parameter is introduced to quantitatively describe the time-dependent segregation behaviors, for example, which exhibits the excellent periodicity of both periodic segregation behaviors. With using the order parameter, we also can precisely and conveniently obtain the percolation time and the eruption time in the SPS. With the extracted data, we discuss the relation between the percolation and the eruption time with the vibration acceleration, respectively. Since the eruption stage of the SPS resembles a genuine volcanic eruption in nature, a simplified volcanic model proposed by K. Mogi is used to characterize the profile evolution of the upper surface of the granular volcano, and the experimental evolution of the upper surface agrees well with the expectation of the Mogi model. However, the natural volcanic eruption and the granular eruption, in our experiments, may not share the same physical mechanisms. There could be other theoretical approaches to describe the arching profile of granular eruption by using the continuum theory of surface flows of granular mixtures<xref ref-type="bibr" rid="b8">8</xref><xref ref-type="bibr" rid="b13">13</xref>. Finally, we make a comparison between the SPS and the APS pattern, and qualitatively discuss the air effect on the time-dependent segregation behaviors, as well as on the emergence and the symmetry of the heap.</p></sec><sec disp-level="1"><title>Author Contributions</title><p>P.W. designed and carried out the experiments. P.W., N.Z., L.L. and Q.S. analysed the data and L.L. proposed the model. P.W. and N.Z. contributed to the writing of the manuscript and all authors reviewed the manuscript.</p></sec><sec sec-type="supplementary-material" id="s1"><title>Supplementary Material</title><supplementary-material id="d33e34" content-type="local-data"><caption><title>Supplementary Information</title><p>symmetrically periodic segregation</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="srep06914-s1.avi"/></supplementary-material></sec> |
Re-building Daniell Cell with a Li-ion exchange Film | <p>Daniell cell (i.e. Zn-Cu battery) is widely used in chemistry curricula to illustrate how batteries work, although it has been supplanted in the late 19th century by more modern battery designs because of Cu<sup>2+</sup>-crossover-induced self-discharge and un-rechargeable characteristic. Herein, it is re-built by using a ceramic Li-ion exchange film to separate Cu and Zn electrodes for preventing Cu<sup>2+</sup>-crossover between two electrodes. The re-built Zn-Cu battery can be cycled for 150 times without capacity attenuation and self-discharge, and displays a theoretical energy density of 68.3 Wh kg<sup>−1</sup>. It is more important that both electrodes of the battery are renewable, reusable, low toxicity and environmentally friendly. Owing to these advantages mentioned above, the re-built Daniell cell can be considered as a promising and green stationary power source for large-scale energy storage.</p> | <contrib contrib-type="author"><name><surname>Dong</surname><given-names>Xiaoli</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Wang</surname><given-names>Yonggang</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Xia</surname><given-names>Yongyao</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><aff id="a1"><label>1</label><institution>Department of Chemistry and Shanghai Key Laboratory of Molecular Catalysis and Innovative Materials, Institute of New Energy, Fudan University</institution>, Shanghai 200433, <country>China</country></aff> | Scientific Reports | <p>Daniell cell, invented by the British chemist John Frederic Daniell in 1836, is popularly known as a kind of zinc-copper battery which takes advantage of a porous barrier between the two metals<xref ref-type="bibr" rid="b1">1</xref><xref ref-type="bibr" rid="b2">2</xref>. Once used widely in the European telegraph industry, it was supplanted in the late 19th century by more modern battery designs. Today, it is primarily used in the chemistry curricula to demonstrate how batteries work<xref ref-type="bibr" rid="b1">1</xref><xref ref-type="bibr" rid="b2">2</xref>. As shown in our chemistry curricula, the typical Daniell cell works with a salt bridge connecting the anode electrode of a zinc sulfate solution and an immersed zinc plate as well as the cathode electrode of a copper sulfate solution and an immersed copper plate (<xref ref-type="supplementary-material" rid="s1">Figure S1</xref>). When discharged, the anode of zinc will be oxidized according to the <xref ref-type="disp-formula" rid="m1">equation 1</xref>: <disp-formula id="m1"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e116" xlink:href="srep06916-m1.jpg"/></disp-formula>At the same time, the cathode of copper ions will be reduced following the <xref ref-type="disp-formula" rid="m2">equation 2</xref>: <disp-formula id="m2"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e123" xlink:href="srep06916-m2.jpg"/></disp-formula>Therefore, we can see the total reaction as [Zn(s)] + [Cu<sup>2+</sup>(aq)] → [Zn<sup>2+</sup>(aq)] + [Cu(s)] during the discharge course. These two processes cause copper solid to accumulate at the cathode and the zinc electrode to dissolve into the solution and show a theoretical potential of 1.1 V at 25°C. Without the salt bridge, the reaction will occur directly (i.e. Cu will be deposited on Zn anode) and the electron flow will not be directed through the outer wire to supply power for use. To be exactly, the cations in the salt bridge migrate to the container containing the copper electrode to replace the copper ions being consumed, while the anions in the salt bridge migrate toward the zinc side, where they keep the solution containing the newly formed zinc cations electrically neutral. However, it is a pity to realize that the salt bridge (or porous barrier) can only alleviate the Cu<sup>2+</sup> crossover. At the open circuit condition, Cu<sup>2+</sup> still can slowly diffuse from the Cu electrode room to Zn electrode room through the salt bridge (or porous barrier), and then the Cu<sup>2+</sup> is reduced into metallic Cu on the surface of Zn electrode, indicating a serious self-discharge and/or suicide process (see <xref ref-type="supplementary-material" rid="s1">Figure S2</xref> for detail). It is also undoubted that Daniell Cell is not rechargeable, because recharge would much aggravate the Cu<sup>2+</sup> crossover, indicating a battery-killing process (see <xref ref-type="supplementary-material" rid="s1">Figure S3</xref> for detail).</p><p>Owing to the serious self-discharge and the unrechargeable characteristic, Daniell cell was supplanted by more modern rechargeable battery technologies, such as Lead-acid battery developed in 1859, Ni-Cd (Nickel/Cadmium) battery developed in 1909, Ni-MH (Nickel/Metal hydride) battery developed in 1975 and Li-ion battery developed in 1990<xref ref-type="bibr" rid="b3">3</xref>. Up to present, these aqueous-electrolyte-based rechargeable batteries (i.e. Lead-acid, Ni-Cd and Ni-MH, etc.) and the organic-electrolyte-based Li-ion batteries are still used for diverse range of applications<xref ref-type="bibr" rid="b3">3</xref><xref ref-type="bibr" rid="b4">4</xref>. Due to their higher energy density and cycling stability, the Li-ion batteries using two intercalated compounds (e.g. carbon anode and LiCoO<sub>2</sub> cathode) in an organic solution electrolyte occupy the main market of battery, and are widely used for various electronic devices, from portable devices (such as cellular phones, notebook-size personal computers, and so on) to electric vehicles (EVs)<xref ref-type="bibr" rid="b3">3</xref><xref ref-type="bibr" rid="b4">4</xref>.</p><p>Owing to the accelerated energy consumption and aggravated global warming, it is undoubted that future economy must be based on green and sustainable energy source, such as solar energy, wind energy, etc. As a result, utilization of these sustainable energy sources has been a hot topic. It is well known that the efficient utilization of these sustainable energy sources depends on large-scale stationary energy storage batteries. However, the organic-electrolyte-based Li-ion battery is difficult to play the role of large-scale energy storage device for these sustainable energy sources. Despite the remarkable performance of these organic based systems mentioned above, they suffer from the use of highly toxic and flammable solvents, which can cause safety hazards if used improperly, such as overcharging or short-circuiting<xref ref-type="bibr" rid="b3">3</xref><xref ref-type="bibr" rid="b4">4</xref>. Especially, numerous lithium-ion battery accidents causing fires and explosions have been reported. As a response, these high safety aqueous rechargeable batteries should be the promising candidates for large-scale energy storage, although they display lower energy density. Unfortunately, the application of these commercialized aqueous rechargeable batteries (i.e. Lead-acid, Ni-Cd and Ni-MH, etc.) in large-scale energy storage may be held back by the toxicity of electrode materials (such as Pb and Cd) and/or limited storage of raw materials for electrode (such as Metal hydride). Therefore, aqueous electrolyte Li-ion batteries arrest much attention in recent years, despite their poor cycling stability<xref ref-type="bibr" rid="b5">5</xref><xref ref-type="bibr" rid="b6">6</xref>. However, the raw materials for intercalation compounds (i.e. electrode materials for Li-ion batteries) are not sustainable because cobalt, nickel, manganese and lithium must be natural resources. In addition, most intercalation compounds for Li-ion batteries are prepared by high-temperature solid-state reaction, which also results in consumption of energy and CO<sub>2</sub> emission<xref ref-type="bibr" rid="b3">3</xref>. As we know, the carbon footprint of organic Li-ion batteries even reaches 70 Kg CO<sub>2</sub> per kWh<xref ref-type="bibr" rid="b3">3</xref>. Especially, the reuse of these intercalation compounds in the used batteries is very difficult and complex. As a response, it may be the time for us to reconsider Daniell cell as a candidate for large-scale energy storage, because both Zn and Cu are low toxicity, renewable and reusable. However, the precondition is to well deal with the problem from Cu<sup>2+</sup> crossover on operating process, which obviously is a great challenge.</p><p>Herein a ceramic lithium super-ionic conductor (LATSP, Li<sub>1+x+y</sub>Al<sub>x</sub>Ti<sub>2-x</sub>Si<sub>y</sub>P<sub>3-y</sub>O<sub>12</sub>) thin film, which is recently used in aqueous/non-aqueous double-electrolyte batteries<xref ref-type="bibr" rid="b7">7</xref><xref ref-type="bibr" rid="b8">8</xref><xref ref-type="bibr" rid="b9">9</xref><xref ref-type="bibr" rid="b10">10</xref><xref ref-type="bibr" rid="b11">11</xref><xref ref-type="bibr" rid="b12">12</xref><xref ref-type="bibr" rid="b13">13</xref><xref ref-type="bibr" rid="b14">14</xref><xref ref-type="bibr" rid="b15">15</xref><xref ref-type="bibr" rid="b16">16</xref><xref ref-type="bibr" rid="b17">17</xref><xref ref-type="bibr" rid="b18">18</xref><xref ref-type="bibr" rid="b19">19</xref><xref ref-type="bibr" rid="b20">20</xref><xref ref-type="bibr" rid="b21">21</xref><xref ref-type="bibr" rid="b22">22</xref><xref ref-type="bibr" rid="b23">23</xref>, was used to separate the Cu cathode and Zn anode to build a stable and rechargeable Zn-Cu battery. The resulting Zn-Cu battery can be cycled for 150 times without obvious capacity attenuation, and the open circuit voltage investigation shows that the potential can keep stable for over 100 h without any loss of the capacity.</p><sec disp-level="1" sec-type="results"><title>Results</title><p>The structure and operating mechanism of the rechargeable Zn-Cu battery are illustrated in <xref ref-type="fig" rid="f1">Figure 1</xref>, where it can be detected that a Cu cathode (1 cm<sup>2</sup>) in 0.25 mL 2 M LiNO<sub>3</sub> electrolyte solution and a Zn anode (1 cm<sup>2</sup>) in 0.25 mL 1 M Zn(NO<sub>3</sub>)<sub>2</sub> electrolyte solution are separated by a ceramic LATSP film. Detailed information about the as-prepared rechargeable Zn-Cu battery is given in <xref ref-type="supplementary-material" rid="s1">Figure S4</xref>. On charge, metallic Cu (i.e. cathode) is oxidized into Cu<sup>2+</sup> and the Zn<sup>2+</sup> in anodic room is reduced into metallic Zn at the surface of anode, while Li ions in the cathodic room diffuse to the anodic room through the LATSP ceramic film to balance the charges. Simultaneously, electrons are transferred from Cu cathode to Zn anode through the outer circuit. Discharge reverses the charge process, and is similar with conventional Daniell cell. As a result, the total reaction within the developed rechargeable Zn-Cu battery can be summarized as: <disp-formula id="m3"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e238" xlink:href="srep06916-m3.jpg"/></disp-formula><xref ref-type="fig" rid="f2">Figure 2</xref> gives the cyclic profile of the rechargeable Zn-Cu battery with an applied current of 0.25 mA. In this investigation, a battery is charged for 6 hours to reach a charge capacity of 1.5 mAh, and then the battery is discharged to 0.2 V. As shown in <xref ref-type="fig" rid="f2">Figure 2a</xref>, the battery displays a flat charge voltage of 1.25 V for 6 hours and a flat discharge voltage of about 0.8 V for about 6 hours with the applied current of 0.25 mA. About 1.78 mg metallic Cu is transformed into Cu<sup>2+</sup> in cathodic room through the 6 h charge with a current of 0.25 mA, and simultaneously Zn<sup>2+</sup> in the electrolyte of anodic room is reduced into metallic Zn. At the same time, Li-ions diffuse from cathodic room to anodic room to balance the charge. In order to further clarify the coulombic efficiency, we calculated the capacity (mAh g<sub>Cu</sub><sup>−1</sup>) based on the mass of consumed metallic Cu (or generated Cu<sup>2+</sup>) over charge process. As shown in <xref ref-type="fig" rid="f2">Figure 2b</xref>, the achieved discharge capacity of 843 mAh g<sub>Cu</sub><sup>−1</sup> is equal to the charge capacity at initial cycle. However, the discharge capacity at 20th, 75th and 150th cycle is slightly smaller than corresponding charge capacity, indicating that the Coulombic efficiency of the Zn-Cu battery is only close to 100%. In other words, there is no obvious crossover of Cu<sup>2+</sup> over charge-discharge process. Especially, the battery almost keeps the constant discharge voltage and capacity at various cycles, suggesting perfect cyclic ability (<xref ref-type="fig" rid="f2">Figure 2a and 2b</xref>). The rechargeable characteristic should be attributed to that the ceramic Li-ion exchange film (i.e. LTASP) can efficiently prevent the Cu<sup>2+</sup> crossover, which is confirmed by electrochemical impedance spectroscopic (EIS) investigation (<xref ref-type="supplementary-material" rid="s1">Figure S5</xref>). Furthermore, it also should be noted that there is a clear degradation of the charge voltage curved at the beginning of the charging step. This phenomenon may be ascribed to the formation of Cu<sub>2</sub>O and the growth of dendrite on the surface of cathode, and need further investigation (see <xref ref-type="supplementary-material" rid="s1">supplementary information</xref> for detailed discussion). Herein, we also employed a proton exchange membrane (Nafion 117) to separate the Cu cathode and Zn anode, and investigated its cyclic performance at the same experiment condition for comparison. Unfortunately, being similar to the salt-bridge-based Daniell cell (<xref ref-type="supplementary-material" rid="s1">Figure S3</xref>), the Nafion-film-based Zn-Cu battery can not be recharged (<xref ref-type="supplementary-material" rid="s1">Figure S6</xref>). Although the proton exchange membrane permits the fast pass of Li<sup>+</sup>, it also permits the pass of Cu<sup>2+</sup> (See <xref ref-type="supplementary-material" rid="s1">Figure S7</xref> and corresponding discussion). In order to further clarify the stability of the rechargeable Zn-Cu battery with a LTASP separator, self-discharge investigation of the battery was conducted. In this measurement, the battery was first charged for 10 hours with an applied current density of 0.1 mA. Then, the charged battery was kept at open circuit voltage condition (OCV) for 100 hours (<xref ref-type="fig" rid="f3">Figure 3a</xref>). Finally, the resulting battery was discharged with a current of 0.1 mA (<xref ref-type="fig" rid="f3">Figure 3a</xref>). As shown in <xref ref-type="fig" rid="f3">Figure 3b</xref>, the battery displays a flat discharge voltage for about 10 hours at the current of 0.1 mA (i.e. a discharge capacity of around 843 mAh g<sub>Cu</sub><sup>−1</sup>), indicating that the generated Cu<sup>2+</sup> has been converted into metallic Cu through the electrochemical reduction on discharge process. Herein, we also carried out a control study as a comparison where a Zn-Cu battery was built in the same way as the one described for <xref ref-type="fig" rid="f3">Figure 3a</xref>, and was discharged without OCV storage at the same current of 0.1 mA. In this measurement, the Zn-Cu battery was first charged for 10 hours with an applied current density of 0.1 mA, and then was discharged with a current of 0.1 mA without OCV storage. Discharge curve of the Zn-Cu battery without OCV storage is given in <xref ref-type="fig" rid="f3">Figure 3c</xref>, where it can be observed that the battery displays a flat discharge voltage for about 10 hours, indicating a capacity of around 843 mAh g<sub>Cu</sub><sup>−1</sup>. The comparison between <xref ref-type="fig" rid="f3">Figure 3b and 3c</xref> demonstrates that the conventional Daniell cell can be developed as a rechargeable Zn-Cu battery without self-discharge. In addition, a small voltage plateau arising from the hydrogen evolution can be observed at the end of the discharge, indicating the charge/discharge efficiency does not reach 100%.</p><p><xref ref-type="fig" rid="f4">Figure 4a</xref> gives the rate performance of the rechargeable Zn-Cu battery using a LATSP separator. In this investigation, the battery was charged for 6 h with a current of 1 mA, and then discharged with different currents. The capacities at different currents are also just calculated based on the mass of consumed Cu (or generated Cu<sup>2+</sup>) over charge process. As shown in <xref ref-type="fig" rid="f4">Figure 4a</xref>, the polarization increases obviously with the growth of discharge currents, suggesting a large internal resistance. Obviously, the low conductivity of LATSP will much limit the power output of this kind of LATSP-based Zn-Cu battery. However, this drawback may be solved by adjusting the operating model of this kind of battery. It is well known that proton exchange membrane (i.e. Nafion film) is of high ionic conductivity. For instance, we also investigated the rate performance of Nafion-based Zn-Cu battery at the same condition. In this investigation, a LATSP-based Zn-Cu battery was first charged for 6 hours to form Cu<sup>2+</sup> solution in the cathodic room. Then, the generated Cu<sup>2+</sup> solution is removed to the cathodic room of a Nafion-based Zn-Cu battery (Zn/Nafion/Cu) for discharge measurement. <xref ref-type="fig" rid="f4">Figure 4b</xref> gives the discharge profile of the Nafion-based Zn-Cu battery at different currents. It can be observed from <xref ref-type="fig" rid="f4">Figure 4b</xref> that the Nafion-based Zn-Cu battery displays higher operating voltage even at much higher discharge currents, compared with that of LATSP-based Zn-Cu battery. However, it should be noted that the discharge capacity is lower than the theoretical capacity (i.e. 843 mAh g<sub>Cu</sub><sup>−1</sup>) of metallic Cu (or Cu<sup>2+</sup>), indicating a small amount of Cu<sup>2+</sup> crossover over discharge. Furthermore, it can be observed that an additional voltage plateau (about 0.4 V) appears at the end of discharge of Nafion-based Zn-Cu battery, which should be attributable to the H<sub>2</sub> evolution over discharge process. According to the operating mechanism of the rechargeable Zn-Cu battery (see <xref ref-type="fig" rid="f1">Figure 1</xref>), Li ions should diffuse from anodic room to cathodic room to balance the charge over discharge process. However, the Nafion film also permits the fast pass of H<sup>+</sup>. As a result, the protons also diffuse from anodic room to cathodic room in parallel with the Li ions diffusion on discharge. The proton crossover improves the H<sup>+</sup> concentration in the cathodic room, and thus enhances the H<sub>2</sub> evolution potential over discharge process. The results from <xref ref-type="fig" rid="f4">Figure 4a and 4b</xref> suggest that we can improve the power output of rechargeable Zn-Cu battery through adjusting operating method. As shown in <xref ref-type="fig" rid="f4">Figure 4c</xref>, the LATSP-based Zn-Cu battery can be used for charge storage (i.e. to form Cu<sup>2+</sup> solution and metallic Zn through charge process), and then the charge products (e.g. Cu<sup>2+</sup>) can be flowed to the Nafion-based Zn-Cu battery for high rate discharge when high power output is needed. This technology may be similar with flow batteries. In order to further evaluate the power output of the rechargeable Zn-Cu battery, higher concentration Cu(NO<sub>3</sub>)<sub>2</sub> solution (1 M) was employed as cathodic electrolyte to investigate the power performance of Nafion-based Zn-Cu battery and LATSP-based Zn-Cu battery, respectively. As shown in <xref ref-type="fig" rid="f5">Figure 5</xref>, the discharge voltage of Nafion-based Zn-Cu battery is still higher than 0.5 V even at the high current of 35 mA (i.e. 35 mA cm<sup>−2</sup>). However, the discharge voltage of LATSP-based Zn-Cu battery reduces to 0.5 V at the current of 3 mA (see inset of <xref ref-type="fig" rid="f5">Figure 5</xref>). The result from <xref ref-type="fig" rid="f5">Figure 5</xref> further confirms that Nafion-based Zn-Cu battery can efficiently offset the low power characteristic of LATSP-based Zn-Cu battery.</p></sec><sec disp-level="1" sec-type="discussion"><title>Discussion</title><p>Above results have demonstrated that the new type Zn-Cu battery is of perfect cyclic ability and high stability, and its power output can also be improved through proper operating method. As a result, the next logic step is to evaluate its theoretical energy density. It should be noted that the above capacity (i.e. mAh g<sub>Cu</sub><sup>−1</sup>) calculated from the consumed Cu or Cu<sup>2+</sup> is just used to describe the reversibility of battery, and thus can not be employed to evaluate the true energy density of the battery. According to <xref ref-type="disp-formula" rid="m3">Equation 3</xref>, the active materials of the new type Zn-Cu battery include Cu, LiNO<sub>3</sub> and Zn(NO<sub>3</sub>)<sub>2</sub>. Furthermore, the solubility in water of LiNO<sub>3</sub> and Zn(NO<sub>3</sub>)<sub>2</sub> should also be considered in the calculation of energy density. Accordingly, the theoretical energy density of the rechargeable Zn-Cu battery based on the total weight of the Cu cathode, the LiNO<sub>3</sub> electrolyte and the aqueous Zn(NO<sub>3</sub>)<sub>2</sub> anode can be calculated from <xref ref-type="disp-formula" rid="m4">equation (4)</xref>
<disp-formula id="m4"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e468" xlink:href="srep06916-m4.jpg"/></disp-formula>where <italic>E</italic> is the energy density (Wh kg<sup>−1</sup>), <italic>V</italic> is the discharge voltage (~0.8 V), <italic>Q</italic> is the theoretical capacity of Cu (843 mAh g<sub>Cu</sub><sup>−1</sup>), <italic>m<sub>cu</sub></italic> is the mass of <italic>Cu</italic> cathode (63.55 g; 1 mol), <italic>m<sub>LiNO3</sub></italic> is the mass of LiNO<sub>3</sub> in electrolyte (2 × 68.95 g; 2 mol), and <italic>m<sub>Zn(NO3)2</sub></italic> is the mass of Zn(NO<sub>3</sub>)<sub>2</sub> in aqueous anode (189.39 g; 1 mol). The <italic>m</italic><sup><italic>1</italic></sup><italic><sub>H2O</sub></italic> and <italic>m<sup>2</sup><sub>H2O</sub></italic> are the masses of water for LiNO<sub>3</sub> dissolution (100 g H<sub>2</sub>O) and Zn(NO<sub>3</sub>)<sub>2</sub> dissolution (137 g H<sub>2</sub>O), which are calculated based on their solubility at 30°C. According to <xref ref-type="disp-formula" rid="m4">equation (4)</xref>, the calculated energy density can reach 68.3 Wh kg<sup>−1</sup>. It should be noted that 2 M LiNO<sub>3</sub> solution and 1 M Zn(NO<sub>3</sub>)<sub>2</sub> solution were employed in our experiment to demonstrate the performance of rechargeable Zn-Cu battery. However, the calculation of theoretical energy of the rechargeable Zn-Cu battery is based on the solubility of LiNO<sub>3</sub> and Zn(NO<sub>3</sub>)<sub>2</sub> at 30°C. Typically, the electrode/electrolyte material weighs about 50% of the total weight of the large-size practical battery. Thus, the practical specific energy of the new type Zn-Cu battery near 34 Wh kg<sup>−1</sup> can be expected, which is compatible with commercialized lead acid battery (30 ~ 40 Wh kg<sup>−1</sup>)<xref ref-type="bibr" rid="b3">3</xref><xref ref-type="bibr" rid="b24">24</xref> and flow battery (25–30 Wh kg<sup>−1</sup>)<xref ref-type="bibr" rid="b3">3</xref><xref ref-type="bibr" rid="b24">24</xref>. It is more important that the electrode reactions of the battery depend on the dissolution–deposition of metallic Cu and Zn, indicating that Cu-cathode and Zn-anode are renewable and reusable. For example, the Cu-cathode or Zn-anode in a used rechargeable Zn–Cu battery can be directly recycled to fabricate a new electrode. However, for present commercialized aqueous battery (e.g. lead acid, Ni-Cd and Ni-MH batteries), the recycle of electrode materials is of a great challenge. Especially, both electrode material and electrolyte of rechargeable Zn-Cu battery are low toxicity, which is quite important for low carbon society. Therefore, the rechargeable Zn-Cu battery can be considered as a promising power source for large-scale energy storage. On the other hand, the charge rate (e.g. energy storage rate) of rechargeable Zn-Cu battery is still much limited by the low conductivity of the ceramic LATSP film, and can not be offset by the above Nafion-based Zn-Cu battery. As a response, this kind of Zn-Cu battery may be only suitable for solar energy storage at present stage. In the further investigation, it is necessary to improve the conductivity of ion-exchange membrane and enhance the operating temperature of the battery. Especially, enhancing the operating temperature can not only improve the conductivity of the ceramic ion-exchange membrane but also increase the solubility of active materials, which thus can improve both energy and power density of the system. It should also be noted that dendrite growth inevitably occurs on the Cu and Zn electrode over cycling process. Although the short circuit hazards in the rechargeable Zn-Cu battery can be eliminated because the rigid ceramic LATSP film is hardly pierced by the dendrites on electrode, the dendrite growth still could limit the efficient utilization of Cu/Zn electrode over long-time cycling. Therefore, more researches should be done to solve the problem, for example, some kind of electrolyte additive might be useful to produce smooth coating during the electrodeposition.</p><p>In summary, the old Daniell cell was re-built as a stable and rechargeable battery through the Li-ion exchange film that can efficiently prevent the crossover of Cu<sup>2+</sup>. The theoretical energy density of the new type Zn-Cu battery can reach 68.3 Wh kg<sup>−1</sup> which is compatible with current aqueous rechargeable batteries. It is more important that both electrodes of the battery are renewable, reusable, low toxicity and environmentally friendly. It can be expected that this investigation not only gives a new birth for the very old Daniell cell, but also provides a new promising and green power source for large-scale energy storage.</p></sec><sec disp-level="1" sec-type="methods"><title>Method</title><sec disp-level="2"><title>Materials and Preparation of Zn-Cu battery</title><p>The reagents [e.g. Zn plate, Cu plate, Zn(NO<sub>3</sub>)<sub>2</sub> and LiNO<sub>3</sub>] were purchased from Sinopharm Chemical Reagent Co. Ltd (Shanghai). Zinc plate (0.2 mm in thickness, 99.9%) and copper plate (0.1 mm in thickness, 99.9%) were polished before battery assembly. The 2.5 × 2.5 cm<sup>2</sup> ceramic lithium super-ionic conductor film (LATSP, Li<sub>1+x+y</sub>Al<sub>x</sub>Ti<sub>2-x</sub>Si<sub>y</sub>P<sub>3-y</sub>O<sub>12</sub> purchased from Ohara Inc., Japan) with a thickness of 0.15 mm and a Li-ion conductivity of 10<sup>−4</sup> S cm<sup>−1</sup> was used as received. The glass microfiber filter was obtained from Whatman® Anodisc Inorganic Membranes. 2 M LiNO<sub>3</sub> solution and 1 M Zn(NO<sub>3</sub>)<sub>2</sub> solution were used as cathodic electrolyte and anodic electrolyte, respectively. Detailed information about the battery assembly was given in <xref ref-type="supplementary-material" rid="s1">Figure S4</xref>.</p></sec><sec disp-level="2"><title>Electrochemical Characterization</title><p>Electrochemical tests were performed on HOKUTO DENKO battery charge/discharge system HJ series (Japan) controlled by a computer. The electrochemical impedance spectroscopy (EIS) was carried out on Solartron Instrument Model 1287 in the frequency range of 10<sup>6</sup>-0.01 Hz with the AC signal amplitude of 10 mV.</p></sec></sec><sec disp-level="1"><title>Author Contributions</title><p>X.L.D. and Y.G.W. designed the experiments and discussed the interpretation of results. X.L.D., Y.G.W. and Y.Y.X. discussed the results, co-wrote the paper and participated in the manuscript revision.</p></sec><sec sec-type="supplementary-material" id="s1"><title>Supplementary Material</title><supplementary-material id="d33e28" content-type="local-data"><caption><title>Supplementary Information</title><p>Supplementary Information</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="srep06916-s1.pdf"/></supplementary-material></sec> |
Preen secretions encode information on MHC similarity in certain sex-dyads in a monogamous seabird | <p>Animals are known to select mates to maximize the genetic diversity of their offspring in order to achieve immunity against a broader range of pathogens. Although several bird species preferentially mate with partners that are dissimilar at the major histocompatibility complex (MHC), it remains unknown whether they can use olfactory cues to assess MHC similarity with potential partners. Here we combined gas chromatography data with genetic similarity indices based on MHC to test whether similarity in preen secretion chemicals correlated with MHC relatedness in the black-legged kittiwake (<italic>Rissa tridactyla</italic>), a species that preferentially mates with genetically dissimilar partners. We found that similarity in preen secretion chemicals was positively correlated with MHC relatedness in male-male and male-female dyads. This study provides the first evidence that preen secretion chemicals can encode information on MHC relatedness and suggests that odor-based mechanisms of MHC-related mate choice may occur in birds.</p> | <contrib contrib-type="author"><name><surname>Leclaire</surname><given-names>Sarah</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a2">2</xref><xref ref-type="author-notes" rid="n1">*</xref></contrib><contrib contrib-type="author"><name><surname>van Dongen</surname><given-names>Wouter F. D.</given-names></name><xref ref-type="aff" rid="a3">3</xref><xref ref-type="aff" rid="a4">4</xref><xref ref-type="author-notes" rid="n1">*</xref></contrib><contrib contrib-type="author"><name><surname>Voccia</surname><given-names>Steeve</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Merkling</surname><given-names>Thomas</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Ducamp</surname><given-names>Christine</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Hatch</surname><given-names>Scott A.</given-names></name><xref ref-type="aff" rid="a5">5</xref></contrib><contrib contrib-type="author"><name><surname>Blanchard</surname><given-names>Pierrick</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Danchin</surname><given-names>Étienne</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Wagner</surname><given-names>Richard H.</given-names></name><xref ref-type="aff" rid="a3">3</xref></contrib><aff id="a1"><label>1</label><institution>CNRS, UPS, ENFA; Laboratoire Évolution et Diversité Biologique (EDB), UMR5174</institution>, 118 route de Narbonne, 31062 Toulouse, <country>France</country></aff><aff id="a2"><label>2</label><institution>Université de Toulouse, EDB, UMR5174</institution>, 31062 Toulouse, <country>France</country></aff><aff id="a3"><label>3</label><institution>Konrad Lorenz Institute of Ethology, Department of Integrative Biology and Evolution, University of Veterinary Medicine Vienna</institution>, Savoyenstrasse 1a, 1160 Vienna, Austria</aff><aff id="a4"><label>4</label><institution>Applied Ecology Research Group and Institute for Sustainability and Innovation, College of Engineering and Science, Victoria University – Footscray Park Campus</institution>, PO Box 14428, Melbourne MC, VIC, <country>Australia 8001</country></aff><aff id="a5"><label>5</label><institution>U.S. Geological Survey, Alaska Science Center, 4210 University Drive</institution>, Anchorage, Alaska 99508, <country>USA</country></aff> | Scientific Reports | <p>The major histocompatibility complex (MHC) is an extraordinarily diverse cluster of genes that play a major role in vertebrate adaptive immunity. MHC heterozygosity offers several fitness advantages, including increased disease resistance and survival<xref ref-type="bibr" rid="b1">1</xref><xref ref-type="bibr" rid="b2">2</xref>. Given that MHC-dissimilar parents are more likely to produce MHC heterozygous offspring, mate choice of MHC-disparate partners is a common strategy in taxa as diverse as mammals, fish, lizards and birds<xref ref-type="bibr" rid="b3">3</xref><xref ref-type="bibr" rid="b4">4</xref><xref ref-type="bibr" rid="b5">5</xref><xref ref-type="bibr" rid="b6">6</xref><xref ref-type="bibr" rid="b7">7</xref>.</p><p>In numerous species, MHC similarity is assessed through olfactory cues<xref ref-type="bibr" rid="b8">8</xref>. For example, rodents, humans and lizards prefer the odour of MHC-dissimilar individuals<xref ref-type="bibr" rid="b4">4</xref><xref ref-type="bibr" rid="b9">9</xref><xref ref-type="bibr" rid="b10">10</xref>. However, while several birds preferentially reproduce with MHC-dissimilar partners<xref ref-type="bibr" rid="b7">7</xref><xref ref-type="bibr" rid="b11">11</xref><xref ref-type="bibr" rid="b12">12</xref>, it remains unknown whether birds can assess MHC relatedness via odour cues. The few studies that have shown that birds can discriminate between their relatives and non-relatives based on odour cues have relied on pedigree relatedness<xref ref-type="bibr" rid="b13">13</xref><xref ref-type="bibr" rid="b14">14</xref><xref ref-type="bibr" rid="b15">15</xref>. Although pedigree is a reliable estimator of genetic relatedness and is useful when investigating inbreeding avoidance<xref ref-type="bibr" rid="b16">16</xref>, MHC relatedness may be a more direct link between odor and genotype that has not yet been explored in birds.</p><p>In a previous study, we had shown that preen secretion chemicals can reflect genetic relatedness at microsatellite loci in the black-legged kittiwake (<italic>Rissa tridactyla</italic>)<xref ref-type="bibr" rid="b17">17</xref>, a species that preferentially mates with genetically dissimilar individuals<xref ref-type="bibr" rid="b18">18</xref>. In contrast to MHC, microsatellites, being neutral markers, are not under selection for high levels of polymorphism. Microsatellite diversity is therefore usually lower than MHC diversity across populations and correlation between microsatellites and MHC may be weak<xref ref-type="bibr" rid="b19">19</xref><xref ref-type="bibr" rid="b20">20</xref><xref ref-type="bibr" rid="b21">21</xref>. Showing that odour is linked to MHC is therefore an important step in the study of odour-based mate choice and immunology in birds. Here, we tested whether the chemical composition of kittiwake scent secretion was related to variation at the MHC, by combining gas chromatography data with indices of relatedness based on MHC.</p><sec disp-level="1" sec-type="results"><title>Results</title><sec disp-level="2"><title>MHC characteristics</title><p>We isolated a maximum of four MHC alleles per individual (range: 2–4 alleles; mean = 3.2 ± 0.7 SD alleles), indicating that we amplified duplicated MHC Class II DRB loci. These two loci were highly polymorphic with 23 alleles being isolated from the 39 individuals (<xref ref-type="table" rid="t1">Table 1</xref>). The amino acid sequences contained characteristic features of functional class II molecules including conserved residues and putative peptide binding regions (<xref ref-type="fig" rid="f1">Fig. 1</xref>). We found no stop codons or frame shift mutations in any allele. The putative peptide binding regions contained 47 segregating sites and a nucleotide diversity (π) of 0.18. The putative non-peptide binding regions contained 24 segregating sites and a nucleotide diversity of 0.05. Codons that were located within putative peptide binding regions had an excess of non-synonymous substitutions (dN = 0.277 ± 0.054, dS = 0.074 ± 0.059; Z = 2.960, p = 0.002), indicating positive selection. This was not the case for codons outside the putative peptide binding regions (dN = 0.058 ± 0.018, dS = 0.041 ± 0.017; Z = 0.665, p = 0.254). Up to three cDNA sequences were isolated per individual confirming that both loci were transcribed in blood cells.</p></sec><sec disp-level="2"><title>MHC and preen secretions</title><p>When chemical distances were mainly influenced by the most abundant compounds, they increased significantly with MHC amino acid distances in male-male dyads (Mantel test: r = 0.22, P = 0.009, n = 210 dyads; <xref ref-type="fig" rid="f2">Fig. 2a</xref>) and male-female dyads (Spearman's correlation permutation test: r = 0.13, P = 0.007, n = 378 dyads; <xref ref-type="fig" rid="f2">Fig. 2b</xref>), while being unrelated to MHC distances in female-female dyads (Mantel test: r = 0.05, P = 0.31, n = 153 dyads; <xref ref-type="fig" rid="f2">Fig. 2c</xref>) and showing a trend when all kittiwake dyads were pooled (Mantel test: r = 0.13, P = 0.051, n = 741 dyads). When chemical distances were calculated when considering all chemical compounds equally, they were not related to MHC distances in any of the dyad groups (all P > 0.38). These results suggest that the relationship between preen secretion and MHC similarities is mainly linked to the most abundant chemical compounds. This is supported by the finding that when analyses were conducted with only the 22 most abundant chemical compounds (i.e., compounds with an average abundance > 1%), the correlations between MHC distances and chemical distances were significant in male-male, male-female and all dyads (r = 0.20, P = 0.015, r = 0.12, P = 0.024, and r = 0.20, P = 0.012 respectively), but not in female-female dyads: r = 0.08, P = 0.25. In contrast, none of the correlations were significant when excluding these 22 compounds (all P > 0.35).</p></sec></sec><sec disp-level="1" sec-type="discussion"><title>Discussion</title><p>Our study provides the first evidence that preen secretion chemicals can encode information on MHC relatedness in birds. The findings suggest that odour cues present in preen secretion may be recognized by birds and allow them to pair MHC-disassortatively. The relationship between preen secretion and MHC dissimilarities in cross-sex dyads and male-male dyads suggests that kittiwakes may recognize related individuals by self-referent or known-kin matching, with individuals avoiding breeding with partners that have scent signatures similar to their own or their known-kin.</p><p>A positive correlation between preen secretion and MHC dissimilarities was detected in male-male dyads, but not in female-female dyads. This finding confirms our previous results using a different dataset, which showed that preen secretion and microsatellite similarity correlated in male-male dyads only<xref ref-type="bibr" rid="b17">17</xref>. Similar sex-differences in the correlation between chemical and genetic similarities were detected in giant pandas (<italic>Ailuropoda melanoleuca</italic>)<xref ref-type="bibr" rid="b22">22</xref>, but not in ring-tailed lemurs (<italic>Lemur catta</italic>)<xref ref-type="bibr" rid="b23">23</xref><xref ref-type="bibr" rid="b24">24</xref> and mandrills (<italic>Mandrillus sphinx</italic>)<xref ref-type="bibr" rid="b25">25</xref>. In kittiwakes, philopatry and intense competition for securing a nesting site is common in males<xref ref-type="bibr" rid="b26">26</xref>. Kin recognition may therefore be favoured in males because it may reduce competition between related males thereby increasing inclusive fitness. In contrast, females being the dispersing sex are much less likely to be surrounded by kin and social interactions amongst females are rare. Kin selection amongst females may thus be under lower selection than amongst males.</p><p>How odours are influenced by MHC genes remains largely unknown<xref ref-type="bibr" rid="b8">8</xref>. Recent studies suggest that in humans and zebrafish (<italic>Danio rerio</italic>), MHC peptides may function as chemical signals for kin recognition<xref ref-type="bibr" rid="b27">27</xref><xref ref-type="bibr" rid="b28">28</xref>. Although in several species, non-peptide compounds, including carboxylic acids, were found to correlate with MHC profiles<xref ref-type="bibr" rid="b29">29</xref><xref ref-type="bibr" rid="b30">30</xref>, it is not known whether a non-peptide mechanism can drive MHC-mediated behavior<xref ref-type="bibr" rid="b31">31</xref>. At the molecular level, the link between MHC and non-peptide odours may stem from excreted odorants becoming conjugated with amino acids and therefore being bound by MHC proteins<xref ref-type="bibr" rid="b29">29</xref>, or from odorants being secondary metabolites of biochemical pathways moderated by MHC or MHC-linked genes. Our study is correlative. Therefore, our results do not allow us to determine the degree to which preen secretion chemicals are influenced by MHC compared to other polymorphic genes whose variations usually covary with those in the MHC, such as the major urinary protein (MUP) genes in natural populations of mice<xref ref-type="bibr" rid="b32">32</xref>. Experiments with MHC-congenic birds, for instance, are needed to disentangle these two hypotheses.</p><p>In our study, we analyzed the composition in preen secretion wax esters. Wax esters are long-chained compounds with low volatility at normal temperatures and pressure. Birds lack a vomeronasal organ<xref ref-type="bibr" rid="b33">33</xref>, and whether they can detect nonvolatile olfactory cues by the main olfactory system, as shown in mice<xref ref-type="bibr" rid="b34">34</xref>, is unknown. Non-exclusively, wax esters may be metabolized by odor-producing bacteria present on feathers or in the preen gland, and lead to the release of volatiles encoding MHC information<xref ref-type="bibr" rid="b35">35</xref>.</p><p>Although preen secretions are spread onto the plumage during preening, they may represent only one component of the body odour emitted by birds. However, we do know that in kittiwakes, chemical profiles of preen oil are highly similar to the chemical profiles of down feathers surrounding the preen gland<xref ref-type="bibr" rid="b36">36</xref> and to the chemical profiles of neck feathers (our unpublished data). Another sampling protocol (e.g., as described in<xref ref-type="bibr" rid="b37">37</xref>) would be necessary to characterise the entire body odour of kittiwakes and to determine to what extent preen secretions contribute to the odour emitted by the birds. In addition, variance in chemical distances was high for any particular MHC distance. Some compounds are likely to be influenced by non-MHC factors, which may have led to the small effect sizes detected in this study. Although our approach has led to new insights into chemical signalling in birds, an important next step is to determine which specific chemical compounds are central in odour-based kin recognition (as suggested in<xref ref-type="bibr" rid="b38">38</xref>).</p><p>In conclusion, our results provide the first evidence that preen secretion can encode information on MHC relatedness in birds. How MHC-related odours influence kittiwake behaviour needs now to be studied. Our findings open the door for further studies that may comprehensively link mate choice and immunity in birds.</p></sec><sec disp-level="1" sec-type="methods"><title>Methods</title><sec disp-level="2"><title>Study site</title><p>Samples were collected in the pre-laying period, between 15 April and 20 May 2011 (first laying in the population: 27 May 2011), in a population of black-legged kittiwakes nesting on an abandoned US Air Force radar tower on Middleton Island (59°26′N,146°20′W), Gulf of Alaska. Preen secretion and blood samples were collected from 18 females and 21 males. Experiments were approved by the US Fish and Wildlife Service and State of Alaska.</p></sec><sec disp-level="2"><title>Preen secretion collection and analyses</title><p>Preen secretion collection, extraction and GC analyses were adapted from our protocol described earlier<xref ref-type="bibr" rid="b36">36</xref>. Preen secretion samples were stored at −20°C until chemical analyses in March 2012. Samples were immersed in 0.5 ml dichloromethane/ nonadecane (internal standard, 20 μg/ml), agitated for 2 h at ambient temperature and then kept frozen until analysis. They were analyzed on a DANI GC-1000 gas chromatograph (DANI Instruments SpA), equipped with a flame-ionization detector and a Restek Rtx®-5MS (30 m × 0.25 mm, 0.25 μm film thickness) capillary column. Helium was used as a carrier gas. The flame-ionization detector was operated at 300°C and the injector was used at 280°C. Samples were injected in splitless mode. The oven was programmed as follows: 7°C/min from 50°C to 200°C and then 3°C/min to 290°C and a 10 min hold-on at 290°C. Blanks were regularly interspersed throughout the sample analyses. We retained peaks that comprised at least 0.1 per cent of the total area of the chromatogram (n = 120 peaks) and analyzed all samples in a short period of time to minimize inter-assay variability.</p><p>In kittiwakes, all individuals of both sexes have the same compounds making gas chromatograph – mass spectrometer (GCMS) analysis not necessary for profile alignment. Two samples were, however, run on a GCMS to further identify the chemical compounds. The analyses were performed on a Finnigan Trace 2000 chromatograph (Thermo Scientific) directly coupled to a mass spectrometer quadrupole detector (electron impact at 70 eV). The temperature source was set at 200°C, the interface between GC and MS modules at 250°C and the splitless injector at 280°C. Helium was the carrier gas and the flow rate was 1.2 ml/min. 1 μl of sample was injected in an apolar capillary column (Restek Rtx®-5MS; 30 m × 0.25 mm, 0.25 μm film thickness, 5% diphenyl and 95% dimethylpolysiloxane). The oven temperature program was as set for the GC analyses. The mass spectra were scanned from 60 to 500 m/z. All identified peaks were wax esters (i.e., esters of long aliphatic carboxylic acids and fatty alcohols) as found in our earlier study<xref ref-type="bibr" rid="b16">16</xref>.</p><p>Because we could not control for the amount of secretion collected, each chromatogram peak was quantified as the relative proportion of the peak size to the overall area of the chromatogram. Chromatograms were analyzed with the Peak Simple integration software (Version 3.77, Buck Scientific Inc.). To measure similarity in preen secretion composition between each dyad of individuals, we calculated the Euclidean distance after chord-transformation (Chord distance)<xref ref-type="bibr" rid="b39">39</xref>. This distance is mainly influenced by compounds with large absolute differences between individuals<xref ref-type="bibr" rid="b40">40</xref> which are, as a rule, compounds with high abundance<xref ref-type="bibr" rid="b41">41</xref> (Pearson correlation between mean and standard deviation of each compound: r = 0.99, P < 0.0001, showing that larger differences are also found in more abundant compounds in our dataset). Therefore, we also calculated pairwise Euclidean distances using prior normalization of the relative abundances, so that all compounds were considered equally. Prior normalization was realized using the “range” method in decostand() function (VEGAN package in the R software)<xref ref-type="bibr" rid="b42">42</xref>.</p></sec><sec disp-level="2"><title>Genetic analyses</title><p>Upon capture, blood was taken from the alar vein. DNA extraction was performed as described in<xref ref-type="bibr" rid="b17">17</xref>. We amplified a 258 bp fragment of exon 2 of the MHC Class II DRB locus using the primers KWMHC2_ex2_F (5′-GCACGAGCAGGGTATTTCCA-3′) and KWMHC2_ex2_R (5′- GTTCTGCCACACACTCACCT-3′), which we designed based on previously-published black-legged kittiwake sequences containing exon 2 and its flanking regions (K.D. McCoy, unpublished data: GenBank Accession numbers: HQ822398- HQ822472). PCR was performed in 25 μl reaction volumes containing the forward and reverse primers (0.2 mM each), 1.25 units of GoTaq Flexi DNA polymerase (Promega), 1× reaction buffer (Promega), 1.5 mM MgCl<sub>2</sub> (Promega), 0.2 mM dNTPs and approximately 50 ng of genomic DNA. The PCR conditions consisted of an initial denaturation step (94°C, 3 min), followed by 34 cycles of 30 s at 94°C, 30 s at 57°C, 45 s at 72°C, and a final extension step for 10 min at 72°C. The success of our amplifications was confirmed on a 1.5% agarose gel stained with ethidium bromide. Primers and excess dNTPs were removed from the amplified products by digesting exonuclease-shrimp alkaline phosphatase (Fermentas Life Sciences).</p><p>As we were expecting multiple MHC alleles per individual, we cloned each PCR product. Cloning was conducted using a TOPO TA Cloning kit (Invitrogen) as per the manufacturer's protocol. We then picked between 24 and 48 transformed colonies for each individual (depending on the success of the cloning) and amplified and sequenced the inserts as described in<xref ref-type="bibr" rid="b43">43</xref>. Sequence editing and alignment was conducted using CLC DNA Workbench 5.7 (CLC bio).</p><p>MHC alleles were classified as unique when they differed by 3 or more bp from all other known alleles and were found more than once within or between samples<xref ref-type="bibr" rid="b44">44</xref>. All potential alleles found only once in one individual were verified by reamplifying the locus for that individual in a new PCR and cloning the product. When we could not verify a particular allele, it was assumed to be a PCR artefact. However, MHC screening was very repeatable across multiple PCRs. We conducted multiple PCRs for 34 individuals and in all cases we identified the same alleles from both PCRs (van Dongen, unpublished data). Ideally, studies should survey a larger region of the MHC than MHC-DRB, but this requires a level of knowledge of MHC structure that is lacking for non-model organisms. However, the MHC region is characterized by strong linkage disequilibrium<xref ref-type="bibr" rid="b45">45</xref>, meaning that relatively small segments of the MHC provide valuable information about the larger complex.</p><p>To confirm that we were genotyping a putatively functional MHC locus and not a pseudogene, we took a number of steps. First, we searched for 1) the presence of frame shift mutations or stop codons in the translated alleles and 2) characteristic features of functional Class II molecules, such as conserved residues and putative peptide binding regions (inferred from<xref ref-type="bibr" rid="b46">46</xref>). We tested for selection by comparing the ratio of non-synonymous (d<sub>N</sub>) over synonymous (d<sub>S</sub>) substitutions using MEGA 5<xref ref-type="bibr" rid="b47">47</xref> both within and outside of the peptide binding regions. We used the Z-test for selection implementing the modified Nei-Gojobori method with Jukes-Cantor correction and calculated standard errors with 1000 bootstraps. Last, to confirm that both loci are transcribed in blood cells, we amplified MHC alleles from the cDNA of two individuals. A RiboPure - Blood kit (Ambion) was used to extract RNA from 250-500 μl of blood that had been stored in RNAlater. The RNA was subsequently treated with DNase that was provided with the RNA extraction kit and reverse transcribed using a QuantiTect Reverse Transcription Kit (Qiagen). The cDNA was subsequently used as a PCR template and cloned, as described above, using the primers KWMHC2_F (5′ CCA ATG GTA CYG AGC GGG TGA 3′) and KWMHC2_R (5′ ACG GCG CAA ACC AGC CTG T 3′), which are located within exon 2 and exon 3, respectively.</p><p>Genetic MHC similarity was calculated based on the amino acid sequences of each allele. To estimate pairwise distances in MHC genotypes between all individuals, we used UniFrac<xref ref-type="bibr" rid="b48">48</xref>, a phylogenetic comparison tool originally developed for measuring phylogenetic distances between microbial communities. Using this approach, two individuals were classified as similar at the MHC if they shared the same alleles or shared alleles that were phylogenetically clustered. A maximum-likelihood phylogenetic tree of all alleles for the UniFrac analysis was inferred using MEGA 5. The maximum likelihood tree was based on a WAG model<xref ref-type="bibr" rid="b49">49</xref>, which was selected as the most appropriate evolutionary model to explain the variability among protein sequences. Evolutionary rate differences among sites were modelled using a discrete Gamma distribution (number of categories = 5).</p></sec><sec disp-level="2"><title>Statistical analyses</title><p>Correlations between preen secretion similarity and MHC relatedness were tested using Mantel tests (VEGAN package<xref ref-type="bibr" rid="b42">42</xref>) and 5000 data randomizations. The correlation between preen secretion similarity and MHC relatedness may stem from one sex only. As Mantel tests cannot handle interactions, we performed Mantel tests in all dyads, in male-male dyads and in female-female dyads separately. The male – female matrix was not square and thus we could not use the Mantel test. Instead, we used a Spearman's correlation permutation test to test for the correlation between chemical distances and MHC distances. All statistical tests were performed with the R statistical software (R Development Core Team 2014).</p></sec></sec><sec disp-level="1"><title>Author Contributions</title><p>S.L., W.F.D.vD. and R.H.W. conceived and designed the study. T.M. collected the samples. S.L., S.V. and C.D. performed the chemical analyses. W.vD. performed the genetic analyses. S.L., W.F.D.vD., S.A.H., P.B., E.D. and R.H.W. wrote the manuscript.</p></sec> |
GWATCH: a web platform for automated gene association discovery analysis | <sec><title>Background</title><p>As genome-wide sequence analyses for complex human disease determinants are expanding, it is increasingly necessary to develop strategies to promote discovery and validation of potential disease-gene associations.</p></sec><sec><title>Findings</title><p>Here we present a dynamic web-based platform – GWATCH – that automates and facilitates four steps in genetic epidemiological discovery: 1) Rapid gene association search and discovery analysis of large genome-wide datasets; 2) Expanded visual display of gene associations for genome-wide variants (SNPs, indels, CNVs), including Manhattan plots, 2D and 3D snapshots of any gene region, and a dynamic genome browser illustrating gene association chromosomal regions; 3) Real-time validation/replication of candidate or putative genes suggested from other sources, limiting Bonferroni genome-wide association study (GWAS) penalties; 4) Open data release and sharing by eliminating privacy constraints (The National Human Genome Research Institute (NHGRI) Institutional Review Board (IRB), informed consent, The Health Insurance Portability and Accountability Act (HIPAA) of 1996 etc.) on unabridged results, which allows for open access comparative and meta-analysis.</p></sec><sec><title>Conclusions</title><p>GWATCH is suitable for both GWAS and whole genome sequence association datasets. We illustrate the utility of GWATCH with three large genome-wide association studies for HIV-AIDS resistance genes screened in large multicenter cohorts; however, association datasets from any study can be uploaded and analyzed by GWATCH.</p></sec> | <contrib contrib-type="author" corresp="yes" equal-contrib="yes" id="A1"><name><surname>Svitin</surname><given-names>Anton</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>anton.svitin@gmail.com</email></contrib><contrib contrib-type="author" equal-contrib="yes" id="A2"><name><surname>Malov</surname><given-names>Sergey</given-names></name><xref ref-type="aff" rid="I1">1</xref><xref ref-type="aff" rid="I2">2</xref><email>malovs@sm14820.spb.edu</email></contrib><contrib contrib-type="author" equal-contrib="yes" id="A3"><name><surname>Cherkasov</surname><given-names>Nikolay</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>x@biomed.spb.ru</email></contrib><contrib contrib-type="author" id="A4"><name><surname>Geerts</surname><given-names>Paul</given-names></name><xref ref-type="aff" rid="I3">3</xref><email>paul.g.geerts@gmail.com</email></contrib><contrib contrib-type="author" id="A5"><name><surname>Rotkevich</surname><given-names>Mikhail</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>rotke@yandex.ru</email></contrib><contrib contrib-type="author" id="A6"><name><surname>Dobrynin</surname><given-names>Pavel</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>pdobrynin@gmail.com</email></contrib><contrib contrib-type="author" id="A7"><name><surname>Shevchenko</surname><given-names>Andrey</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>andrey.k.shevchenko@gmail.com</email></contrib><contrib contrib-type="author" id="A8"><name><surname>Guan</surname><given-names>Li</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>guanl2000@gmail.com</email></contrib><contrib contrib-type="author" id="A9"><name><surname>Troyer</surname><given-names>Jennifer</given-names></name><xref ref-type="aff" rid="I4">4</xref><email>troyerj@mail.nih.gov</email></contrib><contrib contrib-type="author" id="A10"><name><surname>Hendrickson</surname><given-names>Sher</given-names></name><xref ref-type="aff" rid="I5">5</xref><email>shendric@shepherd.edu</email></contrib><contrib contrib-type="author" id="A11"><name><surname>Dilks</surname><given-names>Holli Hutcheson</given-names></name><xref ref-type="aff" rid="I6">6</xref><email>holli.h.dilks@vanderbilt.edu</email></contrib><contrib contrib-type="author" id="A12"><name><surname>Oleksyk</surname><given-names>Taras K</given-names></name><xref ref-type="aff" rid="I7">7</xref><email>dna.lab@upr.edu</email></contrib><contrib contrib-type="author" id="A13"><name><surname>Donfield</surname><given-names>Sharyne</given-names></name><xref ref-type="aff" rid="I8">8</xref><email>sharyne_donfield@rhoworld.com</email></contrib><contrib contrib-type="author" id="A14"><name><surname>Gomperts</surname><given-names>Edward</given-names></name><xref ref-type="aff" rid="I9">9</xref><email>EGomperts@chla.usc.edu</email></contrib><contrib contrib-type="author" id="A15"><name><surname>Jabs</surname><given-names>Douglas A</given-names></name><xref ref-type="aff" rid="I10">10</xref><email>douglas.jabs@mssm.edu</email></contrib><contrib contrib-type="author" id="A16"><name><surname>Sezgin</surname><given-names>Efe</given-names></name><xref ref-type="aff" rid="I11">11</xref><email>efeszgn0@gmail.com</email></contrib><contrib contrib-type="author" id="A17"><name><surname>Van Natta</surname><given-names>Mark</given-names></name><xref ref-type="aff" rid="I11">11</xref><email>mvnatta@jhsph.edu</email></contrib><contrib contrib-type="author" id="A18"><name><surname>Harrigan</surname><given-names>P Richard</given-names></name><xref ref-type="aff" rid="I12">12</xref><xref ref-type="aff" rid="I13">13</xref><email>prharrigan@cfenet.ubc.ca</email></contrib><contrib contrib-type="author" id="A19"><name><surname>Brumme</surname><given-names>Zabrina L</given-names></name><xref ref-type="aff" rid="I14">14</xref><email>zbrumme@sfu.ca</email></contrib><contrib contrib-type="author" corresp="yes" id="A20"><name><surname>O’Brien</surname><given-names>Stephen J</given-names></name><xref ref-type="aff" rid="I1">1</xref><xref ref-type="aff" rid="I15">15</xref><email>lgdchief@gmail.com</email></contrib> | GigaScience | <sec><title>Findings</title><sec><title>Introduction</title><p>Annotations of human genome variation have identified some 60 million single nucleotide polymorphisms (SNPs), which offer the promise of connecting nucleotide and structural variation to hereditary traits [<xref ref-type="bibr" rid="B1">1</xref>-<xref ref-type="bibr" rid="B3">3</xref>]. Genotyping arrays that resolve millions of common SNPs have enabled over 2,000 genome-wide associations studies (GWAS) to discover principal genetic determinants of complex multifactorial human diseases [<xref ref-type="bibr" rid="B4">4</xref>,<xref ref-type="bibr" rid="B5">5</xref>]. Today, whole-genome sequence association has extended the prospects for personalized genomic medicine, capturing rare variants, copy number variation (CNV), indels, epistatic and epigenetic interactions in hopes of achieving individualized genomic assessment, diagnostics, and therapy of complex maladies by interpreting one’s genomic heritage [<xref ref-type="bibr" rid="B6">6</xref>-<xref ref-type="bibr" rid="B9">9</xref>].</p><p>To date, GWAS studies have produced conflicting signals because many SNP associations are not replicated in subsequent studies. Further, GWAS frequently fail to implicate previously-validated gene regions described in candidate gene associations for the same disease, and in most cases offer less than 10% of the explanatory variance for the disease etiology [<xref ref-type="bibr" rid="B9">9</xref>-<xref ref-type="bibr" rid="B13">13</xref>]. In addition, discovered gene variants are frequently nested in noncoding desert regions of the genome that are difficult to interpret. At least part of these weaknesses derive from discounting SNP association “hits” that fail to achieve “genome-wide significance”, a widely accepted, albeit conservative, statistical threshold set to discard the plethora of false positive statistical associations (Type I errors) that derive from the large number of SNPs interrogated [<xref ref-type="bibr" rid="B2">2</xref>,<xref ref-type="bibr" rid="B13">13</xref>-<xref ref-type="bibr" rid="B16">16</xref>].</p><p>A challenge to genetic epidemiology involves disentangling the true functional associations that straddle the genome-wide significance threshold from the myriad of statistical artifacts that also occur. No one has developed a real solution to this conundrum, though some approaches have been offered [<xref ref-type="bibr" rid="B11">11</xref>,<xref ref-type="bibr" rid="B15">15</xref>-<xref ref-type="bibr" rid="B21">21</xref>]. Many researchers agree that more widely practiced open access data sharing of unabridged GWAS data would offer the opportunity for multiple plausible approaches to bear on this question [<xref ref-type="bibr" rid="B22">22</xref>,<xref ref-type="bibr" rid="B23">23</xref>]. However, for many cohorts, especially those developed before the advent of the genomics era, participants were not consented for open access of genome-wide data. Since patient anonymization is virtually impossible with genetic epidemiological data, the prospects of sharing patients’ genotype and clinical data may conflict with ethical concerns over protecting the individual privacy of study subjects [<xref ref-type="bibr" rid="B24">24</xref>-<xref ref-type="bibr" rid="B26">26</xref>]. GWATCH (Genome-Wide Association Tracks Chromosome Highway) addresses this issue through an organized open release of unabridged SNP-test association results from GWAS and whole genome sequencing (WGS) association studies and illustrates its utility using a SNP association analysis for HIV-AIDS in multiple cohorts [<xref ref-type="bibr" rid="B10">10</xref>,<xref ref-type="bibr" rid="B11">11</xref>,<xref ref-type="bibr" rid="B19">19</xref>,<xref ref-type="bibr" rid="B27">27</xref>-<xref ref-type="bibr" rid="B32">32</xref>].</p></sec></sec><sec sec-type="results"><title>Results</title><p>GWATCH is a dynamic genome browser that automates and displays primary analysis results: p-values and Quantitative Association Statistic (QAS, a general term for statistics explaining direction and strength of associations: odds ratio, relative hazard and ez2-transformed correlation coefficient; see Section 2 of Additional file <xref ref-type="supplementary-material" rid="S1">1</xref>: Materials and Methods) from multiple association tests performed for one or more cohorts in a GWAS or WGS association study as a visual array ordered by SNP chromosomal position [<xref ref-type="bibr" rid="B33">33</xref>]. GWATCH offers a number of “features” that allow automated analysis and visualization of multiple test results, rapid discovery, replication and data release of unabridged association results (Table <xref ref-type="table" rid="T1">1</xref>).</p><table-wrap position="float" id="T1"><label>Table 1</label><caption><p>Display feature components of GWATCH</p></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="left"/></colgroup><thead valign="top"><tr><th align="left"><bold>Features displayed</bold></th><th align="left"><bold>Illustration</bold></th></tr></thead><tbody valign="top"><tr><td align="left" valign="bottom">1. <bold>Unabridged data table</bold> of SNP chromosome coordinates, MAF*, p-value and QAS** for each SNP for each test<hr/></td><td align="left" valign="bottom">Additional file <xref ref-type="supplementary-material" rid="S2">2</xref>: Table S1<hr/></td></tr><tr><td align="left" valign="bottom">2. <bold>Association tests list</bold> and <bold>Manhattan plots</bold> for each test across all SNPs<hr/></td><td align="left" valign="bottom">Additional file <xref ref-type="supplementary-material" rid="S1">1</xref>: Figure S1<hr/></td></tr><tr><td align="left" valign="bottom">3. <bold>SNAPSHOTS</bold> of SNP-test results in a chromosome region:<hr/></td><td align="left" valign="bottom"> <hr/></td></tr><tr><td align="left" valign="bottom"> 1. <bold>2D heat plot snapshot</bold> illustrating p-values in any selected chromosome region<hr/></td><td align="left" valign="bottom">Figure <xref ref-type="fig" rid="F1">1</xref>A and Additional file <xref ref-type="supplementary-material" rid="S1">1</xref>: Figure S2<hr/></td></tr><tr><td align="left" valign="bottom"> 2. <bold>3D checkerboard plot snapshot</bold> illustrating p-values and QAS** in any selected chromosome region<hr/></td><td align="left" valign="bottom">Figure <xref ref-type="fig" rid="F1">1</xref>B and Additiona file 1: Figure S3<hr/></td></tr><tr><td align="left" valign="bottom"> 3. <bold>LD-polarized 3D checkerboard snapshot</bold> illustrating p-values and QAS** in any selected chromosome region<hr/></td><td align="left" valign="bottom">Figure <xref ref-type="fig" rid="F1">1</xref>B and Additional file <xref ref-type="supplementary-material" rid="S1">1</xref>: Figure S4<hr/></td></tr><tr><td align="left" valign="bottom">4. <bold>Dynamic HIGHWAY view by chromosome browser</bold> illustrating p-values and QAS**<hr/></td><td align="left" valign="bottom">Figure <xref ref-type="fig" rid="F1">1</xref>C<hr/></td></tr><tr><td align="left" valign="bottom">5. <bold>Top association hits:</bold><hr/></td><td align="left" valign="bottom"> <hr/></td></tr><tr><td align="left" valign="bottom"> 1. <bold>Top hits</bold> based on <bold>ranked -log p-value</bold><hr/></td><td align="left" valign="bottom">Additional file <xref ref-type="supplementary-material" rid="S10">10</xref>: Table S7<hr/></td></tr><tr><td align="left" valign="bottom"> 2. <bold>Top hits</bold> based on <bold>ranked QAS</bold>**<hr/></td><td align="left" valign="bottom">Additional file <xref ref-type="supplementary-material" rid="S10">10</xref>: Table S7<hr/></td></tr><tr><td align="left" valign="bottom"> 3. <bold>Top hits</bold> based on <bold>ranked Density of -log p-value</bold> within a SNP genomic region<hr/></td><td align="left" valign="bottom">Additional file <xref ref-type="supplementary-material" rid="S10">10</xref>: Table S7<hr/></td></tr><tr><td align="left" valign="bottom">6. <bold>TRAX feature:</bold><hr/></td><td align="left" valign="bottom"> <hr/></td></tr><tr><td align="left" valign="bottom"> 1. <bold>TRAX PAGE</bold> – two-page graphic summary illustrating p-values and QAS** for one selected SNP<hr/></td><td align="left" valign="bottom">Additional file <xref ref-type="supplementary-material" rid="S7">7</xref>: Figure S5<hr/></td></tr><tr><td align="left"> 2. <bold>TRAX REPORT</bold> – eleven-page analysis summary with graphs, curves and tables for all association tests for one selected SNP</td><td align="left">Additional file <xref ref-type="supplementary-material" rid="S8">8</xref>: Figure S6</td></tr></tbody></table><table-wrap-foot><p><italic>Abbreviations:</italic><italic>*MAF</italic> minor allele frequency, <italic>**QAS</italic> quantitative association statistic (OR, RH, ez2-transformed correlation coefficient).</p></table-wrap-foot></table-wrap><p>A typical input of a GWAS analysis includes a large unabridged Data Table listing p-values and QASs across multiple SNP association tests performed for a list of ~10,000,000 ordered SNPs (Additional file <xref ref-type="supplementary-material" rid="S2">2</xref>: Table S1). GWATCH displays the Data Table, association tests and various perspectives for results: Manhattan plots for each single test (Additional File <xref ref-type="supplementary-material" rid="S1">1</xref>: Figure S1), 2D and 3D snapshots of test results for chromosome regions of “hits”, and a dynamic chromosome browser that illustrates significant p-values and QASs from the Data Table (Figure <xref ref-type="fig" rid="F1">1</xref>A, B and C). The imagery provides a dynamic traverse along a human chromosome producing a “bird’s eye” view of the strong SNP associations that rise above the chromosome highway surface. The idea is to visualize association results across a gene region (e.g., one that may include a highly significant SNP association) for all the tests performed (on the same or different cohorts) and for all the neighboring, potentially proxy SNPs (i.e., SNPs which track the neighboring causal, disease-affecting SNP due to the linkage disequilibrium [LD]) for the same tests.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p><bold>GWATCH produces different kinds of snapshots and views for selected genomic region. (A)</bold><bold>2D-SNAPSHOT</bold> of <italic>PARD3B</italic> region of Chromosome 2 [<xref ref-type="bibr" rid="B28">28</xref>] tested for the 123 tests in Group A (Table <xref ref-type="table" rid="T2">2</xref>). <bold>(B) POLARIZED 3D-SNAPSHOT</bold> of the <italic>PROX1</italic> region of Chromosome 1 [<xref ref-type="bibr" rid="B31">31</xref>] tested for the same group (Table <xref ref-type="table" rid="T2">2</xref>). <bold>(C)</bold> Dynamic 3D <bold>HIGHWAY</bold> chromosome browser view of <italic>CCR5</italic> region of Chromosome 3 [<xref ref-type="bibr" rid="B32">32</xref>] tested for the same group (Table <xref ref-type="table" rid="T2">2</xref>). See also in Additional file <xref ref-type="supplementary-material" rid="S1">1</xref>: Figures S2-S4.</p></caption><graphic xlink:href="2047-217X-3-18-1"/></fig><p>Top hits are ranked based upon extreme p-values, QASs, or “density” of composite p-value peaks (representing proxy SNPs in linkage disequilibrium and multiple non-independent association tests). A multi-page “TRAX REPORT” produces curves, tables and appropriate statistics for a selected variant (SNP, indel or CNV tracked) on request. As genotyping and clinical data are organized, GWATCH automates the computation and visualization of results allowing instant replication of putative discoveries suggested by outside cohort studies or functional experiments. GWATCH also provides a simple procedure for web release of the association results to interested researchers.</p><p>We illustrate the utility, interpretation, and navigation of GWATCH using a GWAS carried out with study participants enrolled in eight prospective HIV-AIDS cohorts, searching for AIDS Restriction Genes [<xref ref-type="bibr" rid="B10">10</xref>,<xref ref-type="bibr" rid="B11">11</xref>,<xref ref-type="bibr" rid="B19">19</xref>,<xref ref-type="bibr" rid="B27">27</xref>-<xref ref-type="bibr" rid="B32">32</xref>]. We performed a GWAS meta-analysis on 5,922 patients with distinctive clinical outcomes genotyped using an Affymetrix 6.0 genotyping array (700,022 SNPs after quality control [QC] filters) and parsed into three population groups: Group A) A select group of 1,527 European American individuals; Group B) A larger group of 4,462 European American individuals that includes Group A; Group C) An independent group of 1,460 African American individuals (Table <xref ref-type="table" rid="T2">2</xref>). Based upon available clinical information, we performed 123 association tests on Group A, 144 association tests on Group B, and 60 association tests on Group C (Table <xref ref-type="table" rid="T3">3</xref> and Additional file <xref ref-type="supplementary-material" rid="S3">3</xref>: Table S2, Additional file <xref ref-type="supplementary-material" rid="S4">4</xref>: Table S3, Additional file <xref ref-type="supplementary-material" rid="S5">5</xref>: Table S4, Additional file <xref ref-type="supplementary-material" rid="S6">6</xref>: Table S5). The tests include allele and genotype associations for four stages of AIDS: HIV acquisition/infection, AIDS progression (including categorical and survival analyses), AIDS-defining conditions and Highly Active AntiRetroviral Therapy (HAART) outcomes as described previously [<xref ref-type="bibr" rid="B27">27</xref>-<xref ref-type="bibr" rid="B32">32</xref>]; however, the unabridged dataset displayed in GWATCH-AIDS is far richer. For example, in references [<xref ref-type="bibr" rid="B28">28</xref>,<xref ref-type="bibr" rid="B31">31</xref>,<xref ref-type="bibr" rid="B32">32</xref>] each describes one association test (implicating the <italic>PARD3B</italic>, <italic>PROX1</italic>, and <italic>CCR5-∆32</italic> AIDS restriction genes respectively); [<xref ref-type="bibr" rid="B29">29</xref>,<xref ref-type="bibr" rid="B30">30</xref>] analyze small subsets of the SNPs tested within NEMP and HDF gene groups, respectively. GWATCH-AIDS presents complete results for 700,022 SNPs for 327 tests (Table <xref ref-type="table" rid="T3">3</xref>) for 5,922 study participants listed in Table <xref ref-type="table" rid="T2">2</xref>.</p><table-wrap position="float" id="T2"><label>Table 2</label><caption><p>Categories and numbers of patients genotyped in AIDS GWAS meta-analysis</p></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="left"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/></colgroup><thead valign="top"><tr><th rowspan="3" align="left" valign="top"><bold>Abbreviations</bold></th><th rowspan="3" align="left" valign="top"><bold>Risk groups</bold></th><th colspan="3" align="center" valign="bottom"><bold>Number of patients for each group</bold><hr/></th><th align="center" valign="bottom"><bold>Total</bold><hr/></th></tr><tr><th align="center" valign="bottom"><bold>Group A</bold><hr/></th><th align="center" valign="bottom"><bold>Group B</bold><hr/></th><th align="center" valign="bottom"><bold>Group C</bold><hr/></th><th align="center" valign="bottom"><bold>B + C</bold><hr/></th></tr><tr><th align="center"><bold>EA*-I</bold></th><th align="center"><bold>EA*-Total</bold></th><th align="center"><bold>AA**</bold></th><th align="center"> </th></tr></thead><tbody valign="top"><tr><td align="left" valign="bottom">HREU<hr/></td><td align="left" valign="bottom">High Risk Exposed HIV Uninfected<hr/></td><td align="center" valign="bottom">254<hr/></td><td align="center" valign="bottom">300<hr/></td><td align="center" valign="bottom">148<hr/></td><td align="center" valign="bottom">448<hr/></td></tr><tr><td align="left" valign="bottom">EU (except HREU)<hr/></td><td align="left" valign="bottom">Exposed HIV Uninfected (all risks)<hr/></td><td align="center" valign="bottom">1<hr/></td><td align="center" valign="bottom">351<hr/></td><td align="center" valign="bottom">267<hr/></td><td align="center" valign="bottom">618<hr/></td></tr><tr><td align="left" valign="bottom">SC<hr/></td><td align="left" valign="bottom">Sero-Convertor<hr/></td><td align="center" valign="bottom">703<hr/></td><td align="center" valign="bottom">767<hr/></td><td align="center" valign="bottom">288<hr/></td><td align="center" valign="bottom">1 055<hr/></td></tr><tr><td align="left" valign="bottom">SP-LTS<hr/></td><td align="left" valign="bottom">Sero-Prevalent-Long-Term-Survivor (no AIDS for >10 years)<hr/></td><td align="center" valign="bottom">444<hr/></td><td align="center" valign="bottom">831<hr/></td><td align="center" valign="bottom">170<hr/></td><td align="center" valign="bottom">1 001<hr/></td></tr><tr><td align="left" valign="bottom">Sequelae<hr/></td><td align="left" valign="bottom">AIDS sequelae diagnosis<hr/></td><td align="center" valign="bottom">461<hr/></td><td align="center" valign="bottom">1 848<hr/></td><td align="center" valign="bottom">0<hr/></td><td align="center" valign="bottom">1 848<hr/></td></tr><tr><td align="left" valign="bottom">HAART<hr/></td><td align="left" valign="bottom">Anti-retroviral treatment<hr/></td><td align="center" valign="bottom">485<hr/></td><td align="center" valign="bottom">1 319<hr/></td><td align="center" valign="bottom">65<hr/></td><td align="center" valign="bottom">1 384<hr/></td></tr><tr><td align="left">Total study participants</td><td align="left"> </td><td align="center">1 527</td><td align="center">4 462</td><td align="center">1 460</td><td align="center">5 922</td></tr></tbody></table><table-wrap-foot><p><italic>Abbreviations: *EA</italic> European Americans, <italic>**AA </italic>African Americans.</p></table-wrap-foot></table-wrap><table-wrap position="float" id="T3"><label>Table 3</label><caption><p><bold>Statistical tests performed on 3 HIV-AIDS cohort Study Groups A-C (see Table </bold><xref ref-type="table" rid="T2">2</xref><bold>)</bold></p></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="left"/><col align="center"/><col align="center"/><col align="center"/></colgroup><thead valign="top"><tr><th rowspan="2" align="left" valign="top"><bold>Clinical stage</bold></th><th rowspan="2" align="left" valign="top"><bold>Test type</bold></th><th colspan="3" align="center" valign="bottom"><bold>Number of tests for each group</bold><hr/></th></tr><tr><th align="center"><bold>Group A</bold></th><th align="center"><bold>Group B</bold></th><th align="center"><bold>Group C</bold></th></tr></thead><tbody valign="top"><tr><td align="left" valign="bottom">I. HIV Infection<hr/></td><td align="left" valign="bottom">Ia. Infection - categorical<hr/></td><td align="center" valign="bottom">3<hr/></td><td align="center" valign="bottom">12<hr/></td><td align="center" valign="bottom">12<hr/></td></tr><tr><td rowspan="3" align="left" valign="top">II. HIV Progression<hr/></td><td align="left" valign="bottom">IIa. Progression - categorical dichotomous<hr/></td><td align="center" valign="bottom">12<hr/></td><td align="center" valign="bottom">12<hr/></td><td align="center" valign="bottom">12<hr/></td></tr><tr><td align="left" valign="bottom">IIb. Progression - categorical multipoint<hr/></td><td align="center" valign="bottom">12<hr/></td><td align="center" valign="bottom">12<hr/></td><td align="center" valign="bottom">12<hr/></td></tr><tr><td align="left" valign="bottom">IIc. Progression - survival<hr/></td><td align="center" valign="bottom">48<hr/></td><td align="center" valign="bottom">48<hr/></td><td align="center" valign="bottom">24<hr/></td></tr><tr><td rowspan="4" align="left" valign="top">III. AIDS-defining Conditions<hr/></td><td align="left" valign="bottom">IIIa. Sequelae - categorical first sequela<hr/></td><td align="center" valign="bottom">9<hr/></td><td align="center" valign="bottom">9<hr/></td><td align="center" valign="bottom">-<hr/></td></tr><tr><td align="left" valign="bottom">IIIb. Sequelae - survival first sequela<hr/></td><td align="center" valign="bottom">9<hr/></td><td align="center" valign="bottom">-<hr/></td><td align="center" valign="bottom">-<hr/></td></tr><tr><td align="left" valign="bottom">IIIc. Sequelae - categorical any sequelae<hr/></td><td align="center" valign="bottom">9<hr/></td><td align="center" valign="bottom">33<hr/></td><td align="center" valign="bottom">-<hr/></td></tr><tr><td align="left" valign="bottom">IIId. Sequelae - survival any sequelae<hr/></td><td align="center" valign="bottom">9<hr/></td><td align="center" valign="bottom">6<hr/></td><td align="center" valign="bottom">-<hr/></td></tr><tr><td rowspan="2" align="left" valign="top">IV. Treatment with ARV<hr/></td><td align="left" valign="bottom">IVa. HAART - categorical<hr/></td><td align="center" valign="bottom">6<hr/></td><td align="center" valign="bottom">-<hr/></td><td align="center" valign="bottom">-<hr/></td></tr><tr><td align="left" valign="bottom">IVb. HAART - survival<hr/></td><td align="center" valign="bottom">6<hr/></td><td align="center" valign="bottom">12<hr/></td><td align="center" valign="bottom">-<hr/></td></tr><tr><td align="left">Total</td><td align="left"> </td><td align="center">123</td><td align="center">144</td><td align="center">60</td></tr></tbody></table><table-wrap-foot><p>See Additional file <xref ref-type="supplementary-material" rid="S3">3</xref>: Table S2, Additional file <xref ref-type="supplementary-material" rid="S4">4</xref>: Table S3, Additional file <xref ref-type="supplementary-material" rid="S5">5</xref>: Table S4, Additional file <xref ref-type="supplementary-material" rid="S6">6</xref>: Table S5, for detailed description of statistical association tests performed in each group.</p></table-wrap-foot></table-wrap><p>The first step of data analysis using GWATCH is to produce a large Data Table listing all SNP names, chromosome coordinates and minor allele frequency (MAF), with p-values and QASs for each test (Additional file <xref ref-type="supplementary-material" rid="S2">2</xref>: Table S1) plus a description of each test. Results in this Table are displayed as familiar Manhattan plots for each test as well as by <bold>SNAPSHOT</bold> views of chromosome regions. <bold>2D-SNAPSHOT</bold> is a heat plot of ordered SNP-test results (e.g., ~80 SNPs at 4 kb average distance for 123 tests in Group A (Table <xref ref-type="table" rid="T2">2</xref>) equaling ~10,000 SNP-test combinations) indexed by the p-values from p > 0.05 (light grey) to richer colors for decreasing p-values, assuring that significant region clusters are more densely colorful (Figure <xref ref-type="fig" rid="F1">1</xref>A and Additional file <xref ref-type="supplementary-material" rid="S1">1</xref>: Figure S2). Similarly a <bold>3D-SNAPSHOT</bold> presents a checkerboard view of a chromosome region whereby the blocks rising above the surface reflect –log p-value and the color intensity reflects the QAS values with green indicating “resistant” associations (QAS < 1.0) and red showing “susceptible” ones (QAS > 1.0) (Figure <xref ref-type="fig" rid="F1">1</xref>B and Additional file <xref ref-type="supplementary-material" rid="S1">1</xref>: Figure S3 and S4). The moving browser <bold>HIGHWAY</bold>, a major feature of GWATCH, scrolls across the entire chromosomes in the 3D view of background statistical “noise” plus interesting regions of dense elevated blocks (Figure <xref ref-type="fig" rid="F1">1</xref>C).</p><p>Since susceptible/resistant colors are initially indexed by the minor (less common) allele at any locus, color discordance will arise in a region when minor allele at a given locus is tracked in LD by the common allele at an adjacent locus. The <bold>POLARIZE</bold> option corrects this computational artifact by inverting the QAS in locus pairs that show discrepant (common and minor allele tracking as proxies) LD polarity. When the entire association signal for a region, driving the non-independent SNPs and non-independent tests, derives from a single causal allele within the region, the blocks of associated SNPs in the viewed region should be the same color after polarization (Figure <xref ref-type="fig" rid="F1">1</xref>B and Additional file <xref ref-type="supplementary-material" rid="S1">1</xref>: Figure S4).</p><p>Automated searches for extreme locus “hits” revealing remarkable associations across the genome can be performed for each stage of disease (see above) screening for extreme p-values, QAS values and/or density of extreme p-values. For loci of particular interest, a detailed <bold>TRAX REPORT</bold> is generated to display each curve, table and statistic that had driven the association discovery (Additional file <xref ref-type="supplementary-material" rid="S7">7</xref>: Figure S5 and Additional file <xref ref-type="supplementary-material" rid="S8">8</xref>: Figure S6). TRAX REPORT is available for 641 SNPs in 241 genes listed in Additional file <xref ref-type="supplementary-material" rid="S9">9</xref>: Table S6. For the rest of the SNPs, the TRAX PAGE (shorter version of TRAX REPORT) is available.</p><p>To demonstrate GWATCH, three previously validated AIDS resistance gene regions, <italic>CCR5-∆32, PROX1</italic> and <italic>PARD3B</italic>, can be examined by simple entering rs-number, gene name or chromosome coordinates in the search option (see also 2D and 3D snapshots in Additional file <xref ref-type="supplementary-material" rid="S1">1</xref>: Figures S2-S4). GWATCH moves HIGHWAY to the selected region so one can visualize the signal with the 2D and 3D-SNAPSHOTS plus the TRAX REPORTS. Lastly, we also include a listing of discovered regions that showed AIDS association signals that, though they did not reach genome-wide significance, represented outlier values for several related tests and linked SNPs (Additional file <xref ref-type="supplementary-material" rid="S10">10</xref>: Table S7). These regions then would be considered as candidates for future evaluation and replication in independent cohort studies.</p><p>Finally, GWATCH is a generalizable web tool suitable for GWAS and/or WGS dataset for any complex disease. The “finished“or “processed” data (ones containing a final Data Table of p-values and QAS for completed association tests) can be uploaded directly by following instructions for dataset upload on the GWATCH website. “Primary” or “unfinished” data (ones with genotypes and clinical data for which tests need to be constructed and calculated) will be uploaded with our assistance in custom development of a disease-specific GWATCH-based analysis.</p></sec><sec sec-type="discussion"><title>Discussion</title><p>GWATCH is designed to enable investigators and users not connected to the original study to access the results of SNP association (from the whole genome sequence or SNP array genotyping) in order to view and share their study design and results openly. It can be used for visualization of regions with low p-values to inspect the pattern of variation across linked SNPs and also at different stages of disease (e.g., HIV infection, AIDS progression and treatment outcome).</p><p>As a primary discovery approach, screening across unabridged test results poses large statistical penalties for multiple tests eroding confidence in associations that fail to achieve genome-wide significance [<xref ref-type="bibr" rid="B2">2</xref>,<xref ref-type="bibr" rid="B13">13</xref>-<xref ref-type="bibr" rid="B18">18</xref>,<xref ref-type="bibr" rid="B21">21</xref>]. For this reason, one should use caution in inspection of putative regions of significance. Nonetheless, wholesale discarding of marginally significant “hits” will discount some true associations within the mix of statistical artifacts. GWATCH offers an opportunity to screen the genome for disease-associated regions, which may contain causal SNP variants included (or not) in the SNP array used for genotyping, as well as proxy SNPs tracking the causal variant. Further, in complex diseases for which there are many different cohorts being studied (e.g., in HIV-AIDS there are at least twenty different groups conducting AIDS GWAS on small, well-defined cohorts that may differ in genetic background and clinical data available for association testing) [<xref ref-type="bibr" rid="B34">34</xref>] GWATCH offers rapid replication opportunities with an independent dataset.</p><p>There are several websites that aim at cataloguing and displaying SNP associations. For example, GWAS Central [<xref ref-type="bibr" rid="B35">35</xref>] is a valuable resource for releasing and accessing GWAS data [<xref ref-type="bibr" rid="B36">36</xref>]. At the same time, we believe that GWATCH can be advantageous in some cases for the following reasons: 1) GWATCH utilizes (while not revealing directly) primary unabridged clinical/phenotypic data providing detailed analytical reports, like TRAX, not offered in GWAS Central; 2) GWATCH contains summary tools, such as top hits tables, and performs calculation of density that allows for identification, inspection and replication of putative association hits; 3) GWAS Central reports traditional Manhattan plots while GWATCH extends these to 2D and 3D static and dynamic region visuals that expands user comprehension and perception for better grasp of large data.</p><p>The GWATCH web browser provides a dynamic visual journey, similar to driving a video game along human chromosomes to view patterns of GWAS- or WGS-based variant association with any complex disease. It is meant to be appealing, intuitive, and accessible to non-experts and experts alike, including the various contributors to today’s exciting gene association studies. The format and open web access allows for importing new data from any disease-gene association study with multiple disease stages or genetic models of analysis. The wide breadth of test associations displayed is particularly suited to complex disease cohorts with detailed clinical parameters over distinct disorder stages. Further, although GWATCH is potentially useful for initial gene discovery, an important corollary lies in providing rapid replication of gene discoveries from independent cohort studies by simply keying in the putative gene region and inspecting the many test results of the posted dataset. Since replication screens are hypothesis-driven, they avoid the stringent multiple test correction penalties of a GWAS/WGS (p < 10<sup>-8</sup>). Finally, different cohort studies can be compared directly or combined to build meta-analyses.</p><p>Should many cohort investigators release their unabridged results, then association discoveries will be replicated (or not) in a rapid, open and productive manner, allowing for large meta-analyses as have been proposed for HIV-AIDS and other complex diseases [<xref ref-type="bibr" rid="B22">22</xref>,<xref ref-type="bibr" rid="B23">23</xref>,<xref ref-type="bibr" rid="B34">34</xref>]. Unlike other methods of data sharing, this results-based open data sharing/release approach avoids any violation of patient privacy, IRB (institutional review board) and HIPAA (Health Insurance Portability and Accountability Act of 1996) concerns, or informed consent constraints, since the primary clinical and genotype data remain confidential while the derivative results (p-values, QASs, plots) of multiple conceivable analytical approaches are openly released. In this approach, we hope to considerably expand discovery and replication opportunities in important biomedical research. To us, this ensures the maximum benefit of open access data sharing while protecting patients who prefer privacy (many do), but wish to see their volunteerism fulfilled.</p></sec><sec sec-type="materials|methods"><title>Materials and methods</title><sec><title>GWATCH implementation</title><p>GWATCH is a web-based application that integrates several technologies and programming languages. Server-side is represented by Apache web server, which employs PHP engine and Java-based toolkit Batik. R-project functions and modules are used for performing statistical tests, polarization and density calculation. MySQL database component of GWATCH allows access, retrieval and management of genotypes, clinical information and test results. On the frontend, GWATCH employs HTML5, Javascript, jQuery and WebGL for HIGHWAY browser interface, and Ajax and JSON technologies for data exchange between server and client.</p></sec><sec><title>GWATCH tools</title><sec><title>TRAX REPORTS</title><p>After screening for associations of clinical traits and genotypes one may be interested in a closer review of certain SNPs. The TRAX REPORT (Additional file <xref ref-type="supplementary-material" rid="S7">7</xref>: Figure S5 and Additional file <xref ref-type="supplementary-material" rid="S8">8</xref>: Figure S6) tool allows the production of reports on extended statistical analysis for any single SNP if the corresponding genotype and clinical information is available for all individuals. Important genotype information is given in the header on the TRAX front page: SNP identifier, SNP coordinate, chromosome, alleles and their frequencies. The header also lists information on populations involved in the analysis. In addition to the header, front page also contains a summary for all tests with p-values, as well as values of QAS represented in the bar plot form. The following pages of TRAX REPORT contain detailed information, such as contingency tables (that are produced in the form of corresponding bar plots for any categorical test, including progression categorical tests), and Kaplan–Meier survival curves that are reported for all three genotypes for all survival tests.</p></sec><sec><title>Polarization</title><p>The polarization tool enables the inversion of test results for minor and common SNP-alleles around some fixed SNP (called index SNP) for better approximation of true associations. A polarization table is produced using linkage disequilibrium coefficients (<italic>D’</italic>) between neighboring SNPs. Linkage disequilibrium coefficients are calculated for 80 SNPs upstream and 80 SNPs downstream of the index SNP. In the case of a sufficiently large positive value of linkage disequilibrium (<italic>D’</italic> > 0.9), the polarization mark is assigned to 1, whereas in the case of a sufficiently large negative linkage disequilibrium (<italic>D’</italic> < -0.9) the polarization mark is assigned to -1. If the linkage disequilibrium is sufficiently small, the polarization mark is assigned to 0. In the process of polarization, QAS values for test results of neighboring SNPs are inverted if the polarization mark is -1 implying the inversion of direction of disease association for such SNPs.</p></sec><sec><title>Density</title><p>Density top scoring that identifies regions of concentration of small p-values is calculated for each SNP in two steps:</p><p>1) in the window of specified size (<italic>n</italic> SNPs upstream and downstream or <italic>n</italic> Kbp upstream and downstream) average -log p-value is computed for each test (lane of the Highway)</p><p>2) these per-test (per-lane) averages are used for calculating density at this SNP either by averaging them or by finding the largest one (depending on the option chosen)</p><p>The second step can be performed for all the tests or for the group of tests by the disease stage (e.g., all tests for HIV infection, all tests for AIDS progression etc.).</p></sec></sec><sec><title>Statistical tests and data used for complex AIDS study</title><p>General types of statistical data and tests relevant to GWATCH are described in Additional file <xref ref-type="supplementary-material" rid="S1">1</xref>: Materials and Methods. Below we describe particular tests and data types used in the exemplary analysis of HIV/AIDS study data.</p><p>To illustrate GWATCH utility in the analysis of GWAS results we used data from multicenter longitudinal studies of several cohorts of patients exposed to the risk of HIV infection and/or already infected with HIV: ALIVE, DCG, HGDS, HOMER, LSOCA, MACS, MHCS and SFCC [<xref ref-type="bibr" rid="B11">11</xref>,<xref ref-type="bibr" rid="B34">34</xref>,<xref ref-type="bibr" rid="B37">37</xref>,<xref ref-type="bibr" rid="B38">38</xref>]. The total pool of patients was divided into three groups A, B and C based on ethnicity and timing of data development (see Table <xref ref-type="table" rid="T2">2</xref>). A total of 5,922 patients were analyzed in all 3 groups.</p><p>All patient samples and genotypes were subjected to QC filtering depicted in Additional file <xref ref-type="supplementary-material" rid="S1">1</xref>: Table S8 as described previously [<xref ref-type="bibr" rid="B28">28</xref>,<xref ref-type="bibr" rid="B31">31</xref>]. Once final genotypes were obtained, population structure was assessed using the Principal Components Analysis module of <italic>Eigensoft</italic> software in European and African American populations [<xref ref-type="bibr" rid="B39">39</xref>] and structured SNP variants were excluded [<xref ref-type="bibr" rid="B28">28</xref>,<xref ref-type="bibr" rid="B39">39</xref>].</p><p>The statistical tests described below and listed in Table <xref ref-type="table" rid="T3">3</xref> and Additional file <xref ref-type="supplementary-material" rid="S3">3</xref>: Table S2, Additional file <xref ref-type="supplementary-material" rid="S4">4</xref>: Table S3, Additional file <xref ref-type="supplementary-material" rid="S5">5</xref>: Table S4 and Additional file <xref ref-type="supplementary-material" rid="S6">6</xref>: Table S5, were applied to the three patient study groups A, B and C (see Table <xref ref-type="table" rid="T2">2</xref>). For each of the tests described below three genetic models were used (D, R and CD, see in Section 1 of Supplementary Materials and Methods under “Genotype classification” in Additional file <xref ref-type="supplementary-material" rid="S1">1</xref>) unless stated otherwise.</p><sec><title>Infection tests (INF)</title><p>The aim of infection tests is to specify association of any selected genotype with HIV infection. The original clinical data is of categorical type based on the population of seronegatives (SN, individuals which stay HIV-negative throughout the whole study) at the baseline with the response variable indicating serostatus at the endpoint and having three levels: “high risk exposed uninfected” (HREU) seronegatives, “other seronegatives” (OSN) and “seroconverters” (SC, individuals which entered the study as HIV-negative, but became HIV-positive during the study). Three combinations of HIV status classifications were used to perform the categorical tests: “SC” vs. “HREU”, “SC” vs. “HREU” plus “OSN” and “SC” vs. “HREU” vs. “OSN”. In addition to the three genotype classifications described above (D, R and CD), allelic model (A) was also used for this test. One more group of individuals based on infection status, “seroprevalents” (SP, individuals which entered the study already being HIV-positive), was not informative for this type of test and therefore was not included in it.</p></sec><sec><title>Disease progression tests</title><p>The disease progression tests were used for screening significant associations between AIDS progression and genotype. The original data were of right-censored survival type under four different criteria of AIDS disease: CD4 < 200 (level of CD4+ cells falling below 200 cells/mm<sup>3</sup>), AIDS-1987 (patient meeting criteria of 1987 CDC definition of AIDS), AIDS-1993 (patient meeting criteria of 1993 CDC definition of AIDS) and Death from AIDS. Only SC and SP individuals were included in this analysis. SC individuals were included into analysis with HIV infection date (date of seroconvertion) as the baseline. SP individuals were included into categorical analysis with the date of the first visit as the baseline with some warnings.</p><p><italic>Disease progression categorical analysis</italic> (PDCA) used the categorical tests for survival data (CTSD) approach described in Section 2 of Additional file <xref ref-type="supplementary-material" rid="S1">1</xref>: Materials and Methods. The CTSD were performed in dichotomous (PDCA2, two groups by the survival time or current status data) and multipoint (PDCAM, more than two groups by the survival time) forms. All individuals censored before the breakpoint were removed from the PDCA dichotomous analysis, as well as the SP individuals who failed before the breakpoint. All remaining individuals censored or failed after the breakpoint were classified into the group of long-term survivors (LTS, those who do not show AIDS symptoms before the breakpoint). The breakpoints used for classification in multipoint PDCA are stated in Additional file <xref ref-type="supplementary-material" rid="S3">3</xref>: Table S2, Additional file <xref ref-type="supplementary-material" rid="S4">4</xref>: Table S3, Additional file <xref ref-type="supplementary-material" rid="S5">5</xref>: Table S4.</p><p><italic>Proportional hazard (PHAZ</italic>) analysis of disease progression used the proportional hazards survival tests (PHST) approach described in Section 2 of Additional file <xref ref-type="supplementary-material" rid="S1">1</xref>: Materials and Methods. These tests were performed for all four criteria of AIDS. Only SC individuals were included into PHAZ analysis.</p></sec><sec><title>Sequelae tests</title><p>Survival and categorical tests were performed for survival data on Kaposi’s sarcoma (KS), <italic>Pneumocystis carnii</italic> pneumonia (PCP), cytomegalovirus infection (CM), lymphoma (LY), mycobacterial infection (MYC) and other opportunistic infections (OOI). As in progression disease tests, survival sequelae tests included seroconverters only, while categorical sequelae tests included both seroconverters and seroprevalents.</p><p><italic>Sequelae tests for any infection order</italic> classify patients based on whether specific sequela occurred at all, irrespectively of its order (i.e., whether it was the first sequela to occur for patient). The survival tests (SEQSA) under proportional hazards model as well as the progression categorical tests (SEQCA) were performed separately for each of the diseases described above.</p><p><italic>Sequelae tests for the first infection</italic> classify patients based on whether specific sequela occurred first or not. The survival tests (SEQS1) under proportional hazards model as well as the progression categorical tests (SEQC1) were performed separately for each of the diseases described above.</p></sec><sec><title>Highly active antiretroviral therapy (HAART) tests</title><p>HAART tests were performed for the cohorts of patients who were subject to this type of treatment. Patients were classified based on either the level of suppression of HIV viral load or on the rebound of viral load following its suppression. Both survival (HRTS) and progression categorical (HRTC) tests were used for this analysis.</p></sec><sec><title>Hardy–Weinberg equilibrium (HWE) tests</title><p>The HWE tests are performed to control for the quality of data used for the screening of associations. Large deviations from HWE are not typical for the large populations and thus signal the genotyping error or some other type of data quality breach.</p></sec></sec></sec><sec><title>Availability and requirements</title><p><bold>Project name:</bold> GWATCH</p><p><bold>Project home page:</bold> gen-watch.org</p><p><ext-link ext-link-type="uri" xlink:href="https://github.com/DobzhanskyCenter/GWATCH">https://github.com/DobzhanskyCenter/GWATCH</ext-link></p><p><bold>Operating system(s):</bold> Platform independent (runs in the web browser)</p><p><bold>Programming language:</bold> HTML5, Javascript, PHP, Java, R, MySQL</p><p><bold>Other requirements:</bold> WebGL-supporting web browser (Firefox 4.0 and above; Chrome 12 and above; under OS X runs also in Safari 5.1 and above)</p><p><bold>License:</bold> GPL v2.0</p><p><bold>Any restrictions to use by non-academics:</bold> no</p></sec><sec><title>Availability of supporting data</title><p>Archive of the version of GWATCH used in this paper is available from the <italic>GigaScience</italic> database [<xref ref-type="bibr" rid="B40">40</xref>], and for the most recent version please see our GitHub repository.</p></sec><sec><title>Abbreviations</title><p>AIDS: Acquired immunodeficiency syndrome; CDC: Centers for Disease Control and Prevention; CNV: Copy-number variation; CTSD: categorical tests for survival data; GWAS: Genome-wide association study; GWATCH: Genome-Wide Association Tracks Chromosome Highway; HAART: Highly Active Antiretroviral Therapy; HIPAA: The Health Insurance Portability and Accountability Act of 1996; HIV: Human immunodeficiency virus; HREU: High risk exposed uninfected; HTML5: Hypertext markup language, revision 5; IRB: Institutional Review Board; HWE: Hardy-Weinberg equilibrium; LD: Linkage disequilibrium; LTS: Long-term survivor; MAF: Minor allele frequency; OSN: Other seronegatives; QAS: Quantitative Association Statistic; QC: Quality control; PDCA: Disease progression categorical analysis; PHAZ: Proportional hazard; PHP: Hypertext Preprocessor; SC: Seroconverter; SN: Seronegative; SNP: Single nucleotide polymorphism; SP: Seroprevalent; WGS: Whole genome sequencing.</p></sec><sec><title>Competing interests</title><p>ASv, SM, NC, PG and SJO are authors of the provisional application for patent US 61/897,524 “Visualization, sharing and analysis of large data sets” filed on 10/30/2013.</p></sec><sec><title>Authors’ contributions</title><p>ASv, SM, NC, PG, MR, PD, ASh, TKO and SJO developed GWATCH. LG, JT, SH, HHD, ES and SJO performed the original GWAS studies. SD, EG, DAJ, MVN, RH and ZLB contributed new epidemiological data from their AIDS cohorts. ASv, SM, NC and SJO wrote the manuscript. All authors read and approved the final manuscript.</p></sec><sec sec-type="supplementary-material"><title>Supplementary Material</title><supplementary-material content-type="local-data" id="S1"><caption><title>Additional file 1</title><p><bold>Supplementary Information.</bold> Contains Materials and Methods, Figures S1–S4, legends for Figure S5 and S6, legends for Table S1–S7, Table S8 and References.</p></caption><media xlink:href="2047-217X-3-18-S1.docx"><caption><p>Click here for file</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="S2"><caption><title>Additional file 2: Table S1</title><p>Data Table of GWAS results: 100 rows of the Data Table containing SNPs, p-values and QASs for AIDS Restriction Genes dataset in Study Group A in the <italic>PARD3B</italic> region of chromosome 2. Full unabridged data tables for Groups A-C are available on the GWATCH web portal [<xref ref-type="bibr" rid="B33">33</xref>].</p></caption><media xlink:href="2047-217X-3-18-S2.xlsx"><caption><p>Click here for file</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="S3"><caption><title>Additional file 3: Table S2</title><p>List of SNP association statistical tests and patient counts for Study Group A.</p></caption><media xlink:href="2047-217X-3-18-S3.xlsx"><caption><p>Click here for file</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="S4"><caption><title>Additional file 4: Table S3</title><p>List of SNP association statistical tests and patient counts for Study Group B.</p></caption><media xlink:href="2047-217X-3-18-S4.xlsx"><caption><p>Click here for file</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="S5"><caption><title>Additional file 5: Table S4</title><p>List of SNP association statistical tests and patient counts for Study Group C.</p></caption><media xlink:href="2047-217X-3-18-S5.xlsx"><caption><p>Click here for file</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="S6"><caption><title>Additional file 6: Table S5</title><p>Summary of SNP association tests performed for each Study Group.</p></caption><media xlink:href="2047-217X-3-18-S6.xlsx"><caption><p>Click here for file</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="S7"><caption><title>Additional file 7: Figure S5</title><p>TRAX PAGE, 2 page summary or all test results for a single SNP for a study group (e.g. p-values and QASs for HIV infection, AIDS progression using categorical and survival tests, AIDS sequelae, and HAART outcomes can be viewed and compared). TRAX PAGE can be generated <italic>de novo</italic> for any SNP of interest by placing mouse tip over a significant tower/block in the HIGHWAY and selecting the TRAX PAGE option from the data window that appears (SNPs for which TRAX REPORT is available do not have separate TRAX PAGE option in data window since TRAX REPORT includes TRAX PAGE content).</p></caption><media xlink:href="2047-217X-3-18-S7.xlsx"><caption><p>Click here for file</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="S8"><caption><title>Additional file 8: Figure S6</title><p>Detailed 11 page TRAX REPORT of derived statistics for all the tests accomplished including tables, bar graphs, survival curves and additional parameters for each test. TRAX REPORT can be generated <italic>de novo</italic> for the SNP of interest by placing mouse tip over a significant tower/block in HIGHWAY and selecting the TRAX REPORT option from the data window that appears. TRAX REPORTs are available for 641 SNPs in 241 human genes that were genotyped to replicate the GWAS associations for Study Groups A-C (Additional file <xref ref-type="supplementary-material" rid="S9">9</xref>: Table S6).</p></caption><media xlink:href="2047-217X-3-18-S8.xlsx"><caption><p>Click here for file</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="S9"><caption><title>Additional file 9: Table S6</title><p>List of 641 SNPs within 241 human genes that were assessed to replicate the GWAS associations for Study Groups A-C. For each of these SNPs a full TRAX REPORT (11 page report of figures and tables for each test) is available on the GWATCH web portal [<xref ref-type="bibr" rid="B33">33</xref>] as illustrated in Additional file <xref ref-type="supplementary-material" rid="S8">8</xref>: Figure S6.</p></caption><media xlink:href="2047-217X-3-18-S9.pdf"><caption><p>Click here for file</p></caption></media></supplementary-material><supplementary-material content-type="local-data" id="S10"><caption><title>Additional file 10: Table S7</title><p>Genomic regions of remarkable statistical association (HITS) identified in ARG-GWAS by the screen for extreme p-values.</p></caption><media xlink:href="2047-217X-3-18-S10.pdf"><caption><p>Click here for file</p></caption></media></supplementary-material></sec> |
Epidemiology of in-hospital trauma deaths in a Brazilian university hospital | <sec><title>Background</title><p>The analysis of patterns of trauma deaths may improve the evaluation of a trauma system and identify areas that may benefit from more resources. The objective of this study was to analyze the epidemiology of trauma deaths in a Brazilian university hospital in order to assess the profile of these fatalities over a 16-year period.</p></sec><sec><title>Method</title><p>Retrospective study of time series using database records. The research subjects were in-hospital deaths from external causes during the years 1995, 2000, 2005 and 2010. The following variables were analyzed: cause of injury, trauma scores, time and cause of death.</p></sec><sec><title>Results</title><p>467 cases were studied, being 325 patients (69.6%) admitted with signs of life and 142 (30.4%) considered dead on arrival. The mean age was 35.35 ± 18.03 years. 85.4% were males. Blunt trauma occurred in 73.0% of cases and penetrating mechanism in 27.0%. There was a significant increase (p < 0.001) in deaths from motorcycle crashes over the years, which went from 7.3% in 1995 to 31.5% in 2010. In contrast, there was a significant decrease (p = 0.030) in firearm-injury victims; from 21.0% in 1995 to 9.6% in 2010. About 60% of deaths occurred less than 24 hours after admission. The main causes of death were lesions of the central nervous system (56.3% of the total), followed by hemorrhagic shock (18.1%) and sepsis/multiple organ dysfunction syndrome (17.1%). The mean Injury Severity Score (ISS) of patients with signs of life was 26.41 ± 9.00, 71.3% of whom had ISS >25. The mean Revised Trauma Score (RTS) was 5.24 ± 2.05. Only 25.8% of the deaths had TRISS <0.50.</p></sec><sec><title>Conclusion</title><p>There was a shift in the profile of causes of death from trauma in this university teaching hospital, with a large decrease in penetrating injuries and a higher incidence of deaths of motorcycle riders.</p></sec> | <contrib contrib-type="author" corresp="yes" id="A1"><name><surname>Trajano</surname><given-names>Adriano D</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>adrianotrajano@gmail.com</email></contrib><contrib contrib-type="author" id="A2"><name><surname>Pereira</surname><given-names>Bruno M</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>drbrunompereira@gmail.com</email></contrib><contrib contrib-type="author" id="A3"><name><surname>Fraga</surname><given-names>Gustavo P</given-names></name><xref ref-type="aff" rid="I1">1</xref><email>fragagp2008@hotmail.com</email></contrib> | BMC Emergency Medicine | <sec><title>Background</title><p>Globally, external causes of death are a major challenge to public health authorities
[<xref ref-type="bibr" rid="B1">1</xref>]. They account for more than five million deaths annually worldwide and cause temporary or permanent disability to millions more, representing about 9% of global mortality. For every death, it is estimated that there are dozens of hospitalizations, hundreds of visits to emergency and thousands of medical appointments
[<xref ref-type="bibr" rid="B2">2</xref>]. External causes, including trauma, mainly affect people from 5 to 44 years old, usually males, living in poor and developing countries
[<xref ref-type="bibr" rid="B3">3</xref>].</p><p>In 2010, Brazil registered 143,256 deaths from external causes, 21% more than in 2000 (118,397 deaths). External causes account for 12.9% of registered deaths in the country, being the third cause of death among Brazilians, losing only to cardiovascular diseases (29%) and cancers (16%)
[<xref ref-type="bibr" rid="B4">4</xref>]. The pattern in Brazil differs from elsewhere in the world because most deaths are caused by homicide or are related to traffic accidents, unlike most WHO-member countries, where 51% of deaths related to external causes are suicides and 11% are due to warfare
[<xref ref-type="bibr" rid="B5">5</xref>].</p><p>Studies have shown that the analysis of the epidemiology of traumatic deaths can improve the assessment of a trauma system and identify the critical areas that may benefit from education provision, research and allocation of resources. It is known that the distribution of times and locations of in-hospital deaths from trauma are influenced by cause of injury, age and injured body areas
[<xref ref-type="bibr" rid="B6">6</xref>-<xref ref-type="bibr" rid="B12">12</xref>].</p><p>Lesions of the central nervous system (CNS) appear as the main cause of death among trauma patients, being present in about 50% of the victims, and with a mortality peak in 24 to 72 hours after hospital admission. Hemorrhagic shock is the second most frequent cause of death, ranging from 20% to 40% in some series, depending on the rate of penetrating trauma victims at the specific region
[<xref ref-type="bibr" rid="B6">6</xref>,<xref ref-type="bibr" rid="B7">7</xref>,<xref ref-type="bibr" rid="B10">10</xref>-<xref ref-type="bibr" rid="B12">12</xref>]. Traumatic deaths occurring beyond the expected time have been used as a parameter to assess the quality of care of trauma patients. Exsanguination is the leading cause of in-hospital deaths and is deemed as preventable by the delay between diagnosis and definitive treatment. Several studies confirm that the percentage of avoidable deaths from trauma is significantly higher when there is no adequate structure for treatment and when there is no medical expertise available to work on issues related to trauma patients
[<xref ref-type="bibr" rid="B13">13</xref>-<xref ref-type="bibr" rid="B16">16</xref>].</p><p>In Brazil there is only rudimentary information on mortality due to lack of programs to collect data and assess information related to trauma. Trauma records are essential to guide the decisions and actions relevant to trauma victims and to serve as a link between what needs to be known about the causes and outcomes of trauma, and the development of local, regional and national intervention plans. A computerized system is essential to store these data, so that information can be easily accessed and the results analyzed practically and rapidly. Knowledge of this information is crucial for the mapping of trauma incidence in the country
[<xref ref-type="bibr" rid="B17">17</xref>-<xref ref-type="bibr" rid="B20">20</xref>].</p><p>The aim of this study is to analyze time-series of deaths by trauma at a University Teaching Hospital (HC-UNICAMP), according to the causes of injury, time and causes of in-hospital deaths, injury severity and body-affected areas. We hypothesized that the pattern of the trauma deaths has changed in recent years at our specific hospital.</p></sec><sec sec-type="methods"><title>Methods</title><p>The HC-Unicamp has about 500 beds and serves patients predominantly with high complexity health issues from within the entire metropolitan area of Campinas, which corresponds to a population of about 3 million inhabitants. It is one of the referenced centers for trauma patients in this region. Currently the city of Campinas has 1.2 million inhabitants, and has two other public hospitals (with 200 beds each) for trauma patients. The Division of Trauma (DCT) of HC-UNICAMP was established in 1987 and is responsible for traumatic and non-traumatic surgical emergencies for adults, admitting about 5,000 trauma patients per year. We retrospectively reviewed the charts of all in-hospital deaths from external causes (codes V01-Y98 in Chapter XX of the 10th revision of International Classification of Diseases, ICD-10) during the years 1995, 2000, 2005 and 2010. The 5-year intervals enabled the demonstration of death trends. We included all patients admitted to the emergency room of HC-Unicamp who died, including those dead on arrival. All lesions present, major or minor, were considered. In-hospital deaths by drowning, poisoning, burns, falling with isolated long-bone fracture and medical complications were excluded. Patients with incomplete charts, mostly from the year 1995, were also excluded. The causes of death were determined by clinical evidence, laboratory tests, imaging methods and surgical findings. In Brazil, by law, medico-legal autopsies are performed in all cases of sudden, suspicious or external-cause-related deaths, although there were no autopsy data analyses in this study.</p><p>Patients declared dead at the scene while under pre-hospital care were not transported to the hospital. Some patients had signs of life (SOL) in the ambulance or helicopter and were admitted to the trauma bay with traumatic cardiopulmonary arrest (no pulses, no electrical activity) and subsequently considered dead on arrival (DOA). These patients, except for those with stab wounds in the cardiac area, were not submitted to Emergency Department thoracotomy and were declared deceased.</p><p>The pre-hospital care system in the metropolitan area of Campinas is mature, and few patients are transported using their own means. Data regarding the pre-hospital care of all patients were obtained from all years since the year 2000.</p><p>For each patient admitted with SOL, a data register was created, containing the following information: age, gender, transport mode to hospital, pre-hospital time, time between admission and death (death less than 24 hours was called early death), length of stay, location of death inside the hospital, cause of injury, cause of death, surgical procedures performed, systolic blood pressure (SBP), respiratory rate (RR) and Glasgow Coma Scale (GCS) on admission. Severity of injury was determined using the trauma scores: Revised Trauma Score (RTS), Abbreviated Injury Scale (AIS), Injury Severity Score (ISS) and the probability of survival (TRISS).</p><p>Causes of death were classified as lesions to CNS, hemorrhagic shock (HS), sepsis and multiple organ dysfunction syndrome (MODS). The locations of death considered were DOA, emergency room, operative room, intensive care unit (ICU) and ward (in some cases there were not ICU beds available for all trauma patients).</p><p>The research protocol of this study was approved without restrictions by the Committee of Research Ethics and Institutional Review Board (IRB), School of Medical Sciences, Unicamp, protocol number 652/2011. The authors had complete access to patients’ charts and kept the database information anonymous.</p><p>Statistical analyses were performed using chi-square test, Fisher exact test, Mann–Whitney test and the Cochran-Armitage trend test. Analyses of univariate and multivariate logistic regressions were used to identify the risk factors of death within 24 hours of admission. P values of less than 0.05 were considered significant. All data were analyzed using the SAS system (Release 8.2, SAS Institute Inc).</p></sec><sec sec-type="results"><title>Results</title><p>From a total of 7,258 deaths registered in HC-Unicamp as a result of various causes and pathologies in the defined periods (years 1995, 2000, 2005 and 2010), 549 deaths (7.56% of the total) were caused by trauma. During these years, there was a relative and absolute decrease in the cases of traumatic deaths, falling from 240 deaths (9.2% of the total) in 1995 to 89 deaths (5.0% of the total) in 2010. Looking at the number of deaths from trauma in relation to the number of hospital admissions for trauma ICD, there was a decrease from 3.1% in 1995 to 1.4% in 2010. From a total of 549 deaths due to trauma, 82 were excluded (15.0%) due to incomplete data in patients charts.</p><p>467 cases (85.0% of the total sample) were studied, the vast majority being male (85.4%, n = 399). The average age of the patients was 35.35 ± 18.03 years, varying from 1 to 99 years of age (Table 
<xref ref-type="table" rid="T1">1</xref>). It was observed that the predominant age group was 25–39 years, with 163 patients (34.9%), followed by the age group 15–24 years (24.6%) (Figure 
<xref ref-type="fig" rid="F1">1</xref>).</p><table-wrap position="float" id="T1"><label>Table 1</label><caption><p>Study population characteristics (N = 467)</p></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="left"/><col align="left"/></colgroup><thead valign="top"><tr><th align="left"> </th><th align="left"><bold>Frequency (N and %)</bold></th><th align="left"><bold>Average ± SD</bold></th></tr></thead><tbody><tr><td align="left" valign="bottom">Age (years)<hr/></td><td align="left" valign="bottom"> <hr/></td><td align="left" valign="bottom"> <hr/></td></tr><tr><td align="left" valign="bottom">≤ 60<hr/></td><td align="left" valign="bottom">417 (91.3)<hr/></td><td rowspan="2" align="left" valign="top">35.35 ± 18.03<hr/></td></tr><tr><td align="left" valign="bottom">> 60<hr/></td><td align="left" valign="bottom">50 (10.7)<hr/></td></tr><tr><td align="left" valign="bottom">Gender<hr/></td><td align="left" valign="bottom"> <hr/></td><td rowspan="3" align="left" valign="top"> <hr/></td></tr><tr><td align="left" valign="bottom">Male<hr/></td><td align="left" valign="bottom">399 (85.4)<hr/></td></tr><tr><td align="left" valign="bottom">Female<hr/></td><td align="left" valign="bottom">68 (14.6)<hr/></td></tr><tr><td align="left" valign="bottom">Mechanism of injury<hr/></td><td align="left" valign="bottom"> <hr/></td><td rowspan="11" align="left" valign="top"> <hr/></td></tr><tr><td align="left" valign="bottom">Blunt<hr/></td><td align="left" valign="bottom">341 (73.0)<hr/></td></tr><tr><td align="left" valign="bottom">  - Running over<hr/></td><td align="left" valign="bottom">113 (24.2)<hr/></td></tr><tr><td align="left" valign="bottom">  - Motorcycle crash<hr/></td><td align="left" valign="bottom">70 (15.0)<hr/></td></tr><tr><td align="left" valign="bottom">  - Car crash<hr/></td><td align="left" valign="bottom">66 (14.2)<hr/></td></tr><tr><td align="left" valign="bottom">  - Fall<hr/></td><td align="left" valign="bottom">58 (12.4)<hr/></td></tr><tr><td align="left" valign="bottom">  - Bicycle<hr/></td><td align="left" valign="bottom">17 (3.6)<hr/></td></tr><tr><td align="left" valign="bottom">  - Assault<hr/></td><td align="left" valign="bottom">8 (1.7)<hr/></td></tr><tr><td align="left" valign="bottom">  - Others<hr/></td><td align="left" valign="bottom">9 (1.9)<hr/></td></tr><tr><td align="left" valign="bottom">Gunshot wound<hr/></td><td align="left" valign="bottom">108 (23.2)<hr/></td></tr><tr><td align="left" valign="bottom">Stab wound<hr/></td><td align="left" valign="bottom">18 (3.5)<hr/></td></tr><tr><td align="left" valign="bottom">Signs of life on arrival<hr/></td><td align="left" valign="bottom"> <hr/></td><td rowspan="3" align="left" valign="top"> <hr/></td></tr><tr><td align="left" valign="bottom">Yes<hr/></td><td align="left" valign="bottom">325 (69.6)<hr/></td></tr><tr><td align="left" valign="bottom">No (DOA)<hr/></td><td align="left" valign="bottom">142 (30.4)<hr/></td></tr><tr><td align="left" valign="bottom">Revised Trauma Score (RTS)<hr/></td><td align="left" valign="bottom"> <hr/></td><td align="left" valign="bottom">5.24 ± 2.05<hr/></td></tr><tr><td align="left">Injury Severity Score (ISS)</td><td align="left"> </td><td align="left">26.41 ± 9.00</td></tr></tbody></table></table-wrap><fig id="F1" position="float"><label>Figure 1</label><caption><p>Percentage of cases according to age group.</p></caption><graphic xlink:href="1471-227X-14-22-1"/></fig><p>The most common type of injury was blunt trauma with 341 cases (73.0%) (Table 
<xref ref-type="table" rid="T1">1</xref>), but there was no statistical difference between the mechanisms of trauma over the years (p = 0.181) (Figure 
<xref ref-type="fig" rid="F2">2</xref>).</p><fig id="F2" position="float"><label>Figure 2</label><caption><p>Types of trauma, in percentage, over the years.</p></caption><graphic xlink:href="1471-227X-14-22-2"/></fig><p>Of the study group, 142 (30.4%) were declared DOA and 325 cases (69.6%) were admitted with SOL to the emergency room. There is a statistical difference between the SOL and DOA groups over time, and a decrease in the DOA group (p < 0.001). Comparing variables between the SOL and DOA groups, there is no statistical difference between the groups for: age (lower in DOA, 32.31 ± 15.36 years, versus 36.68 ± 18.94 years, p = 0.031), method of pre-hospital transport (higher frequency of rescue and own means in DOA), type of trauma (higher frequency of penetrating in DOA), surgery performed (lower frequency in DOA), and cause of injury (higher frequency of motorcycles, bicycles and falls in SOL and of gunshots in DOA). Comparative analysis of the DOA and SOL groups showed that the age range ≤60 years was statistically significant (p = 0.019) in the DOA group.</p><p>Regarding the causes of trauma, in general, the most frequent were: PHBC with 113 cases (24.20%) and GSW with 108 cases (23.13%), but over the years there was a significant increase (p < 0.001) of motorcycle accidents and stab wounds (p = 0.030) in the total sample.</p><p>Of the 325 patients (69.6%) who were admitted with SOL to the emergency room, the mean ISS was 26.41 ± 9.00, with 93.1% (303 cases) having ISS greater than 15. The mean RTS was 5.24 ± 2.05. Regarding the body segments, the most affected (with AIS score greater than or equal to four) were: head with 222 cases (68.3%), followed by chest with 44 cases (13.5%) and abdomen with 39 cases (12.0%). The average TRISS in every sample year was 0.68 ± 0.33, and 74.1% (241cases) had TRISS ≥50. 29.2% of patients (95 cases) suffered multiple traumas (three or more body segments with affected AIS score), 60.3% (169) of patients were admitted with GCS less than or equal to 8, while 27.6% (90) of the patients were admitted with SBP less than 90 mmHg. The most common causes of death were: lesion of the CNS with 183 cases (56.3%), hemorrhagic shock (HS) with 59 cases (18.1%) and sepsis and MODS with 56 cases (17.1%) together. Patients who died of HS had the following body segments most affected: abdomen (32.8%), chest (28.4%) and pelvis (14.9%).</p><p>With respect to the time of death after hospital admission, considering the dead upon arrival, we found that 267 patients (57.18%) died within 24 hours of hospital admission. We performed a comparative analysis between the patients who died within 24 hours (early death) and those who didn’t. Statistically significant (p < 0.001) were the early deaths in penetrating injuries, SBP less than 90 mmHg on admission, chest and abdomen injury with AIS ≥ 4, hemorrhagic shock as cause of death and TRISS < 0.50. The CNS injury mortality peaked within 24–72 hours of hospital admission (33.9%) and the peak of death due to HS occurred between 6–24 hours (37.3%) and sepsis/MODS after 7 days (89.3%).</p><p>Tables 
<xref ref-type="table" rid="T2">2</xref> and
<xref ref-type="table" rid="T3">3</xref> present the results of analyses of univariate and multivariate logistic regression to study the risk factors associated with early death in the SOL group. From the results of the multivariate analysis with Stepwise criterion variable selection, it appears that the variables place of death, head injury, cause of death and avoidable death (TRISS) were associated significantly with early death in the SOL group. The patients at greatest risk of death within 24 hours were: those in the emergency room and operating room (risks of 4.6 and 14.2 times higher, respectively), those with head injury with AIS < 4 (7.1 times greater risk), those whose trauma causes were CNS injury/HS and acute respiratory failure (risks of 30.4 and 17.7 times, respectively), and those presenting avoidable death (TRISS <0.50) (risk of 3.3 times higher).</p><table-wrap position="float" id="T2"><label>Table 2</label><caption><p>Significant risk factors for early death (SOL) using univariate logistic regression analysis</p></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/></colgroup><thead valign="top"><tr><th align="center"><bold>Variable*</bold></th><th align="center"><bold>Categories</bold></th><th align="center"><bold>P-value</bold></th><th align="center"><bold>OR**</bold></th><th align="center"><bold>IC 95% OR</bold></th></tr></thead><tbody><tr><td rowspan="2" align="center" valign="top">Mechanism of Trauma<hr/></td><td align="center" valign="bottom">Blunt (ref.)<hr/></td><td align="center" valign="bottom">—<hr/></td><td align="center" valign="bottom">1.00<hr/></td><td align="center" valign="bottom">—<hr/></td></tr><tr><td align="center" valign="bottom">Penetrating<hr/></td><td align="center" valign="bottom"><0.001<hr/></td><td align="center" valign="bottom">3.20<hr/></td><td align="center" valign="bottom">1.81 – 5.67<hr/></td></tr><tr><td rowspan="4" align="center" valign="top">Cause of death<hr/></td><td align="center" valign="bottom">Sepsis/MODS (ref.)<hr/></td><td align="center" valign="bottom">—<hr/></td><td align="center" valign="bottom">1.00<hr/></td><td align="center" valign="bottom">—<hr/></td></tr><tr><td align="center" valign="bottom">CNS/Shock<hr/></td><td align="center" valign="bottom"><0.001<hr/></td><td align="center" valign="bottom">22.83<hr/></td><td align="center" valign="bottom">5.45 – 95.69<hr/></td></tr><tr><td align="center" valign="bottom">Respiratory failure<hr/></td><td align="center" valign="bottom">0.001<hr/></td><td align="center" valign="bottom">27.00<hr/></td><td align="center" valign="bottom">3.74 – 195.17<hr/></td></tr><tr><td align="center" valign="bottom">Others<hr/></td><td align="center" valign="bottom">0.003<hr/></td><td align="center" valign="bottom">18.00<hr/></td><td align="center" valign="bottom">2.71 – 119.79<hr/></td></tr><tr><td rowspan="4" align="center" valign="top">Place of death<hr/></td><td align="center" valign="bottom">Ward (ref.)<hr/></td><td align="center" valign="bottom">—<hr/></td><td align="center" valign="bottom">1.00<hr/></td><td align="center" valign="bottom">—<hr/></td></tr><tr><td align="center" valign="bottom">Emergency room<hr/></td><td align="center" valign="bottom"><0.001<hr/></td><td align="center" valign="bottom">5.96<hr/></td><td align="center" valign="bottom">3.18 – 11.18<hr/></td></tr><tr><td align="center" valign="bottom">Operating room<hr/></td><td align="center" valign="bottom"><0.001<hr/></td><td align="center" valign="bottom">150.04<hr/></td><td align="center" valign="bottom">8.91 – 2527.03<hr/></td></tr><tr><td align="center" valign="bottom">ICU<hr/></td><td align="center" valign="bottom">0.123<hr/></td><td align="center" valign="bottom">0.59<hr/></td><td align="center" valign="bottom">0.31 – 1.15<hr/></td></tr><tr><td rowspan="2" align="center" valign="top">PAS<hr/></td><td align="center" valign="bottom">> 89 (ref.)<hr/></td><td align="center" valign="bottom">—<hr/></td><td align="center" valign="bottom">1.00<hr/></td><td align="center" valign="bottom">—<hr/></td></tr><tr><td align="center" valign="bottom">≤ 89<hr/></td><td align="center" valign="bottom"><0.001<hr/></td><td align="center" valign="bottom">4.09<hr/></td><td align="center" valign="bottom">2.46 – 6.81<hr/></td></tr><tr><td rowspan="2" align="center" valign="top">Head Injury<hr/></td><td align="center" valign="bottom">< 4 (ref.)<hr/></td><td align="center" valign="bottom">—<hr/></td><td align="center" valign="bottom">1.00<hr/></td><td align="center" valign="bottom">—<hr/></td></tr><tr><td align="center" valign="bottom">≥4<hr/></td><td align="center" valign="bottom"><0.001<hr/></td><td align="center" valign="bottom">0.28<hr/></td><td align="center" valign="bottom">0.17 – 0.46<hr/></td></tr><tr><td rowspan="2" align="center" valign="top">Chest Injury<hr/></td><td align="center" valign="bottom">< 4 (ref.)<hr/></td><td align="center" valign="bottom">—<hr/></td><td align="center" valign="bottom">1.00<hr/></td><td align="center" valign="bottom">—<hr/></td></tr><tr><td align="center" valign="bottom">≥ 4<hr/></td><td align="center" valign="bottom"><0.001<hr/></td><td align="center" valign="bottom">4.20<hr/></td><td align="center" valign="bottom">2.12 – 8.29<hr/></td></tr><tr><td rowspan="2" align="center" valign="top">Abdomen Injury<hr/></td><td align="center" valign="bottom">< 4 (ref.)<hr/></td><td align="center" valign="bottom">—<hr/></td><td align="center" valign="bottom">1.00<hr/></td><td align="center" valign="bottom">—<hr/></td></tr><tr><td align="center" valign="bottom">≥ 4<hr/></td><td align="center" valign="bottom">0.006<hr/></td><td align="center" valign="bottom">2.59<hr/></td><td align="center" valign="bottom">1.31 – 5.13<hr/></td></tr><tr><td rowspan="2" align="center" valign="top">Avoidable death TRISS<hr/></td><td align="center" valign="bottom">≥ 0,50 (ref.)<hr/></td><td align="center" valign="bottom">—<hr/></td><td align="center" valign="bottom">1.00<hr/></td><td align="center" valign="bottom">—<hr/></td></tr><tr><td align="center" valign="bottom">< 0,5<hr/></td><td align="center" valign="bottom"><0.001<hr/></td><td align="center" valign="bottom">4.01<hr/></td><td align="center" valign="bottom">2.38 – 6.76<hr/></td></tr><tr><td align="center" valign="bottom">RTS<hr/></td><td align="center" valign="bottom">Continuous variable<hr/></td><td align="center" valign="bottom"><0.001<hr/></td><td align="center" valign="bottom">0.782<hr/></td><td align="center" valign="bottom">0.698 – 0.877<hr/></td></tr><tr><td align="center">TRISS</td><td align="center">Continuous variable</td><td align="center"><0.001</td><td align="center">0.155</td><td align="center">0.076 – 0.315</td></tr></tbody></table><table-wrap-foot><p>*No early death (n =200); With early death (n =125). Ref: reference level. **OR = Reason for risk of early death; IC 95% OR = 95% confidence interval for OR (Odds Ratio).</p></table-wrap-foot></table-wrap><table-wrap position="float" id="T3"><label>Table 3</label><caption><p>Multivariate logistic regression analysis for early death (SOL)</p></caption><table frame="hsides" rules="groups" border="1"><colgroup><col align="center"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/></colgroup><thead valign="top"><tr><th align="center"><bold>Selected variables*</bold></th><th align="center"><bold>Categories</bold></th><th align="center"><bold>P-value</bold></th><th align="center"><bold>OR**</bold></th><th align="center"><bold>IC 95% OR</bold></th></tr></thead><tbody><tr><td rowspan="4" align="left" valign="top">1. Place of death<hr/></td><td align="center" valign="bottom">Ward (ref.)<hr/></td><td align="center" valign="bottom">—<hr/></td><td align="center" valign="bottom">1.00<hr/></td><td align="center" valign="bottom">—<hr/></td></tr><tr><td align="center" valign="bottom">Emergency room<hr/></td><td align="center" valign="bottom"><0.001<hr/></td><td align="center" valign="bottom">4.64<hr/></td><td align="center" valign="bottom">2.25 – 9.54<hr/></td></tr><tr><td align="center" valign="bottom">Operating room<hr/></td><td align="center" valign="bottom"><0.001<hr/></td><td align="center" valign="bottom">14.19<hr/></td><td align="center" valign="bottom">3.00 – 67.16<hr/></td></tr><tr><td align="center" valign="bottom">ICU<hr/></td><td align="center" valign="bottom">0.168<hr/></td><td align="center" valign="bottom">0.59<hr/></td><td align="center" valign="bottom">0.28 – 1.25<hr/></td></tr><tr><td rowspan="2" align="left" valign="top">2. Head Injury<hr/></td><td align="center" valign="bottom">< 4 (ref.)<hr/></td><td align="center" valign="bottom">—<hr/></td><td align="center" valign="bottom">1.00<hr/></td><td align="center" valign="bottom">—<hr/></td></tr><tr><td align="center" valign="bottom">≥ 4<hr/></td><td align="center" valign="bottom"><0.001<hr/></td><td align="center" valign="bottom">0.14<hr/></td><td align="center" valign="bottom">0.07 – 0.29<hr/></td></tr><tr><td rowspan="4" align="left" valign="top">3. Cause of death<hr/></td><td align="center" valign="bottom">Sepsis/MODS (ref.)<hr/></td><td align="center" valign="bottom">—<hr/></td><td align="center" valign="bottom">1.00<hr/></td><td align="center" valign="bottom">—<hr/></td></tr><tr><td align="center" valign="bottom">CNS/Shock<hr/></td><td align="center" valign="bottom"><0.001<hr/></td><td align="center" valign="bottom">30.39<hr/></td><td align="center" valign="bottom">6.60 – 140.00<hr/></td></tr><tr><td align="center" valign="bottom">Respiratory failure<hr/></td><td align="center" valign="bottom">0.038<hr/></td><td align="center" valign="bottom">17.67<hr/></td><td align="center" valign="bottom">1.17 – 267.44<hr/></td></tr><tr><td align="center" valign="bottom">Others<hr/></td><td align="center" valign="bottom">0.101<hr/></td><td align="center" valign="bottom">6.58<hr/></td><td align="center" valign="bottom">0.70 – 62.22<hr/></td></tr><tr><td rowspan="2" align="left" valign="top">4. TRISS</td><td align="center" valign="bottom">≥ 0,50 (ref.)<hr/></td><td align="center" valign="bottom">—<hr/></td><td align="center" valign="bottom">1.00<hr/></td><td align="center" valign="bottom">—<hr/></td></tr><tr><td align="center">< 0,5</td><td align="center"><0.001</td><td align="center">3.31</td><td align="center">1.64 – 6.68</td></tr></tbody></table><table-wrap-foot><p>*No early death (n = 200); With early death (n = 125). Stepwise selection criterion variables. Ref: reference level. **OR = Reason for risk of early death; IC 95% OR = 95% confidence interval for OR (Odds Ratio).</p></table-wrap-foot></table-wrap><p>Regarding the location of death in the hospital, most patients died in the ICU or ward. It was observed that death from penetrating trauma was statistically significant in the operating room and death from blunt trauma in the ICU (p = 0.014). The average hospital stay was 9.68 ± 48.67 days.</p><p>In the years 2000, 2005 and 2010, from a total of 222 patients (47.53% of the total sample), the hospital received 89 patients (40.09%) brought in by EMS (rescue services) and 133 patients (59.90%) were brought in by non-rescue services (ambulances, transferred from other Campinas health services). The mean pre-hospital times in the “rescue services” group were 55.38 ± 38.43 minutes in 2000, 44.40 ± 30.86 minutes in 2005 and 46.00 ± 30.98 minutes in 2010. The mean pre-hospital times in the “ambulance” group were similar within the time series with an overall mean of 145.61 ± 165.70 minutes. If we compare the “rescue services” and “ambulance” groups, there were no statistical differences between the mechanisms of injury (blunt versus penetrating), causes of death (CNS injury/HS versus sepsis/MODS), severity scores (ISS ISS ≤ 15 versus ISS > 15), head injuries (AIS < 4 versus AIS ≥ 4) and causes of trauma.</p></sec><sec sec-type="discussion"><title>Discussion</title><p>This study not only reveals the complex scenario of trauma in a specific local region, but that trauma remains the leading cause of death in the younger population. We observed a decrease in the cases of traumatic deaths recorded in the hospital for various diseases during the years studied. Traffic accidents and violence accounted for about 9% of in-hospital deaths in 1995, falling to 5% in 2010. This also explains the decrease in the number of trauma admissions in HC-Unicamp by around 30% during the same period.</p><p>In this study, 66% of recorded deaths occurred in patients under 40 years of age with the predominant age range being 25–39 (34.9%). The vast majority of deaths were male (85.4%) which is also observed in literature
[<xref ref-type="bibr" rid="B21">21</xref>-<xref ref-type="bibr" rid="B23">23</xref>]. It can be noted that deaths caused by penetrating trauma occurred in younger individuals, but in the elderly, over 60 years of age, it was noted that almost all patients were victims of blunt trauma, mainly caused by road accidents. This also agrees with international literature
[<xref ref-type="bibr" rid="B6">6</xref>,<xref ref-type="bibr" rid="B15">15</xref>].</p><p>Regarding the causes of injury, there was a significant increase in deaths of motorcycle riders over time, increasing from 7.3% in 1995 to 31.5% in 2010. Carrasco and colleagues
[<xref ref-type="bibr" rid="B21">21</xref>] studied all fatal motorcycle crashes between 2001 and 2009 in Campinas, Brazil, and in 479 autopsies it was observed that the number of deaths from traffic accidents exceeded that of homicides and other external causes of death, and motorcycles play a significant role in these statistics. Gawryszewski and colleagues
[<xref ref-type="bibr" rid="B23">23</xref>], studying responses to land transport accidents in the State of São Paulo, noted that motorcycle accidents were the majority, representing 29.8% of cases, followed by automotive (25.7%) and pedestrians (24.1%). Marín-León and colleagues
[<xref ref-type="bibr" rid="B24">24</xref>], studying the trend of traffic accidents in Campinas, found an increase of 241% in the motorcycle fleet in just over a decade, and which represented nearly 50% of all fatal accidents on public roads in 2008. Reichenheim and colleagues
[<xref ref-type="bibr" rid="B25">25</xref>] observed that the proportion of deaths from motorcycles, within the total of traffic-related deaths in Brazil, rose from 4.1% in 1996 to 28.4% in 2007, showing an increase in risk of 820%. This study concluded that the increase in motorcycle deaths is due to the immense expansion of the fleet in the country, which doubled between 2001 and 2005.</p><p>The deaths considered as dead on arrival (DOA) accounted for about 30% of the total. We observed a significant decrease (p < 0.001) in the number of cases of DOA over time, which can be mainly explained by the decrease in the number of gunshot victims in Campinas, where the homicide rate fell from 46.2 per 100,000 in 2000 to 14.5 per 100,000 in 2010
[<xref ref-type="bibr" rid="B24">24</xref>]. The high homicide rates were explained by the strong associations between social disorganization, due to rapid urbanization since the 1990s, arms and drugs trafficking, illegal possession of weapons and police violence. In the last decade, there has been a decrease in mortality from homicide in southeastern Brazil, mainly due to economic growth associated with disarmament policies and public security
[<xref ref-type="bibr" rid="B25">25</xref>]. In the study on motorcycle deaths in Campinas, it was found that 50.3% of the victims died before receiving adequate medical attention
[<xref ref-type="bibr" rid="B21">21</xref>].</p><p>As for the times of death, the peak of late mortality was observed after 72 hours when 25% of the deaths occurred due to sepsis and MODS. The in-hospital deaths, which occurred within less than 24 hours, accounted for 57% of the sample. Comparing the data with the Demetriades
[<xref ref-type="bibr" rid="B15">15</xref>] study, there was a smaller number of deaths from 1 to 24 hours and a greater number after 72 hours. This may represent a lack of organized trauma system care in the region. The deaths that took place within 24 hours were statistically significant (p < 0.001) in patients with penetrating injury, SBP less than 90 mmHg on admission, AIS ≥4 in the chest and abdomen, HS as a cause of death and TRISS < 0.50. Patients at the greatest risk of death within 24 hours were: those in the local emergency room and operating room (risk 4.6 and 14.2 times higher, respectively), those with head injury with AIS < 4 (7.1 times greater risk), those with CNS injury/hemorrhage shock and acute respiratory failure (risks 30.4 and 17.7 times greater, respectively), and those with TRISS < 0.50 (3.3 times greater risk). As for the location of death, it is understandable that is higher in the OR because critical patients with HS are transferred to this area quickly. It was also high in the ED, compared with the ICU and ward.</p><p>Periodic assessment of time and place of traumatic deaths can provide a valuable assessment of a trauma system or trauma center
[<xref ref-type="bibr" rid="B15">15</xref>]. The time of traumatic deaths was described in 1977, during the development of the American trauma system. Baker and colleagues
[<xref ref-type="bibr" rid="B8">8</xref>] conducted a study of trauma deaths in the city of San Francisco during a period of one year when they analyzed 437 autopsies and described the classic trimodal distribution of deaths by trauma. The authors concluded that the deaths occurred during one of three peaks: immediately, early hospital (less than 48 hours) and late hospital (more than 48 hours). In 1992, Sauaia and colleagues
[<xref ref-type="bibr" rid="B9">9</xref>] reevaluated the study by the Baker group and concluded that there was no longer a trimodal distribution. There was a shift from immediate to early-hospital deaths that was attributed to the improvement of pre-hospital care. Between 1993 and 2002, Demetriades
[<xref ref-type="bibr" rid="B15">15</xref>] also showed that the trimodal distribution did not apply to his trauma system and that the improvement of pre-hospital emergency service in his region over the years increased the rate of admission of patients considered "in extremis" (extremely serious patients) arriving at the trauma center, but he also noticed the disappearance of the peak of late mortality caused by sepsis and MODS, as cited by the Baker study. This is primarily due to the improvements in pre-hospital, hospital and intensive care. The authors advocated that a more efficient emergency medical service could have been responsible for this change. It is known that cause of injury, age and injured body region are all factors having an influence on the temporal distribution of trauma deaths, and related statistics would be a reflection of improving care in each region, and of the capacities of their trauma systems. With regard to the location of death, 28.9% of patients died in the ward beds, 19.9% in the Intensive Care Unit (ICU), 15.2% in the emergency room and 5.5% in the operating room. This result does not coincide with existing literature, although the metropolitan region of Campinas is one of the most socio-economically developed in the country, and has a low percentage of deaths occurring post-24-hours in intensive care beds. The lack of beds in the ICU is due to a high demand of patients with diverse pathologies treated in a multidisciplinary hospital, chronic underfunding and a lack of specific beds for trauma patients.</p><p>With regard to the causes of death, injuries to the central nervous system resulting from traumatic brain injury were the most frequent at 56.3% of the total. Brain injuries also appear as the main cause of death in other publications with rates of between 21% and 71%
[<xref ref-type="bibr" rid="B21">21</xref>,<xref ref-type="bibr" rid="B26">26</xref>,<xref ref-type="bibr" rid="B27">27</xref>]. In this study, death by head trauma occurred mainly between 24 and 72 hours after trauma. HS was the second cause of death (18.1%), with a peak occurring between 6 and 24 hours. The areas most affected by bleeding were abdomen (32%), chest (28%) and pelvic region (10%). As reported in literature, some studies have shown that deaths from hemorrhage occur mainly in the first 6 hours after injury and others in up to 24 hours
[<xref ref-type="bibr" rid="B15">15</xref>,<xref ref-type="bibr" rid="B16">16</xref>,<xref ref-type="bibr" rid="B26">26</xref>]. Sepsis and MODS corresponded, respectively, to the third and fourth most frequent causes of death, totaling 17.1% and occurring 7 days after trauma. There was a significant increase (p = 0.023) of sepsis/MODS compared to CNS injury/HS over the years studied, and this caught our attention, since, in literature, there has been a decrease. This may be related to the lack of organization within the trauma care system, and consequently to a long pre-hospital time for patients transferred from other health services – the group called “ambulance”. About 70% of patients taken by pre-hospital emergency care – the “rescue services” group - died within 72 hours, which corroborates the thesis that death from sepsis/MODS was higher in the “ambulance” group. The most common cause of death in this group remains CNS injury at 30.1% (28 cases), followed by HS at 23.6% (22 cases) and sepsis/MODS at 11.8% (11 cases). This concurs with international literature
[<xref ref-type="bibr" rid="B14">14</xref>,<xref ref-type="bibr" rid="B16">16</xref>,<xref ref-type="bibr" rid="B21">21</xref>,<xref ref-type="bibr" rid="B27">27</xref>]. Comparing the causes and locations of death, the OR was the most common place for patients who died of HS in 30.5% of cases, and for those who died of CNS injury or sepsis/MODS, the ward beds were the most common places of death, at 44.8% and 60.7% respectively.</p><p>With respect to rates of trauma, the neurological level on admission showed a lower RTS, and 60.3% of patients admitted with signs of life (SOL) had a GCS less than or equal to eight. In this study, the RTS analysis resulted in a mean of 5.24 ± 2.05. The analysis of ISS resulted in a mean of 26.41 ± 9.00, having 71.3% with a value greater than 25 (considered very severe cases). The mean ISS amongst the blunt trauma cases was 26 and for the penetrating cases, 27. Other publications have described an average of 38–40 in cases of deaths by trauma
[<xref ref-type="bibr" rid="B28">28</xref>,<xref ref-type="bibr" rid="B29">29</xref>]. The lower average in our hospital may be explained by the fact that in most of the other publications, patients with isolated lesions were excluded, even if they were potentially fatal, and some studies took into consideration the rates of pre-hospital deaths, which are gathered by way of autopsies.</p><p>Of the 325 patients for which the trauma indices were calculated, in 135 (41.5%), injuries occurred in a single body segment, and only 91 patients (28.0%) had lesions in three or more segments. For the body area with AIS (Abbreviated Injury Score) greater than or equal to four, the head was the part most affected, with 222 cases (68.3%), followed by the chest with 44 cases (13.5%) and abdomen with 39 cases (12.0%). The TRISS results, showing an average of 0.68 ± 0.33 and with only 25.8% of patients showing TRISS < 0.50, revealed the limitation of this method in this sample, when analyzed in isolation. The initial objective of TRISS was to develop norms for the treatment of trauma that could be adopted in hospitals, in order to ensure a certain quality of care. Thus it would be then possible for hospitals to compare their results for groups of patients with similar severity, and identify, for further analysis, patients who died unexpectedly. The identification of these patients, whose results deviate from the established norm, allows assessment of preventable or possibly-preventable deaths, in an attempt to identify any eventual flaws in diagnosis, interpretation or application of techniques, motivating the medical staff for change in eventual conduct
[<xref ref-type="bibr" rid="B18">18</xref>].</p><p>Many studies have shown that an organized system of trauma care is essential in reducing mortality and sequelae resulting from trauma
[<xref ref-type="bibr" rid="B30">30</xref>-<xref ref-type="bibr" rid="B33">33</xref>]. Thus, measures ranging from prevention to the investment of resources in the health sector are necessary. Fraga
[<xref ref-type="bibr" rid="B18">18</xref>] points out that, in Brazil, there is not yet an organized system of trauma care with coverage of the different phases of care, and also that there are no epidemiological studies or trauma registries at the municipal, state and federal levels. There is little information regarding pre-hospital care, and a disintegration among hospitals of different complexities and the legal Medical Institute, leads to a lack of information which could be used for a comprehensive study on the reasons of death from external causes. Anyway, in our region, systems of trauma care are still undergoing maturation, requiring more research, education and financial resources, in order to reduce deaths from trauma.</p><p>This study has the limitation of analyzing the deaths in only one hospital in Campinas, and it doesn’t represent the total mortality for external causes in the city. Other limitations were: it was retrospective (15% of patients were excluded due to incomplete data) and no autopsy analyses were considered.</p></sec><sec sec-type="conclusions"><title>Conclusions</title><p>It is concluded that the deaths due to trauma occurred predominantly in young people and those of the male gender, and that penetrating trauma was more common among the young and blunt trauma the elderly, where being run over constituted the major cause of death. In this hospital there was a decrease in cases of traumatic deaths over the years, associated with the decline in the number of hospital visits for trauma and gunshot victims. There was a significant increase in deaths by motorcycle accident, which in 2010, became the leading cause of traumatic death. The deaths occurring within 24 hours were associated with penetrating injuries, SBP less than 90 mmHg on admission, AIS greater than or equal to four in the chest and abdomen, hemorrhagic shock and TRISS below 0.50. CNS injury remains the most common cause of death, and hemorrhagic shock was the second most frequent, especially of victims of penetrating trauma. A late mortality peak persists in the present study, and was caused by sepsis and MODS, which may be a reflection of a lack of organization and maturation within the trauma care system in which the HC-Unicamp finds itself.</p><sec><title>Key messages</title><p>• There was a decrease in cases of traumatic deaths over the years, associated with the decline in the number of hospital visits for trauma and gunshot victims.</p><p>• There was a significant increase in deaths by motorcycle accident, which in 2010 became the leading cause of traumatic death.</p></sec></sec><sec><title>Abbreviations</title><p>AIS: Abbreviated injury scale; CNS: Central nervous system; DOA: Dead on arrival; DCT: Division of trauma surgery; GCS: Glasgow coma scale; HC: Unicamp University Hospital of the University of Campinas; HS: Hemorrhagic shock; ICD: International classification of diseases; ICU: Intensive care unit; ISS: Injury severity score; MODS: Multiple organ dysfunction syndrome; RR: Respiratory rate; RTS: Revised trauma score; SOL: Admitted with signs of life; SPB: Systolic blood pressure; TRISS: Trauma score and injury severity score; WHO: World Health Organization.</p></sec><sec><title>Competing interests</title><p>The authors declare that they have no competing interests.</p></sec><sec><title>Authors' contributions</title><p>ADT participated in the acquisition, statistical analysis and interpretation of data, participated in the study’s design and drafted the manuscript. GPF conceived the study, and participated in its design and coordination and helped to draft the manuscript. BMP contributed to the study’s design and the revision of the manuscript. All authors read and approved the final manuscript.</p></sec><sec><title>Authors' information</title><p>ADT: Division of Trauma Surgery, Department of Surgery, School of Medical Sciences, University of Campinas (Unicamp), Campinas, São Paulo, Brazil.</p><p>GPF: Professor, Head, Division of Trauma Surgery, Department of Surgery, School of Medical Sciences, University of Campinas (Unicamp), Campinas, São Paulo, Brazil; and Postdoctoral Fellow at University of California, San Diego (UCSD).</p><p>BMP: Division of Trauma Surgery, Department of Surgery, School of Medical Sciences, University of Campinas (Unicamp), Campinas, São Paulo, Brazil.</p></sec><sec><title>Pre-publication history</title><p>The pre-publication history for this paper can be accessed here:</p><p><ext-link ext-link-type="uri" xlink:href="http://www.biomedcentral.com/1471-227X/14/22/prepub">http://www.biomedcentral.com/1471-227X/14/22/prepub</ext-link></p></sec> |
Frequency-Controls of Electromagnetic Multi-Beam Scanning by Metasurfaces | <p>We propose a method to control electromagnetic (EM) radiations by holographic metasurfaces, including to producing multi-beam scanning in one dimension (1D) and two dimensions (2D) with the change of frequency. The metasurfaces are composed of subwavelength metallic patches on grounded dielectric substrate. We present a combined theory of holography and leaky wave to realize the multi-beam radiations by exciting the surface interference patterns, which are generated by interference between the excitation source and required radiation waves. As the frequency changes, we show that the main lobes of EM radiation beams could accomplish 1D or 2D scans regularly by using the proposed holographic metasurfaces shaped with different interference patterns. This is the first time to realize 2D scans of antennas by changing the frequency. Full-wave simulations and experimental results validate the proposed theory and confirm the corresponding physical phenomena.</p> | <contrib contrib-type="author"><name><surname>Li</surname><given-names>Yun Bo</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Wan</surname><given-names>Xiang</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Cai</surname><given-names>Ben Geng</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Cheng</surname><given-names>Qiang</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Cui</surname><given-names>Tie Jun</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a1">1</xref></contrib><aff id="a1"><label>1</label><institution>State Key Laboratory of Millimeter Waves, School of Information Science and Engineering, Southeast University</institution>, Nanjing 210096, <country>China</country></aff> | Scientific Reports | <p>In recent years, it has been shown that extreme controls of electromagnetic (EM) waves can be achieved by using metasurfaces. Due to advantages of smaller physical space and less lossy structures than bulk metamaterial<xref ref-type="bibr" rid="b1">1</xref><xref ref-type="bibr" rid="b2">2</xref><xref ref-type="bibr" rid="b3">3</xref><xref ref-type="bibr" rid="b4">4</xref><xref ref-type="bibr" rid="b5">5</xref><xref ref-type="bibr" rid="b6">6</xref>, metasurfaces have found great potential applications in both microwave and optical frequencies. Similar to metamaterials, the metasurfaces are also modeled as sub-wavelength textures and several analysis methods have been established. Typically, the surface impedance or effective surface refractive index can be calculated analytically using the transverse resonance approach<xref ref-type="bibr" rid="b7">7</xref>, from which surface-wave waveguides<xref ref-type="bibr" rid="b8">8</xref> and lenses<xref ref-type="bibr" rid="b9">9</xref><xref ref-type="bibr" rid="b10">10</xref><xref ref-type="bibr" rid="b11">11</xref> have been designed. To reduce the computational time in eigen-mode simulations, a fast method was presented to calculate the surface impedance efficiently<xref ref-type="bibr" rid="b12">12</xref>. Later, the printed-circuit tensor impedance surface<xref ref-type="bibr" rid="b13">13</xref> has been analyzed using the modified transverse resonance technique and idealized tensor impedance boundary condition. Another method to model metasurface or metafilm is the generalized sheet transition condition<xref ref-type="bibr" rid="b14">14</xref>, in which the small scatterers are characterized as electric and magnetic polarization densities. Based on this method, reflection and transmission coefficients of metafilms or metasurfaces have been obtained<xref ref-type="bibr" rid="b15">15</xref><xref ref-type="bibr" rid="b16">16</xref>, promoting the study of meta-transmission arrays<xref ref-type="bibr" rid="b17">17</xref><xref ref-type="bibr" rid="b18">18</xref><xref ref-type="bibr" rid="b19">19</xref>. To control EM waves with more flexibility, the generalized Snell's law was proposed<xref ref-type="bibr" rid="b20">20</xref> by introducing the abrupt phases on the interface of the two media. Based on similar ideas, a gradient-index metasurface was presented to convert propagating waves to surface waves efficiently<xref ref-type="bibr" rid="b21">21</xref> and convert the circularly-polarized light to cross-polarized light<xref ref-type="bibr" rid="b22">22</xref><xref ref-type="bibr" rid="b23">23</xref>.</p><p>On the other hand, due to the low profile and low loss, metasurfaces have great advantages in the design of surface aperture antennas. Based on the original analysis of sinusoidally-modulated reactance surface<xref ref-type="bibr" rid="b24">24</xref>, the holographic antenna composed of surface impedance was proposed<xref ref-type="bibr" rid="b25">25</xref>, which shapes the monopole source as a pencil beam in the far-field region and changes the linearly-polarized source to circularly-polarized radiation by using anisotropic surface impedance units. Later, a spiral leaky-wave antenna<xref ref-type="bibr" rid="b26">26</xref> based on the modulated isotropic surface impedance implemented by corrugated dielectric or metallic patches on a grounded substrate can generate circularly polarized waves. Then the anisotropic tensor surface impedance formed by isotropic textures was used to realize circularly-polarized isoflux radiations<xref ref-type="bibr" rid="b27">27</xref> for space-to-ground data link applications in the X-band. In study of microwave imaging, a concept of metamaterial aperture has been presented for computational imaging<xref ref-type="bibr" rid="b28">28</xref><xref ref-type="bibr" rid="b29">29</xref>, in which the random-aperture leaky-wave antennas composed of complementary electric-inductor-capacitor elements have been used to generate multi- beam radiation patterns. The metasurface antennas can also be tunable<xref ref-type="bibr" rid="b30">30</xref><xref ref-type="bibr" rid="b31">31</xref> and conformal<xref ref-type="bibr" rid="b32">32</xref><xref ref-type="bibr" rid="b33">33</xref> so that they are more functional and practical in many engineering applications.</p><p>In this work, we propose to control extreme EM radiations by using holographic metasurfaces, which can radiate multiple beams with the frequency sweeping in one dimension (1D) or two dimensions (2D) without using the complicated beam-forming network<xref ref-type="bibr" rid="b34">34</xref> and anisotropic structures<xref ref-type="bibr" rid="b35">35</xref><xref ref-type="bibr" rid="b36">36</xref>. The proposed metasurfaces are composed of sub-wavelength quasi-periodic metallic patches on a grounded dielectric substrate, which can be shaped by mapping the variation of patch gaps to the surface impedance under the particular holographic interference. Once the interference pattern is recorded by the interference between reference waves and multi-beam radiations on the metasurface aperture, according to microwave holography theory, the metasurface excited by the reference waves generated by a monopole antenna can reproduce the multiple beams as we desire. We demonstrate that the radiation waves generated by the holographic metasurface can accomplish 2D scans regularly with the change of frequency, which has not been realized in the earlier holographic metasurfaces and leaky-wave antennas.</p><sec disp-level="1" sec-type="results"><title>Results</title><sec disp-level="2"><title>Theory and analysis</title><p>The surface impedance is defined as the ratio of electric to magnetic fields near a surface. Hence, the perfectly electric conducting (PEC) and perfectly magnetic conducting (PMC) surfaces are physically zero-impedance and high-impedance surfaces, respectively. We can design a metasurface by forming quasi-periodic gradient distribution of surface impedance. According to microwave holography theory, the desired radiation beam can be produced by using a reference wave to excite holographic interferogram, which is generated by the interference between the reference wave and radiation wave. The interferogram equation is given as<xref ref-type="bibr" rid="b37">37</xref>
<disp-formula id="m1"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e207" xlink:href="srep06921-m1.jpg"/></disp-formula>in which <italic>Ψ<sub>ref</sub></italic> is the reference wave and <italic>Ψ<sub>obj</sub></italic> is the object wave. To design the surface-aperture radiations, we use the distribution of surface impedance to embody the whole surface interferogram. We rewrite Eq. (1) to describe the distribution of surface impedance as <disp-formula id="m2"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e220" xlink:href="srep06921-m2.jpg"/></disp-formula>where <italic>X</italic> and <italic>M</italic> indicate the average value of surface impedance and the modulation depth, respectively, and “*” represents the conjugate operation. To build up the object wave (or radiation wave), we should use the reference wave to excite the interference pattern in determining <inline-formula id="m22"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e230" xlink:href="srep06921-m22.jpg"/></inline-formula>.</p><p>In our design, the basic unit of metasurface is a sub-wavelength metallic patch on a grounded substrate, as shown in <xref ref-type="fig" rid="f1">Fig. 1(a)</xref>. We extract the surface impedance of the metasurface via its dispersion curve. When the surface-wave vector <italic>k<sub>t</sub></italic>passes through a unit cell, the phase difference across the unit is <italic>ϕ</italic> = <italic>k<sub>t</sub>a</italic>, which can be acquired under eigen-mode simulations, where <italic>a</italic> is the period of the unit cell. Then the surface refractive index is given as <italic>n</italic> = <italic>c</italic>/<italic>v<sub>t</sub></italic> = <italic>k<sub>t</sub>c</italic>/<italic>ω<sub>t</sub></italic>, where <italic>c</italic> is the light speed in free space and <italic>ω<sub>t</sub></italic> is the surface angular frequency. Combining with the equation of surface impedance <italic>Z</italic> = <italic>jZ</italic><sub>0</sub><italic>k<sub>z</sub></italic>/<italic>k</italic> of the transverse-magnetic (TM) modes, we obtain the relation between the unit phase difference and surface impedance as <disp-formula id="m3"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e304" xlink:href="srep06921-m3.jpg"/></disp-formula>where <italic>Z</italic><sub>0</sub> is the impedance in free space. Therefore, once the unit phase difference is acquired through the eigen-mode simulation, we can calculate the surface impedance. The gap between square patches (see the orange area in <xref ref-type="fig" rid="f1">Fig. 1(a)</xref>) determines the value of surface impedance. When slowly varying the gap sizes to obtain different surface impedances, the structure is considered as quasi-periodic.</p><p>From the nine dispersion curves with changing gap sizes shown in <xref ref-type="fig" rid="f1">Fig. 1(b)</xref>, we calculate nine values of surface impedance at 17 GHz, from which the following equation is fitted using cubic polynomials to describe the relationship between surface impedance and gap size: <disp-formula id="m4"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e321" xlink:href="srep06921-m4.jpg"/></disp-formula>Then we determine the values of <italic>X</italic> and <italic>M</italic> as 197.5 and 36.5, respectively, from the simulated values of surface impedance.</p><p>According to the holographic antenna theory, once the interference pattern is recorded by the interaction between reference wave and multi-beam radiation wave on the metasurface, we can reconstruct the multiple beams as we desire. We choose a monopole antenna placed in the center of metasurface to produce the reference wave, which is written as <disp-formula id="m5"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e332" xlink:href="srep06921-m5.jpg"/></disp-formula>where <italic>n</italic> is the effective surface refractive index and <italic>r</italic> is the distance from the origin to the surface radiation unit. For double-beam radiation, the object wave (i.e. the desired radiation wave) can be defined as <disp-formula id="m6"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e341" xlink:href="srep06921-m6.jpg"/></disp-formula>in which <inline-formula id="m23"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e344" xlink:href="srep06921-m23.jpg"/></inline-formula> and <inline-formula id="m24"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e348" xlink:href="srep06921-m24.jpg"/></inline-formula> are wave-number vectors of the two beams with the elevation and azimuth angles <italic>θ</italic><sub>1/2</sub> and <italic>ϕ</italic><sub>1/2</sub>, respectively, and <inline-formula id="m25"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e361" xlink:href="srep06921-m25.jpg"/></inline-formula> is the spatial location vector. In particular, when <italic>θ</italic><sub>1</sub> = <italic>θ</italic><sub>2</sub> = 30° and <italic>φ</italic><sub>1</sub> = 45°, <italic>φ</italic><sub>2</sub> = 135°, Eq. (6) is rewritten as <disp-formula id="m7"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e386" xlink:href="srep06921-m7.jpg"/></disp-formula>in which the positive and negative exponential terms indicate the radiation waves propagating to the two sides of the normal direction on the metasurface. To obtain the double-beam radiation shaped by Eq. (7), the interferogram generated by Eq. (2) should be excited by the reference wave defined in Eq. (5).</p><p>We will show later that the proposed holographic metasurface is equivalent to the 2D leaky-wave structure. To radiate EM waves more efficiently, we define the “-1” order leaky-wave radiation without other Floquet modes participating the interference by synthetically considering the unit design and radiation frequency. In fact, the forward and backward waves in leaky waves mean that the radiation directions are uniform and non-uniform with the propagation of surface waves. If we place the monopole antenna in the center of metasurface as excitation, the full-wave simulation results reveal that the leaky-wave (object-wave) radiation pattern may generate the sag like “rabbit's ears” in the far fields, as shown in <xref ref-type="fig" rid="f2">Fig. 2(a)</xref>. This phenomenon is caused by the slightly radiating deviation of forward and backward modes, which is attributed to the inaccuracy of effective surface refractive index that determines the distribution of surface currents. The surface currents excited by the monopole antenna will not be completely the same as the reference wave so that the forward and backward waves have different radiation directions, as illustrated in <xref ref-type="fig" rid="f2">Fig. 2(a)</xref>.</p><p>The optimization of surface refractive index <italic>n</italic> can avoid the “rabbit's ear” phenomenon by enforcing <italic>θ</italic><sub>1</sub> = <italic>θ</italic><sub>2</sub> in <xref ref-type="fig" rid="f2">Fig. 2(a)</xref>, but it is more complicated. Here, we present a side-feeding method, as shown in <xref ref-type="fig" rid="f2">Fig. 2(b)</xref>. The interferogram shaped by the holographic metasurface is generated by Eq. (2), which is combined with Eq. (4) to determine the relation to describe the gap size versus position on the metasurface. <xref ref-type="fig" rid="f3">Fig. 3(a)</xref> illustrates the interferogram generated by the interference between double-beam radiation (Eq. (7)) and reference (Eq. (5)) waves at 17 GHz. Based on the full-wave simulations by commercial software, CST Microwave Studio, the reproduced waves are demonstrated in <xref ref-type="fig" rid="f3">Fig. 3(c)</xref>. We also show that the double-beam radiations can make 1D scan with the change of frequency (16–18 GHz), as clearly observed in <xref ref-type="fig" rid="f3">Figs. 3(b) – (d)</xref>.</p><p>To explain the phenomenon of frequency scanning by the 2D metasurface, we now show the equivalence of the holographic reproduction and “-1”-order leaky-wave radiation. For simplicity, we analyze a single-beam radiation defined by <italic>Ψ<sub>obj</sub></italic> = <italic>e</italic><sup>−<italic>jkx</italic>sin(<italic>θ</italic>)</sup>, which will interference with reference wave <italic>Ψ<sub>ref</sub></italic> = <italic>e<sup>−jknx</sup></italic> to generate the interferogram as <disp-formula id="m8"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e456" xlink:href="srep06921-m8.jpg"/></disp-formula>To obtain periodicity of the holographic distribution of surface impedance along the <italic>x</italic> direction, we define <italic>k</italic><sub>0</sub><italic>nx</italic>-<italic>k</italic><sub>0</sub><italic>x</italic> sin(<italic>θ</italic>) = 2<italic>π</italic>, and the corresponding period is determined as <disp-formula id="m9"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e484" xlink:href="srep06921-m9.jpg"/></disp-formula>which can be rewritten as <disp-formula id="m10"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e487" xlink:href="srep06921-m10.jpg"/></disp-formula>The above result is exactly the same as that in the “-1”-order leaky-wave radiation<xref ref-type="bibr" rid="b12">12</xref>. Here, <italic>θ</italic> is the radiation direction of the holographic reproduction. It is obvious that the object wave can accomplish the beam scanning controlled by frequency, and the corresponding radiation direction is given as <disp-formula id="m11"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e495" xlink:href="srep06921-m11.jpg"/></disp-formula>where <italic>k</italic>′, <italic>n</italic>′ and <italic>θ</italic>′ are the same as those in Eq. (8) but under different operating frequencies.</p><p>We introduce the concept of surface-wave phase gradient to explain the above phenomenon. For simplicity, we still analyze the single-beam radiation defined by <inline-formula id="m26"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e510" xlink:href="srep06921-m26.jpg"/></inline-formula> and <inline-formula id="m27"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e513" xlink:href="srep06921-m27.jpg"/></inline-formula>, which generate the interferogram as <disp-formula id="m12"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e516" xlink:href="srep06921-m12.jpg"/></disp-formula>Here, we assume that the sinusoidal phase distribution (<inline-formula id="m28"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e519" xlink:href="srep06921-m28.jpg"/></inline-formula>) of interferogram generated by the impedance units is approximately invariant by changing frequencies since the higher- and lower-impedance areas of the interferogram are fixed. Hence we only need to concern the object wave term in processing the holographic reproduction. If we excite the interferogram by reference wave defined as <inline-formula id="m29"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e522" xlink:href="srep06921-m29.jpg"/></inline-formula>, where <italic>k</italic>′ and <italic>n</italic>′are the wave number in free space and effective refractive index, then the phase of surface-wave front <inline-formula id="m30"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e532" xlink:href="srep06921-m30.jpg"/></inline-formula> becomes Φ = <italic>k</italic>′<italic>n</italic>′<italic>r</italic>−<italic>k</italic><sub>0</sub><italic>nr</italic> + sin(<italic>θ</italic>)<italic>k</italic><sub>0</sub>[<italic>x</italic> cos(<italic>φ</italic>) + <italic>y</italic> sin(<italic>φ</italic>)]. By calculations, the surface-wave phase gradient ∇Φ is given as <disp-formula id="m13"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e573" xlink:href="srep06921-m13.jpg"/></disp-formula>where <inline-formula id="m31"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e576" xlink:href="srep06921-m31.jpg"/></inline-formula>, <inline-formula id="m32"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e579" xlink:href="srep06921-m32.jpg"/></inline-formula> and <inline-formula id="m33"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e583" xlink:href="srep06921-m33.jpg"/></inline-formula> are unit vectors. Hence the surface wave front is generated by the interference of two surface waves <inline-formula id="m34"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e586" xlink:href="srep06921-m34.jpg"/></inline-formula> and <inline-formula id="m35"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e589" xlink:href="srep06921-m35.jpg"/></inline-formula>, and the phase of wave front in the <italic>x</italic> direction is written as <disp-formula id="m14"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e595" xlink:href="srep06921-m14.jpg"/></disp-formula>where <italic>φ</italic> is the angle between <italic>r</italic> and <italic>x</italic> directions. Thus, the <italic>x</italic> component of effective surface wave number at the particular frequency is <disp-formula id="m15"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e611" xlink:href="srep06921-m15.jpg"/></disp-formula>which is corresponding to the wave number of the “-1” order leaky-wave. Likewise, the <italic>y</italic> component of effective surface wave number at the particular frequency is <disp-formula id="m16"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e617" xlink:href="srep06921-m16.jpg"/></disp-formula>Thus, the amplitude and azimuth angle of the effective surface wave number are given as <disp-formula id="m17"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e621" xlink:href="srep06921-m17.jpg"/></disp-formula><disp-formula id="m18"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e623" xlink:href="srep06921-m18.jpg"/></disp-formula></p><p>As a consequence, the elevation angle of the main radiation direction is expressed as <disp-formula id="m19"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e627" xlink:href="srep06921-m19.jpg"/></disp-formula>According to Eq. (18), if we change the object wave as <inline-formula id="m36"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e630" xlink:href="srep06921-m36.jpg"/></inline-formula> (<italic>φ</italic> = 45°; the interferogram is defined in the area of x>0 and y>0), in which “+” and “−” represent the backward and forward modes of leaky waves, we find that the azimuth angle of main radiation direction will be unchanged with frequencies under the circumstance of <inline-formula id="m37"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e636" xlink:href="srep06921-m37.jpg"/></inline-formula>, which can accomplish 1D scanning controlled by frequency, as shown in the simulation results in <xref ref-type="fig" rid="f3">Fig. 3</xref>.</p><p>This method can be generalized to produce multi-beam radiations by recording interferograms in several subareas, each of which radiates a single beam. For instance, in four-beam radiations, the overall interferogram is generated by mirroring the metasurface pattern in the first quadrant to other quadrants. The object wave is then given as <disp-formula id="m20"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e644" xlink:href="srep06921-m20.jpg"/></disp-formula>in which we determine <italic>φ</italic> = 45° to satisfy the condition of <inline-formula id="m38"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e650" xlink:href="srep06921-m38.jpg"/></inline-formula>, and the azimuth angles of the desired four beams are 45°, 135°, 225°, and 315°, respectively. When we set <italic>θ</italic> = 45°, the corresponding interferograms and simulation results for forward and backward modes are presented in <xref ref-type="fig" rid="f4">Figs. 4(a) and (b)</xref>. Although the feeding point is located in the center of the overall metasurface, for each radiation beam, it is the side feeding in the subarea. Hence the earlier “rabbit's ears” phenomena are avoided. From <xref ref-type="fig" rid="f4">Fig. 4</xref>, we notice that the four-beam radiations in the backward mode are scanned in 1D direction with increasing elevation angles as the frequency becomes larger; while the four beams in the forward mode are scanned with decreasing elevation angles.</p><p>From Eq. (18) and (19), if <inline-formula id="m39"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e666" xlink:href="srep06921-m39.jpg"/></inline-formula>, we can realize extreme controls of the multi-beam radiations, in which the azimuth angle (<italic>ϕ</italic>′) and elevation angle (<italic>θ</italic>′) of the leaky waves can be simultaneously scanned with the change of frequency. In this case, the object wave with four-beam radiations is defined as <disp-formula id="m21"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e675" xlink:href="srep06921-m21.jpg"/></disp-formula>in which we determine <italic>φ</italic> = 135°and <italic>θ</italic> = 45° to satisfied the condition <inline-formula id="m40"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e685" xlink:href="srep06921-m40.jpg"/></inline-formula>. The corresponding interferogram and simulation results of the four-beam radiations with 2D frequency scanning are demonstrated in <xref ref-type="fig" rid="f5">Fig. 5</xref>. We clearly observe that the four-beam radiation patterns are scanned in both azimuth and elevation directions as the operating frequency changes. The detailed scanning properties are summarized in <xref ref-type="table" rid="t1">Table 1</xref>, from which we notice that the simulation results have good agreements to the theoretical analysis. The little deviation may be caused by the inaccuracy of effective surface refractive index and approximate condition of sinusoidal phase distribution under different frequencies. Compared to the conventional holographic leaky-wave radiations that can only accomplish 1D frequency scanning, the proposed method and holographic metasurface have greatly enhanced the capabilities to reach 2D frequency scanning.</p></sec><sec disp-level="2"><title>Fabrication and Measurement</title><p>To validate the proposed method experimentally, we fabricate a sample of the holographic leaky-wave metasurface shown in <xref ref-type="fig" rid="f3">Fig. 3</xref> to show the frequency scanning properties of two beams. The sample of the double-beam metasurface has a dimension of 240 × 240 mm<sup>2</sup>, containing 6400 unit cells. In fabrication, we choose commercial printed circuit board (FR4) as the dielectric substrate and choose copper with tinning as the ground. The experimental setup in anechoic chamber to measure the far fields is illustrated in <xref ref-type="fig" rid="f6">Fig. 6(a)</xref>, and the measured far-field radiation patterns with the 1D frequency scanning in azimuth direction of the double-beam holographic metasurface are demonstrated in <xref ref-type="fig" rid="f6">Fig. 6(b)</xref>.</p><p>The detailed double-beam scanning parameters of the holographic metasurface from theoretical analysis, simulation and experimental results are presented in <xref ref-type="table" rid="t2">Table 2</xref>. We clearly observe that the experimental results have good agreements to numerical simulation and theoretical predictions. We do not measure the four-beam radiations with 2D frequency scanning due to the much complicated experimental process based on our current measurement system, but the simulation results in <xref ref-type="fig" rid="f5">Fig. 5</xref> and <xref ref-type="table" rid="t1">Table 1</xref> have verified the good performance to make extreme controls of EM radiations only by frequency.</p></sec></sec><sec disp-level="1" sec-type="discussion"><title>Discussion</title><p>We proposed an efficient method to design holographic leaky-wave metasurfaces to perform complicated multi-beam radiations with frequency scanning. We presented two approaches to record the desired interferograms, one of which is recorded on the whole metasurface with side feeding, and the other of which is recorded on several subdomains of the metasurface with central feeding. According to theoretical analysis, the first approach can accomplish 1D frequency scanning, while the second can reach 1D and/or 2D frequency scanning. Numerical simulation and experiment results show that the radiation beam directions in the far fields can be coded by frequency instead of complicated feeding network. The proposed method has potential applications in the satellite communications and radar systems, and can be extended to the millimeter wave and THz regimes.</p></sec><sec disp-level="1"><title>Author Contributions</title><p>Y.B.L. and T.J.C. conceived the idea, did the theoretical calculations, and wrote the manuscript. Y.B. Li designed the samples and performed the measurements. X.W., B.G.C. and Q.C. involved in the simulations and measurement. All authors contributed to the discussions.</p></sec> |
Level Anticrossing of Impurity States in Semiconductor Nanocrystals | <p>The size dependence of the quantized energies of elementary excitations is an essential feature of quantum nanostructures, underlying most of their applications in science and technology. Here we report on a fundamental property of impurity states in semiconductor nanocrystals that appears to have been overlooked—the anticrossing of energy levels exhibiting different size dependencies. We show that this property is inherent to the energy spectra of charge carriers whose spatial motion is simultaneously affected by the Coulomb potential of the impurity ion and the confining potential of the nanocrystal. The coupling of impurity states, which leads to the anticrossing, can be induced by interactions with elementary excitations residing inside the nanocrystal or an external electromagnetic field. We formulate physical conditions that allow a straightforward interpretation of level anticrossings in the nanocrystal energy spectrum and an accurate estimation of the states' coupling strength.</p> | <contrib contrib-type="author"><name><surname>Baimuratov</surname><given-names>Anvar S.</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Rukhlenko</surname><given-names>Ivan D.</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Turkov</surname><given-names>Vadim K.</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Ponomareva</surname><given-names>Irina O.</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Leonov</surname><given-names>Mikhail Yu.</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Perova</surname><given-names>Tatiana S.</given-names></name><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Berwick</surname><given-names>Kevin</given-names></name><xref ref-type="aff" rid="a4">4</xref></contrib><contrib contrib-type="author"><name><surname>Baranov</surname><given-names>Alexander V.</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Fedorov</surname><given-names>Anatoly V.</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><aff id="a1"><label>1</label><institution>ITMO University</institution>, 49 Kronverksky Avenue, 197101 Saint Petersburg, <country>Russia</country></aff><aff id="a2"><label>2</label><institution>Monash University, Clayton Campus</institution>, Victoria 3800, <country>Australia</country></aff><aff id="a3"><label>3</label><institution>Trinity College Dublin, The University of Dublin</institution>, Dublin 2, <country>Ireland</country></aff><aff id="a4"><label>4</label><institution>Dublin Institute of Technology</institution>, Dublin 8, <country>Ireland</country></aff> | Scientific Reports | <p>Colloidal semiconductor nanocrystals, also known as quantum dots, have unique optical and electronic properties that are controlled by their size, shape and chemical composition<xref ref-type="bibr" rid="b1">1</xref><xref ref-type="bibr" rid="b2">2</xref><xref ref-type="bibr" rid="b3">3</xref><xref ref-type="bibr" rid="b4">4</xref><xref ref-type="bibr" rid="b5">5</xref>. Doping of nanocrystals with impurity atoms or ions is another method of tuning their physical properties, giving rise to many useful optical phenomena<xref ref-type="bibr" rid="b6">6</xref><xref ref-type="bibr" rid="b7">7</xref><xref ref-type="bibr" rid="b8">8</xref><xref ref-type="bibr" rid="b9">9</xref>. The presence of various impurities in semiconductor nanocrystals is responsible <italic>e.g.</italic> for the Stark effect<xref ref-type="bibr" rid="b10">10</xref>, the spin Hall effect<xref ref-type="bibr" rid="b11">11</xref>, the Kondo effect<xref ref-type="bibr" rid="b12">12</xref>, giant Zeeman splitting<xref ref-type="bibr" rid="b13">13</xref>, and light-induced spontaneous magnetization<xref ref-type="bibr" rid="b14">14</xref>. This provides the basis for applications of semiconductor nanocrystals in nanodevices such as transistors<xref ref-type="bibr" rid="b15">15</xref><xref ref-type="bibr" rid="b16">16</xref>, light-emitting diodes<xref ref-type="bibr" rid="b17">17</xref>, bioimaging labels<xref ref-type="bibr" rid="b18">18</xref>, chemical sensors<xref ref-type="bibr" rid="b19">19</xref>, solar cells<xref ref-type="bibr" rid="b20">20</xref> and spintronic devices<xref ref-type="bibr" rid="b21">21</xref>.</p><p>There are several methods of doping nanocrystals based on two different fabrication strategies: nucleation doping and growth doping<xref ref-type="bibr" rid="b22">22</xref><xref ref-type="bibr" rid="b23">23</xref>. Nucleation doping is realized by mixing the dopant and host precursor during nanocrystal formation<xref ref-type="bibr" rid="b24">24</xref>, whereas the growth-doping strategy requires absorption of dopant precursors by the nanocrystal surface, followed by encapsulation of the precursors through overgrowth of an isocrystalline or heterocrystalline shell. The radial position of impurities inside a semiconductor nanocrystal can be controlled by adding the precursors at different stages of the nanocrystal formation<xref ref-type="bibr" rid="b25">25</xref>. This control can be performed with angstrom precision, enabling extremely fine tuning of the optical and magnetic properties of the nanocrystals<xref ref-type="bibr" rid="b26">26</xref>.</p><p>Nanocrystal impurities are studied theoretically using two major approaches, based on the effective mass approximation. The first approach, relying on an exact solution of the Schrödinger equation for a given confining potential, yields the impurity's wave functions and energy spectrum in quadratures<xref ref-type="bibr" rid="b27">27</xref><xref ref-type="bibr" rid="b28">28</xref><xref ref-type="bibr" rid="b29">29</xref>. The second technique, employing the variational method, allows one to find an approximate solution to the Schrödinger equation as a linear sum of the exact wave functions corresponding to the nanocrystals with and without an impurity<xref ref-type="bibr" rid="b30">30</xref><xref ref-type="bibr" rid="b31">31</xref>. Most of the theoretical work based on the effective mass approximation is devoted to studies of size dependencies of the energy levels and binding energies of impurity states in simple nanocrystals<xref ref-type="bibr" rid="b27">27</xref><xref ref-type="bibr" rid="b29">29</xref><xref ref-type="bibr" rid="b32">32</xref>. A few were focused on modifications of energy spectrum by external electric and magnetic fields<xref ref-type="bibr" rid="b33">33</xref><xref ref-type="bibr" rid="b34">34</xref>, multilayer nanocrystals with different numbers of shells<xref ref-type="bibr" rid="b35">35</xref><xref ref-type="bibr" rid="b36">36</xref>, and nanocrystals with off-center impurities<xref ref-type="bibr" rid="b37">37</xref><xref ref-type="bibr" rid="b38">38</xref>.</p><p>Since most of the optical properties of doped nanostructures strongly depend on their size, the study of the size dependencies of nanostructure impurity levels is a problem of both fundamental and applied significance. Precise, yet rather complex and time-consuming methods of single quantum-dot spectroscopy<xref ref-type="bibr" rid="b39">39</xref><xref ref-type="bibr" rid="b40">40</xref><xref ref-type="bibr" rid="b41">41</xref> allows one to resolve this problem experimentally. In this paper, we theoretically show that the simultaneous exposure of charge carriers to the Coulomb and confining potentials results in two different contributions to the size dependence of the nanocrystal energy spectrum. The confining potential ceases to determine the structure of the spectrum with an increase of the nanocrystal size while the Coulomb potential starts to dominate. As a consequence, the energies of some impurity states can accidentally coincide in nanocrystals of a particular size. By examining the ordering of the energy levels for two limiting cases of small and large spherical nanocrystals, we show that this accidental degeneracy is inherent to doped nanocrystals regardless of their shape and composition. The degeneracy is removed by any interaction between the degenerate states, leading to the anticrossings in the size dependencies of the respective energy levels. Using an illustrative example of electron–phonon interaction, we show how the accidental degeneracy is removed and identify which anticrossings are easiest to interpret experimentally. We also discuss which anticrossings are convenient to use for the estimation of interaction strengths in semiconductor nanocrystals.</p><sec disp-level="1" sec-type="results"><title>Results</title><sec disp-level="2"><title>Energy spectrum of hydrogenic impurity</title><p>Consider a donor hydrogenic impurity in a spherical semiconductor nanocrystal of radius <italic>R</italic>, as shown in <xref ref-type="fig" rid="f1">Fig. 1(a)</xref>. The impurity comprises a positive ion of charge <italic>Ze</italic>, which is located at the nanocrystal center and coupled through the Coulomb interaction to an electron of charge −<italic>e</italic> and effective mass <italic>m<sub>e</sub></italic>. By assuming that the nanocrystal boundary is impenetrable to the electron, one can find the wave functions and energy spectrum of the impurity from the Schrödinger equation <disp-formula id="m1"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e298" xlink:href="srep06917-m1.jpg"/></disp-formula>in which the confining potential is given by <disp-formula id="m2"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e301" xlink:href="srep06917-m2.jpg"/></disp-formula>with <italic>α</italic> = <italic>Ze</italic><sup>2</sup>/<italic>ε</italic><sub>0</sub> and <italic>ε</italic><sub>0</sub> being the low-frequency permittivity of the nanocrystal.</p><p>Owing to the spherical symmetry of the confining potential, <xref ref-type="disp-formula" rid="m1">Eq. (1)</xref> allows an analytical solution<xref ref-type="bibr" rid="b27">27</xref><xref ref-type="bibr" rid="b28">28</xref><xref ref-type="bibr" rid="b29">29</xref> (see Methods), which enables a simple analysis of the absorption and photoluminescence spectra of nanocrystals with donor impurities<xref ref-type="bibr" rid="b42">42</xref><xref ref-type="bibr" rid="b43">43</xref><xref ref-type="bibr" rid="b44">44</xref><xref ref-type="bibr" rid="b45">45</xref><xref ref-type="bibr" rid="b46">46</xref>. It is possible, in particular, to estimate the nanocrystal size by finding the number of absorption peaks lying below the fundamental absorption band. The peaks are centered at the energies of the lowest states in the nanocrystal energy spectrum <italic>E<sub>n,l</sub></italic>, corresponding to different principal quantum numbers and angular momenta, <italic>n</italic> = 1, 2, 3, … and <italic>l</italic> = 0, 1, 2, … A more practical application is the problem of calculating the size of a nanocrystal with a certain number of impurity states within the band gap. For instance, a nanocrystal of radius <italic>R</italic> = 9 <italic>r<sub>B</sub></italic>, where <italic>r<sub>B</sub></italic> = ħ<sup>2</sup>/(<italic>m<sub>e</sub>α</italic>) is the effective Bohr radius, has five impurity states with negative energies <italic>E</italic><sub>1,0</sub>, <italic>E</italic><sub>1,1</sub>, and <italic>E</italic><sub>2,0</sub> (the state with <italic>l</italic> = 1 is three-fold degenerate), whereas the energies of the rest of its states are positive [see <xref ref-type="fig" rid="f1">Fig. 1(b)</xref>].</p><p>To qualitatively understand how the energies and ordering of impurity states depend on the nanocrystal size, we note that the impurity spectrum transforms into the spectrum of an infinitely high potential well without an impurity when <italic>α</italic> = 0, and becomes similar to the hydrogen-like spectrum for <inline-formula id="m18"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e394" xlink:href="srep06917-m18.jpg"/></inline-formula>. The ordering of energy levels <inline-formula id="m19"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e397" xlink:href="srep06917-m19.jpg"/></inline-formula> in the first case, illustrated by <xref ref-type="fig" rid="f1">Fig. 1(c)</xref>, is determined by the zeros <italic>ξ<sub>n,l</sub></italic> of the spherical Bessel functions of the first kind <italic>j<sub>l</sub></italic>(<italic>z</italic>). The hydrogen-like spectrum, shown in <xref ref-type="fig" rid="f1">Fig. 1(d)</xref>, consists of negative- and positive-energy domains<xref ref-type="bibr" rid="b47">47</xref>: the negative-energy spectrum <inline-formula id="m20"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e422" xlink:href="srep06917-m20.jpg"/></inline-formula>, where Ry = <italic>m<sub>e</sub>α</italic><sup>2</sup>/(2ħ<sup>2</sup>) is the effective Rydberg, is discrete; and the positive-energy spectrum <italic>E</italic><sup>(<italic>i</italic>)</sup>(<bold>k</bold>) = Ry <italic>k</italic><sup>2</sup> (<italic>k</italic> > 0) is continuous.</p><p>These limiting cases show that impurity centers should exhibit discrete energy spectra which may extend to negative energies. The negative-energy states are absent in small nanocrystals, emerging one by one and growing in numbers with the nanocrystal radius. The emergence is caused by the weakening of spatial confinement and the reduction of kinetic energy of the impurity electron, and its resulting distancing from the ion<xref ref-type="bibr" rid="b29">29</xref>. The descent of quantum state (<italic>n, l</italic>) from the positive-energy domain to the negative-energy domain occurs when its energy passes through zero in the nanocrystal of radius <disp-formula id="m3"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e464" xlink:href="srep06917-m3.jpg"/></disp-formula>where <italic>ζ<sub>n</sub></italic><sub>,2<italic>l</italic>+1</sub> is the <italic>n</italic>th zero of the cylindrical Bessel function of the first kind <italic>J</italic><sub>2<italic>l</italic>+1</sub>(<italic>x</italic>).</p><p><xref ref-type="fig" rid="f1">Figures 1(e) and 1(f)</xref> show how the energies of the lowest four impurity states vary with <italic>R</italic>. All the energies are seen to be positive for <italic>R</italic> < <italic>R</italic><sub>1,0</sub> ≈ 1.8 <italic>r<sub>B</sub></italic>. The first state of negative energy appears in the spectrum when the nanocrystal radius exceeds <italic>R</italic><sub>1,0</sub>. It is then followed by the higher-energy states with quantum numbers (1, 1), (2, 0), …, and (3, 0), whose energies turn zero for <italic>R</italic> ≈ 5.1 <italic>r<sub>B</sub></italic>, 6.2 <italic>r<sub>B</sub></italic>, …, and 12.9 <italic>r<sub>B</sub></italic>. As the nanocrystal becomes larger, some of these states start forming multiplets due to the ‘accidental' degeneracy of the Coulomb interaction<xref ref-type="bibr" rid="b47">47</xref>. An unlimited increase in <italic>R</italic> leads to a transformation of all orbitally nondegenerate impurity states {<italic>n</italic>, <italic>l</italic>} to hydrogen-like states {<italic>ν</italic>}, which are <italic>ν</italic>-fold degenerate in angular momentum, so that <inline-formula id="m21"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e555" xlink:href="srep06917-m21.jpg"/></inline-formula> for <italic>R</italic> → ∞.</p><p>To find the number of negative-energy impurity states in the nanocrystal of radius <italic>R</italic>, one needs to determine the pair of quantum numbers (<italic>n<sub>R</sub></italic>, <italic>l<sub>R</sub></italic>) such that <inline-formula id="m22"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e576" xlink:href="srep06917-m22.jpg"/></inline-formula>. Some algebra shows that there are <disp-formula id="m4"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e579" xlink:href="srep06917-m4.jpg"/></disp-formula>states of different energies within the interval −Ry ≤ <italic>E<sub>n,l</sub></italic> < 0. These states are (2<italic>l</italic>+1)-fold degenerate with respect to the angular momentum projections, making the total number of negative-energy states equal to (<italic>n<sub>R</sub></italic> + <italic>l<sub>R</sub></italic>)(<italic>n<sub>R</sub></italic> + <italic>l<sub>R</sub></italic> + 1)(2<italic>n<sub>R</sub></italic> + 2<italic>l<sub>R</sub></italic> + 1)/6 − (<italic>l<sub>R</sub></italic> + 1)<sup>2</sup>. <xref ref-type="fig" rid="f2">Figure 2(a)</xref> shows the staircase function <italic>N</italic>(<italic>R</italic>), the steps of which correspond to new negative-energy levels <inline-formula id="m23"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e640" xlink:href="srep06917-m23.jpg"/></inline-formula> successively emerging in the impurity spectrum. The figure enables one to estimate the nanocrystal radius for a known number of negative-energy states, as well as to find the radius required for a desired number of states.</p></sec><sec disp-level="2"><title>Anticrossing of impurity energy levels</title><p>Comparison of the impurity spectrum with the spectrum of the nanocrystal without an ion reveals essentially different orderings of their states. This is seen from the order of the first ten levels of the two spectra shown in <xref ref-type="fig" rid="f2">Fig. 2(b)</xref> for <italic>R</italic> > <italic>R</italic><sub>4,0</sub>. By noticing that the ordering of levels <inline-formula id="m24"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e659" xlink:href="srep06917-m24.jpg"/></inline-formula> coincides with that in small nanocrystals (with <inline-formula id="m25"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e662" xlink:href="srep06917-m25.jpg"/></inline-formula>), and recalling that the impurity states form multiplets in large nanocrystals (with <inline-formula id="m26"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e666" xlink:href="srep06917-m26.jpg"/></inline-formula>), we conclude that the energies of the orbitally nondegenerate impurity states will sometimes coincide as they shift with the nanocrystal radius. Such an ‘accidental' degeneracy occurs <italic>e.g.</italic> for a pair of states (2, 0) and (1, 2) in the nanocrystal with <italic>R</italic> = 2<italic>r<sub>B</sub></italic>. The crossing of the respective energy levels in ZnSe nanocrystals is shown in <xref ref-type="fig" rid="f3">Fig. 3(a)</xref>.</p><p>Accidental degeneracy resulting from different size dependencies of impurity states is removed by the interaction of the impurity with various excitations of the nanocrystal (phonons<xref ref-type="bibr" rid="b48">48</xref><xref ref-type="bibr" rid="b49">49</xref><xref ref-type="bibr" rid="b50">50</xref><xref ref-type="bibr" rid="b51">51</xref>, plasmons<xref ref-type="bibr" rid="b52">52</xref>, excitons<xref ref-type="bibr" rid="b53">53</xref>, <italic>etc.</italic>), external electromagnetic fields<xref ref-type="bibr" rid="b54">54</xref>, or interaction with the environment<xref ref-type="bibr" rid="b55">55</xref><xref ref-type="bibr" rid="b56">56</xref><xref ref-type="bibr" rid="b57">57</xref><xref ref-type="bibr" rid="b58">58</xref><xref ref-type="bibr" rid="b59">59</xref><xref ref-type="bibr" rid="b60">60</xref>. This causes splitting and anticrossing of the degenerate energy levels. The degree of degeneracy of the impurity states modified by the interaction is determined by the quantum numbers of the initial states and the nature of the interaction. One of the simplest level-anticrossing problems, arising for the lowest-energy accidentally degenerate states (2, 0) and (1, 2), requires solving the sixth-order secular equation. If an interaction of strength <italic>V</italic> coupling these states does not couple the states of momentum projections <italic>m</italic> = 0, ±1, and ±2, then this equation has three different roots, two of which, <disp-formula id="m5"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e705" xlink:href="srep06917-m5.jpg"/></disp-formula>correspond to nondegenerate states, and one which corresponds to a four-fold degenerate state of energy <italic>E</italic><sub>1,2</sub> (see Methods). Thus the interaction partially removes the six-fold degeneracy of the impurity states at point A. It should be noted that the degeneracy removal in real nanocrystals is always complete due to the existence of coupling between the states of different momentum projections, splitting the four-fold degenerate state into four components.</p><p><xref ref-type="fig" rid="f3">Figure 3(b)</xref> shows the anticrossing of energy levels <inline-formula id="m27"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e717" xlink:href="srep06917-m27.jpg"/></inline-formula> and <inline-formula id="m28"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e720" xlink:href="srep06917-m28.jpg"/></inline-formula>, caused by the polar interaction of the states in <xref ref-type="fig" rid="f3">Fig. 3(a)</xref> with dispersionless longitudinal optical (LO) phonons<xref ref-type="bibr" rid="b61">61</xref><xref ref-type="bibr" rid="b62">62</xref><xref ref-type="bibr" rid="b63">63</xref><xref ref-type="bibr" rid="b64">64</xref><xref ref-type="bibr" rid="b65">65</xref> confined to the ZnSe nanocrystal. Removal of the accidental degeneracy at point A is seen to result in energy splitting <inline-formula id="m29"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e728" xlink:href="srep06917-m29.jpg"/></inline-formula> of two out of the six initially degenerate states. This splitting is a natural measure of the electron–LO-phonon interaction strength in the nanocrystal. According to the diagram in <xref ref-type="fig" rid="f2">Fig. 2(b)</xref>, the energies <inline-formula id="m30"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e735" xlink:href="srep06917-m30.jpg"/></inline-formula> and <inline-formula id="m31"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e738" xlink:href="srep06917-m31.jpg"/></inline-formula> of the split-off states slowly approach Ry/9 ≈ −2.7 meV and Ry/4 ≈ −6.2 meV with the increase in the nanocrystal radius.</p></sec></sec><sec disp-level="1" sec-type="discussion"><title>Discussion</title><p>Strictly speaking, the accidentally degenerate states are coupled by the electron–phonon interaction not only to each other, but also to all other impurity states of the nanocrystal. Since the interaction strength <italic>V</italic> approximately scales as ∝ <italic>R</italic><sup>−1/2</sup> (see Methods) and the energy-level spacing in small nanocrystals grows as ∝ <italic>R</italic><sup>−2</sup>, the relative contribution of the latter coupling decreases with the reduction of the nanocrystal radius as ∝ <italic>R</italic><sup>3/2</sup>. This implies that considering a pair of accidentally degenerate states independent of the rest of the nanocrystal states is most justified for states (2, 0) and (1, 2), which are degenerate in the smallest nanocrystal. If an impurity-state level becomes accidentally degenerate several times upon variation of the nanocrystal radius, then the resulting anticrossings can be considered independently as long as the interaction of the crossing levels in pairs is much stronger than their interaction with the rest of the impurity levels. Otherwise, an accurate anticrossing description requires taking into account interactions between all the strongly coupled impurity states. This leads to the secular equation of order <inline-formula id="m32"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e765" xlink:href="srep06917-m32.jpg"/></inline-formula>, where <italic>l<sub>j</sub></italic> (<italic>j</italic> = 1, 2, … <italic>η</italic>) are the angular momenta of the strongly coupled states. If <italic>l<sub>z</sub></italic> is the largest angular momentum and <inline-formula id="m33"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e784" xlink:href="srep06917-m33.jpg"/></inline-formula>, then both the accidental degeneracy of the states and their degeneracy in momentum projections are fully removed. The degeneracy removal is partial for <italic>μ</italic> − 4<italic>l<sub>z</sub></italic> < 2, with at least one (4<italic>l<sub>z</sub></italic> + 2 − <italic>μ</italic>)-fold degenerate impurity state of energy <inline-formula id="m34"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e804" xlink:href="srep06917-m34.jpg"/></inline-formula> left.</p><p><xref ref-type="fig" rid="f3">Figures 3(c) and 3(d)</xref> illustrate the anticrossings of three levels <italic>E</italic><sub>1,3</sub>, <italic>E</italic><sub>2,1</sub>, and <italic>E</italic><sub>3,0</sub> coupled through the electron–phonon interaction considered earlier. One can see that the interaction leads to a complex anticrossing pattern, which is not the mere sum of two simple patterns similar to those in <xref ref-type="fig" rid="f3">Fig. 3(b)</xref>. Since <italic>l<sub>z</sub></italic> = 3 and <italic>μ</italic> = 11 in this case, the original energy states transform into eight nondegenerate and one three-fold degenerate states. This example shows that only isolated pairs of impurity states that are strongly coupled to each other and weakly coupled to other states are of interest from an experimental viewpoint owing to their simple anticrossing patterns. Since the coupling between nondegenerate impurity states weakens with nanocrystal size, the most ‘isolated' anticrossing occurs for states (2, 0) and (1, 2). This anticrossing is the most advantageous for the estimation of the strength of various interactions inside semiconductor nanocrystals.</p><p>In conclusion, we have shown that different size dependencies of impurity energy levels in semiconductor nanocrystals result in accidental level degeneracies, the removal of which leads to anticrossings in the size dependencies. Such anticrossings are an important feature inherent to doped nanocrystals, which enables measuring the strength of various interactions of the impurity states with the internal and external elementary excitations of the nanocrystals. We illustrated the removal of the accidental degeneracy of the lowest-energy states by the example of their interaction with optical phonons confined to spherical nanocrystals, and showed that these states are most suited for experimental studies of the nanocrystal anticrossings.</p></sec><sec disp-level="1" sec-type="methods"><title>Methods</title><sec disp-level="2"><title>Wave functions and energy spectrum of hydrogenic impurity</title><p>The solution to <xref ref-type="disp-formula" rid="m1">Eq. (1)</xref> is a product of the radial wave function and a spherical harmonic, Ψ(<bold>r</bold>) = <italic>F</italic>(<italic>r</italic>)<italic>Y<sub>lm</sub></italic>(<italic>ϑ</italic>, <italic>φ</italic>), where <italic>r</italic>, <italic>ϑ</italic>, and <italic>φ</italic> are the spherical coordinates and <italic>l</italic> and <italic>m</italic> are the angular momentum and its projection, respectively. Inside the nanocrystal, the radial wave function obeys the equation <disp-formula id="m6"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e888" xlink:href="srep06917-m6.jpg"/></disp-formula>where radius <italic>r</italic> and energy <italic>E</italic> are expressed in units of effective Bohr radius and effective Rydberg constant. The finite solution to this equation at <italic>r</italic> = 0 is proportional to the Kummer function<xref ref-type="bibr" rid="b66">66</xref>
<italic>M</italic>(<italic>a</italic>, <italic>b</italic>, <italic>c</italic>), <disp-formula id="m7"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e915" xlink:href="srep06917-m7.jpg"/></disp-formula>where <italic>N</italic> = (−<italic>E</italic>)<sup>−1/2</sup> and <italic>K</italic> = <italic>E</italic><sup>1/2</sup>. The uniform Dirichlet boundary condition <italic>F</italic> = 0 at the nanocrystal surface <italic>r</italic> = <italic>R</italic> gives the impurity's energy spectrum <disp-formula id="m8"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e946" xlink:href="srep06917-m8.jpg"/></disp-formula>where <italic>N<sub>n,l</sub></italic> and <italic>K<sub>n,l</sub></italic> are the <italic>n</italic>th positive roots of the equations <disp-formula id="m10"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e963" xlink:href="srep06917-m10.jpg"/></disp-formula><disp-formula id="m9"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e965" xlink:href="srep06917-m9.jpg"/></disp-formula>with <italic>d</italic> = <italic>R</italic>/<italic>r<sub>B</sub></italic>. The radial wave function of the impurity states can be finally written as<xref ref-type="bibr" rid="b47">47</xref>
<disp-formula id="m11"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e982" xlink:href="srep06917-m11.jpg"/></disp-formula>where <italic>A<sub>nl</sub></italic> is the normalization constant, <italic>a<sub>nl</sub></italic> = −<italic>N<sub>nl</sub></italic> and <italic>c<sub>nl</sub></italic> = −2<italic>d</italic>/<italic>N<sub>n,l</sub></italic> for <italic>E<sub>nl</sub></italic> < 0, and <italic>a<sub>nl</sub></italic> = <italic>i</italic>/<italic>K<sub>nl</sub></italic> and <italic>c<sub>nl</sub></italic> = 2<italic>iK<sub>nl</sub> d</italic> for <italic>E<sub>nl</sub></italic> ≥ 0.</p></sec><sec disp-level="2"><title>Degeneracy removal of impurity states</title><p>To determine how a pair of accidentally degenerate impurity states (<italic>n</italic><sub>1</sub>, <italic>l</italic><sub>1</sub>) and (<italic>n</italic><sub>2</sub>, <italic>l</italic><sub>2</sub>) of energies <inline-formula id="m35"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1075" xlink:href="srep06917-m35.jpg"/></inline-formula> and <inline-formula id="m36"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1078" xlink:href="srep06917-m36.jpg"/></inline-formula> split due to an interaction that does not couple the states differing only by the momentum projections, one needs to solve the secular equation <disp-formula id="m12"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1081" xlink:href="srep06917-m12.jpg"/></disp-formula>where <inline-formula id="m37"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1084" xlink:href="srep06917-m37.jpg"/></inline-formula>, <italic>I<sub>l</sub></italic> is the unit matrix of size 2<italic>l</italic> + 1, <disp-formula id="m13"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1096" xlink:href="srep06917-m13.jpg"/></disp-formula>is the (2<italic>l</italic><sub>1</sub> + 1) × (2<italic>l</italic><sub>2</sub> + 1) interaction matrix built of matrix elements <inline-formula id="m38"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1109" xlink:href="srep06917-m38.jpg"/></inline-formula>, in which <italic>q<sub>αβ</sub></italic> denotes the quantum numbers of the interaction quasiparticle coupling the states of momentum projections <italic>m</italic><sub>1</sub> and <italic>m</italic><sub>2</sub>. The <italic>μ</italic> = 2(<italic>l</italic><sub>1</sub> + <italic>l</italic><sub>2</sub> + 1) roots <inline-formula id="m39"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1142" xlink:href="srep06917-m39.jpg"/></inline-formula> of <xref ref-type="disp-formula" rid="m12">Eq. (11)</xref>, some of which may coincide, are the new energy levels resulting from the splitting. The problem of degeneracy removal for more than two degenerate states is solved in a similar fashion.</p><p>The model of longitudinal optical (LO) phonons confined to a spherical semiconductor nanocrystal with a high degree of ionicity yields<xref ref-type="bibr" rid="b61">61</xref><xref ref-type="bibr" rid="b64">64</xref>: <disp-formula id="m14"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1152" xlink:href="srep06917-m14.jpg"/></disp-formula>where <italic>ε</italic>* = <italic>ε</italic><sub>0</sub><italic>ε</italic><sub>∞</sub>/(<italic>ε</italic><sub>∞</sub> − <italic>ε</italic><sub>0</sub>), <italic>ε</italic><sub>∞</sub> and <italic>ε</italic><sub>0</sub> are the high- and low-frequency permittivities, <inline-formula id="m40"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1189" xlink:href="srep06917-m40.jpg"/></inline-formula> is the Clebsch–Gordan coefficient, and <disp-formula id="m15"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1192" xlink:href="srep06917-m15.jpg"/></disp-formula></p><p><xref ref-type="disp-formula" rid="m14">Equation (13)</xref> shows that the five-fold degenerate impurity state (1, 2, <italic>m</italic>) is coupled to the nondegenerate impurity state (2, 0, 0) through the five-fold degenerate phonon mode of quantum numbers (1, 2, <italic>m</italic>). <xref ref-type="disp-formula" rid="m12">Equation (11)</xref> in this case reduces to <disp-formula id="m16"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1207" xlink:href="srep06917-m16.jpg"/></disp-formula>with <disp-formula id="m17"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1210" xlink:href="srep06917-m17.jpg"/></disp-formula></p></sec><sec disp-level="2"><title>Material paramseters</title><p><xref ref-type="fig" rid="f3">Figure 3</xref> was plotted using <xref ref-type="disp-formula" rid="m14">Eqs. (13)</xref>, <xref ref-type="disp-formula" rid="m15">(14)</xref>, and <xref ref-type="disp-formula" rid="m17">(16)</xref>, by assuming that the nanocrystal is made of ZnSe, and using the following parameters: <italic>Z</italic> = 1, <italic>m<sub>e</sub></italic> = 0.15<italic>m</italic><sub>0</sub>, <italic>ε</italic><sub>0</sub> = 9.1, <italic>ε</italic><sub>∞</sub> = 6.3, ħΩ = 31.7 meV (the energy of the bulk LO phonon at the Brillouin zone center), <italic>r<sub>B</sub></italic>≈ 3.21 nm, and Ry ≈ 24.6 meV<xref ref-type="bibr" rid="b67">67</xref>.</p></sec></sec><sec disp-level="1"><title>Author Contributions</title><p>I.D.R., A.V.B. and A.V.F. jointly suggested the study conducted by A.S.B., V.K.T., M.Yu.L. and I.O.P. Namely, A.S.B., V.K.T., M.Yu.L. and I.O.P. performed analytical calculations and analyzed the obtained expressions, drew the figures, and prepared the first draft of the manuscript. I.D.R., A.V.B., T.S.P., K.B. and A.V.F. supervised the study, contributed to the analysis and interpretation of the results, helped to formulate and present the research outcomes, and thoroughly edited the manuscript.</p></sec> |
Genetic markers for toxicity of adjuvant oxaliplatin and fluoropyrimidines in the phase III TOSCA trial in high-risk colon cancer patients | <p>We investigated 17 polymorphisms in 11 genes (<italic>TS, MTHFR, ERCC1, XRCC1, XRCC3, XPD, GSTT1, GSTP1, GSTM1, ABCC1, ABCC2</italic>) for their association with the toxicity of fluoropyrimidines and oxaliplatin in colorectal cancer patients enrolled in a prospective randomized trial of adjuvant chemotherapy. The TOSCA Italian adjuvant trial was conducted in high-risk stage II–III colorectal cancer patients treated with 6 or 3 months of either FOLFOX-4 or XELOX adjuvant chemotherapy. In the concomitant ancillary pharmacogenetic study, the primary endpoint was the association of polymorphisms with grade 3–4 CTCAE toxicity events (grade 2–4 for neurotoxicity). In 517 analyzed patients, grade ≥ 3 neutropenia and grade ≥ 2 neurotoxicity events occurred in 150 (29%) and in 132 patients (24.8%), respectively. Diarrhea grade ≥ 3 events occurred in 34 (6.5%) patients. None of the studied polymorphisms showed clinically relevant association with toxicity. Hopefully, genome-wide association studies will identify new and more promising genetic variants to be tested in future studies.</p> | <contrib contrib-type="author"><name><surname>Ruzzo</surname><given-names>Annamaria</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a1">1</xref><xref ref-type="author-notes" rid="n1">*</xref></contrib><contrib contrib-type="author"><name><surname>Graziano</surname><given-names>Francesco</given-names></name><xref ref-type="aff" rid="a2">2</xref><xref ref-type="author-notes" rid="n1">*</xref></contrib><contrib contrib-type="author"><name><surname>Galli</surname><given-names>Fabio</given-names></name><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Giacomini</surname><given-names>Elisa</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Floriani</surname><given-names>Irene</given-names></name><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Galli</surname><given-names>Francesca</given-names></name><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Rulli</surname><given-names>Eliana</given-names></name><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Lonardi</surname><given-names>Sara</given-names></name><xref ref-type="aff" rid="a4">4</xref></contrib><contrib contrib-type="author"><name><surname>Ronzoni</surname><given-names>Monica</given-names></name><xref ref-type="aff" rid="a5">5</xref></contrib><contrib contrib-type="author"><name><surname>Massidda</surname><given-names>Bruno</given-names></name><xref ref-type="aff" rid="a6">6</xref></contrib><contrib contrib-type="author"><name><surname>Zagonel</surname><given-names>Vittorina</given-names></name><xref ref-type="aff" rid="a4">4</xref></contrib><contrib contrib-type="author"><name><surname>Pella</surname><given-names>Nicoletta</given-names></name><xref ref-type="aff" rid="a7">7</xref></contrib><contrib contrib-type="author"><name><surname>Mucciarini</surname><given-names>Claudia</given-names></name><xref ref-type="aff" rid="a8">8</xref></contrib><contrib contrib-type="author"><name><surname>Labianca</surname><given-names>Roberto</given-names></name><xref ref-type="aff" rid="a9">9</xref></contrib><contrib contrib-type="author"><name><surname>Ionta</surname><given-names>Maria Teresa</given-names></name><xref ref-type="aff" rid="a6">6</xref></contrib><contrib contrib-type="author"><name><surname>Veltri</surname><given-names>Enzo</given-names></name><xref ref-type="aff" rid="a10">10</xref></contrib><contrib contrib-type="author"><name><surname>Sozzi</surname><given-names>Pietro</given-names></name><xref ref-type="aff" rid="a11">11</xref></contrib><contrib contrib-type="author"><name><surname>Barni</surname><given-names>Sandro</given-names></name><xref ref-type="aff" rid="a12">12</xref></contrib><contrib contrib-type="author"><name><surname>Ricci</surname><given-names>Vincenzo</given-names></name><xref ref-type="aff" rid="a5">5</xref></contrib><contrib contrib-type="author"><name><surname>Foltran</surname><given-names>Luisa</given-names></name><xref ref-type="aff" rid="a7">7</xref></contrib><contrib contrib-type="author"><name><surname>Nicolini</surname><given-names>Mario</given-names></name><xref ref-type="aff" rid="a13">13</xref></contrib><contrib contrib-type="author"><name><surname>Biondi</surname><given-names>Edoardo</given-names></name><xref ref-type="aff" rid="a14">14</xref></contrib><contrib contrib-type="author"><name><surname>Bramati</surname><given-names>Annalisa</given-names></name><xref ref-type="aff" rid="a15">15</xref></contrib><contrib contrib-type="author"><name><surname>Turci</surname><given-names>Daniele</given-names></name><xref ref-type="aff" rid="a16">16</xref></contrib><contrib contrib-type="author"><name><surname>Lazzarelli</surname><given-names>Silvia</given-names></name><xref ref-type="aff" rid="a17">17</xref></contrib><contrib contrib-type="author"><name><surname>Verusio</surname><given-names>Claudio</given-names></name><xref ref-type="aff" rid="a18">18</xref></contrib><contrib contrib-type="author"><name><surname>Bergamo</surname><given-names>Francesca</given-names></name><xref ref-type="aff" rid="a4">4</xref></contrib><contrib contrib-type="author"><name><surname>Sobrero</surname><given-names>Alberto</given-names></name><xref ref-type="aff" rid="a19">19</xref></contrib><contrib contrib-type="author"><name><surname>Frontini</surname><given-names>Luciano</given-names></name><xref ref-type="aff" rid="a20">20</xref></contrib><contrib contrib-type="author"><name><surname>Magnani</surname><given-names>Mauro</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><aff id="a1"><label>1</label><institution>Department of Biomolecular Sciences, Università degli Studi di Urbino “Carlo Bo”</institution></aff><aff id="a2"><label>2</label><institution>Azienda Ospedaliera “Ospedali Riuniti Marche Nord”</institution>, Pesaro</aff><aff id="a3"><label>3</label><institution>Laboratorio di Ricerca Clinica, Department of Medical Oncology</institution>, IRCCS-Istituto di Ricerche Farmacologiche “Mario Negri”, Milano</aff><aff id="a4"><label>4</label><institution>IOV- IRCCS</institution>, Padova</aff><aff id="a5"><label>5</label><institution>Ospedale San Raffaele</institution>, Milano</aff><aff id="a6"><label>6</label><institution>Azienda Ospedaliera Universitaria di Cagliari</institution>, P.O. Monserrato</aff><aff id="a7"><label>7</label><institution>Azienda Ospedaliera Universitaria di Udine</institution>, Udine</aff><aff id="a8"><label>8</label><institution>Ospedale “B. Ramazzini”</institution>, Carpi</aff><aff id="a9"><label>9</label><institution>Ospedale Papa Giovanni XXIII</institution>, Bergamo</aff><aff id="a10"><label>10</label><institution>Ospedale di Gaeta ASL Latina</institution></aff><aff id="a11"><label>11</label><institution>Ospedale degli Infermi di Biella</institution></aff><aff id="a12"><label>12</label><institution>Ospedale “Treviglio-Caravaggio”</institution>, Treviglio</aff><aff id="a13"><label>13</label><institution>Azienda Ospedaliera Ospedale “Cervesi”</institution>, Cattolica</aff><aff id="a14"><label>14</label><institution>Ospedale “F</institution>, Renzetti”, Lanciano</aff><aff id="a15"><label>15</label><institution>Azienda Ospedaliera Fatebenefratelli</institution>, Milano</aff><aff id="a16"><label>16</label><institution>AUSL Ospedale di Ravenna</institution></aff><aff id="a17"><label>17</label><institution>Azienda Ospedaliera di Cremona</institution></aff><aff id="a18"><label>18</label><institution>Ospedale di Saronno</institution></aff><aff id="a19"><label>19</label><institution>Azienda Ospedaliera “Ospedale San Martino”</institution>, Genova</aff><aff id="a20"><label>20</label><institution>Fondazione GISCAD</institution></aff> | Scientific Reports | <p>Adjuvant chemotherapy is the standard of care for stage III colorectal cancer patients and an accepted treatment option for high-risk stage II patients<xref ref-type="bibr" rid="b1">1</xref>. Standard regimens include oxaliplatin combined with bolus/infusional 5-fluorouracil (FOLFOX) or capecitabine (XELOX)<xref ref-type="bibr" rid="b1">1</xref>. Unfortunately, several patients experience mild or moderate side effects at some point during treatment. Most frequently reported adverse events of these regimens in randomized adjuvant trials in Western populations are neutropenia (≥grade 3 in 40% to 56% of patients), neurotoxicity (≥grade 3 in 10% to 20% of patients), and diarrhea (≥grade 3 in 10% to 15% of patients)<xref ref-type="bibr" rid="b2">2</xref>. Therefore, the safety profile may be suboptimal and causing treatment delay, reduction, cessation and even death in a minority of patients. This is very important in the adjuvant setting, where potentially cured patients undergo an effective prophylactic treatment strategy<xref ref-type="bibr" rid="b1">1</xref>. Prediction of an individual patients' risk of severe toxicity could allow for an adequate monitoring and improve overall management and quality of care.</p><p>Host non-genetic factors such as medical comorbidity and organ dysfunction may account for differences in the safety profile of adjuvant chemotherapy across populations. However, genetic variability among individuals may play a key role<xref ref-type="bibr" rid="b3">3</xref>. Functional germline polymorphisms may contribute to inter-individual differences in the pharmacokinetic and pharmacodynamics of anti-cancer drugs and this may contribute to the differences in toxicity among patients<xref ref-type="bibr" rid="b3">3</xref>.</p><p>In the last decades, some genetic variants involved in the oxaliplatin and the fluoropyrimidines pathways were identified as potential predictors of toxicity<xref ref-type="bibr" rid="b4">4</xref><xref ref-type="bibr" rid="b5">5</xref>. However, the majority of clinical data have been obtained from retrospective analyses including a limited number of patients. In fact, none of the studied polymorphisms showed sufficient evidence for use in clinical practice<xref ref-type="bibr" rid="b4">4</xref><xref ref-type="bibr" rid="b5">5</xref>. Prospective analyses from randomized clinical trial represent a unique opportunity for evaluating association between genetic variants and clinical outcomes and are necessary for confirming the predictive role for toxicity of candidate polymorphisms<xref ref-type="bibr" rid="b6">6</xref><xref ref-type="bibr" rid="b7">7</xref><xref ref-type="bibr" rid="b8">8</xref><xref ref-type="bibr" rid="b9">9</xref>.</p><p>TOSCA (Three Or Six Colon Adjuvant) is a large randomized trial addressing the role of a shorter duration of an adjuvant oxaliplatin/fluoropyrimidines regimen in surgically resected stage III and high-risk stage II colorectal cancer<xref ref-type="bibr" rid="b10">10</xref>. We adopted this clinical trial for planning a robust pharmacogenetic assessment for toxicity focusing on candidate polymorphisms, which had showed promising associations in previous studies<xref ref-type="bibr" rid="b6">6</xref><xref ref-type="bibr" rid="b7">7</xref><xref ref-type="bibr" rid="b8">8</xref><xref ref-type="bibr" rid="b11">11</xref><xref ref-type="bibr" rid="b12">12</xref><xref ref-type="bibr" rid="b13">13</xref>.</p><sec disp-level="1"><title>Patients, Materials and Methods</title><sec disp-level="2"><title>TOSCA trial</title><p>Patients included in this study represent a subgroup of the 3.759 patients with surgically-resected, stage III and high-risk stage II colorectal cancer recruited in TOSCA trial between 2007 and 2011<xref ref-type="bibr" rid="b10">10</xref>. This is an Italian intergroup, multicentre, randomized, non-inferiority phase III study in high-risk stage II and stage III colon cancer patients treated with 3 or 6 months of either FOLFOX-4 or XELOX adjuvant chemotherapy, sponsored by GISCAD (Italian Group For The Study Of Gastrointestinal Cancer) and supported by Italian Medicines Agency (AIFA)<xref ref-type="bibr" rid="b10">10</xref>. Patients eligible for the TOSCA study were asked to give further and specific written informed consent for the pharmacogenetic study. All experiments were performed in accordance with relevant guidelines and regulations and the Local Ethics Committee of each institution approved the Study.</p></sec><sec disp-level="2"><title>Assessment and management of chemotherapy toxicity</title><p>Selected hematologic and non-hematologic toxicities (anemia, leukopenia, neutropenia, thrombocytopenia, asthenia, diarrhea, mucositis stomatitis, vomiting, nausea, hepatic toxicity, skin toxicity, neurotoxicity) were assessed at the start of each cycle using Common Toxicity Criteria for Adverse Events (CTCAE) version 2.0. All adverse events at any time were monitored and reported. Toxicity was managed as follows: in case of grade ≥ 3 hematologic toxicity or persistent grade 2 the dose of all drugs was reduced by 25%. In case of grade ≥ 3 non-hematologic toxicity the dose of the related drugs was reduced by 50%. In case of grade ≥ 3 or persistent grade 2 neurotoxicity, oxaliplatin dose was reduced by 20%. Oxaliplatin was definitely stopped if grade ≥ 2 neurosensory symptoms persisted between cycles.</p></sec><sec disp-level="2"><title>Molecular assessments</title><p>This prospective study was planned as a confirmatory analysis of genetic variants, which had shown some putative predictive effect for toxicity in previous studies<xref ref-type="bibr" rid="b6">6</xref><xref ref-type="bibr" rid="b7">7</xref><xref ref-type="bibr" rid="b8">8</xref><xref ref-type="bibr" rid="b11">11</xref><xref ref-type="bibr" rid="b12">12</xref><xref ref-type="bibr" rid="b13">13</xref>. Seventeen polymorphisms in eleven genes involved in DNA repair and drug metabolism and as drug targets, were selected from various reports as being potentially predictive of 5-fluorouracil (5-FU) or oxaliplatin toxicity (<xref ref-type="table" rid="t1">Table 1</xref>) in colorectal cancer patients. For each polymorphism, patients were considered in three groups: homozygous wild type (p2); heterozygous (pq); homozygous variant (q2). We also considered the model with merged heterozygous and homoxygous risk variant carriers.</p><p>Blood samples were taken before starting adjuvant chemotherapy. Genomic DNA was extracted by means of QIAmp DNA Blood kit (Qiagen, Valencia, CA). Polymorphisms in <italic>TS</italic> (rs34743033, rs2853542, rs11280056), <italic>XRCC1</italic> (rs25487), <italic>XRCC3</italic> (rs861539), <italic>XPD</italic> (rs1799793, rs13181), <italic>GSTT1</italic> (positive or null), <italic>GSTM1</italic> (positive or null) were assayed as previously reported<xref ref-type="bibr" rid="b11">11</xref>. <italic>ABCC1</italic> (rs2074087) and <italic>ABCC2</italic> (rs3740066, rs1885301, rs4148386) genetic variants were analyzed using HRM (Rotor-Gene 6000®, Corbett Research, Sydney, Australia) or Pyrosequencing (PSQ 96MA®, Biotage AB) technique according to the manufacturer's instructions. Primer sequences and preparative PCR conditions are reported in the <xref ref-type="supplementary-material" rid="s1">supplementary Table S1</xref>. Briefly, all amplification reactions were performed in a volume of 25 ul using 2× PCR Master Mix® (Diatheva, Fano, Italy) kit, 25 ng of gDNA and 200 nM of each primer. The intercalating dye EvaGreen® (Biotium Inc, CA, USA) was added for the HRM analyses. HRM conditions are listed in <xref ref-type="supplementary-material" rid="s1">Table S1</xref>. <italic>ERCC1</italic> (rs11615), <italic>MTHFR</italic> (rs1801133, rs1801131) and <italic>GSTP1</italic> (rs1695) were genotyped with HRM analyses by means of kits containing reagents, enzymes and genotype controls: <italic>ERCC1</italic> Asn118Asn HRM kit, <italic>MTHFR</italic> C677T HRM kit, <italic>MTHFR</italic> A1298C HRM kit and <italic>GSTP1</italic> Ile105Val HRM kit respectively, according to the manufacturer's instructions (Diatheva). All laboratory analyses were performed blind to the patients' treatment and clinical outcomes. Genetic data were then transferred to IRCCS Istituto di Ricerche Farmacologiche “Mario Negri” for statistical analysis.</p></sec><sec disp-level="2"><title>Analysis plan, sample size, and statistics</title><p>According to the planned management of toxicity in TOSCA trial, we chose outcome measures and endpoints, which reflects clinically relevant degrees of both hematologic and non-hematologic toxicity. Primary outcome was defined as the occurrence of a grade 3–4 toxicity (grade 2, 3, 4 for neurotoxicity) considering in each patient the maximum grade of toxicity (MGT) reported during treatment. Secondary outcome was the time to toxicity (TTT), defined as the time from date of randomization in TOSCA trial to the date of first grade ≥ 2 event for neurotoxicity and ≥3 event for other toxicities. Subjects without such a toxicity event at the time of analysis were censored at the date they were last known to be event-free while on treatment.</p><p>The treatment compliance was described in terms of treatment interruption and dose intensity, defined as the dose given in mg/m<sup>2</sup> per week. Logistic regression and Cox proportional hazard models were used to assess the effects of genotypes on MGT and TTT, respectively, adjusting for treatment duration (6 or 3 months). For each polymorphism, toxicity analysis was performed across the three group genotypes (p<sup>2</sup>, pq, q<sup>2</sup>) and after grouping carriers of the heterozygous and homozygous risk genotypes.</p><p>Sample size calculation was based on an expected prevalence of at higher risk allele of at least 30% and assuming a 25% risk of toxicity. Accordingly, 440 patients (105 events) would allow the detection of an odds ratio (OR) of at least 2.0 associated to the group with unfavorable genotypes with a power of 90% and a I type error of 5%, for a bilateral test. Deviation from the Hardy-Weinberg equilibrium was assessed using the Pearson χ<sup>2</sup> test. Analyses were performed with SAS 9.2 (SAS Institute, Cary, NC). All reported p values are two-sided, and confidence intervals (CIs) are at the 95% level. A p value < 0.05 was considered statistically significant.</p></sec></sec><sec disp-level="1" sec-type="results"><title>Results</title><sec disp-level="2"><title>Patient characteristics and toxicity</title><p>From July 2007 to October 2011, 534 patients from 26 experimental centers entered the study. This figure represents 81% of patients randomized in the same period and by the same centers in the main TOSCA trial study. Seventeen patients were not assessable; five patients who were never treated, two patients because of unavailability of treatment data, and ten due to technical problems about blood sampling. Therefore, the analysis was conducted on 517 patients.</p><p>Characteristics of the 517 patients are shown in <xref ref-type="table" rid="t2">Table 2</xref>. Patients' baseline characteristics were consistent with those of the whole trial population (data not reported). Most patients were randomized to FOLFOX-4 because option for XELOX regimen was introduced only during the late phase of accrual of this ancillary study. Toxicity caused by adjuvant chemotherapy is reported in <xref ref-type="table" rid="t3">Table 3</xref>. Again, the spectrum and the frequency of toxicities did not differ from those observed in whole trial population (data on file). The target number of events was reached for neutropenia (150/517 patients, 29%) and neurotoxicity (132/517, 25.5%), only. Dose intensity and treatment interruptions are shown in <xref ref-type="table" rid="t4">Table 4</xref> and <xref ref-type="table" rid="t5">Table 5</xref>, respectively. Dose intensity for patients randomized in 6 months arms is slightly lower than that reported for patients randomized in 3 months arms.</p></sec><sec disp-level="2"><title>Genetic assessments</title><p><xref ref-type="table" rid="t1">Table 1</xref> lists the studied genetic variants and the distribution of genotypes of patients. Consistent with previous observations, genotype frequency did not differ from those observed in Caucasian population. Allele frequencies of all polymorphisms were consistent with the Hardy-Weinberg equilibrium (χ<sup>2</sup>; p > .05) and with values in the published literature.</p></sec><sec disp-level="2"><title>Pharmacogenetics for neutropenia and neurotoxicity</title><p>The results of pharmacogenetic analyses in the 517 patients for neutropenia and neurotoxicity are shown in <xref ref-type="table" rid="t6">Table 6</xref> and <xref ref-type="table" rid="t7">Table 7</xref>, respectively. A weak association was observed between the <italic>GST-T1/M1</italic>
<italic>null/null</italic> genotype (presence of homozygous deletion in both genes) and neutropenia according to MGT (OR 1.99, 95%CI 1.06–3.73; p = 0.03) and TTT (HR 1.70, 95%CI 1.03–2.78; p = 0.04), when compared with wild-type genotype. As far as the <italic>ABCC1</italic> rs2074087 genotype and neurotoxicity is concerned, the planned statistical analyses could not be performed due to low model convergence. Regarding neurotoxicity no evidence of association was found between polymorphisms and MGT and TTT.</p></sec><sec disp-level="2"><title>Other toxicities</title><p>Despite the number of events was less than required, therefore decreasing the power of the tests, the analyses showed some statistically significant association. In presence of the <italic>ABCC2</italic> (rs 4148386) <italic>GG</italic> genotype, there was a greater occurrence of grade 3–4 leukopenia (OR 9.82, 95%CI 1.16–83.02; p = 0.036) and the time to leukopenia was shorter (HR 9.40, 95%CI 1.13–78.10; p = 0.038) in comparison to <italic>ABCC2</italic>
<italic>AA</italic> genotype. <italic>TS</italic> 3′UTR <italic>L</italic> allele showed a protective effect for mucositis for MGT (OR 0.07, 95%CI 0.01–0.65; p = 0.020) and TTT (HR 0.07, 95%CI 0.01–0.67; p = 0.021). Risk of vomiting (MGT) was increased in carriers of the <italic>TS</italic> 5′UTR <italic>2R2R</italic> genotype (OR 8.83, 95%CI 1.01–76.91; p = 0.049) compared to <italic>TS</italic> 5′UTR <italic>3R3R</italic> genotype.</p></sec></sec><sec disp-level="1" sec-type="discussion"><title>Discussion</title><p>This study assessed 17 polymorphisms in 11 genes thought to be associated with toxicity of fluoropyrimidines or oxaliplatin. To the best of our knowledge this is the first and the largest prospective pharmacogenetic analysis in a randomized trial of adjuvant chemotherapy in colorectal cancer. Candidate polymorphisms were selected on the basis of previous promising data from retrospective or single arm studies. The prospective accrual of patients achieved the required number of events for neutropenia and neurotoxicity, however only <italic>GST-T1</italic>/<italic>M1</italic> was statistically associated to neutropenia and the strength of this association was very low. Therefore, no polymorphism showed a clinically relevant association with neurotoxicity and neutropenia. The results on the other toxicities should be looked at with caution because of the low number of events.</p><p>To date, five randomized clinical trials in colorectal cancer have incorporated pharmacogenetic analysis<xref ref-type="bibr" rid="b6">6</xref><xref ref-type="bibr" rid="b7">7</xref><xref ref-type="bibr" rid="b8">8</xref><xref ref-type="bibr" rid="b9">9</xref>, but only one study in the adjuvant setting<xref ref-type="bibr" rid="b14">14</xref>. In the US Intergroup N9741 pharmacogenetic analysis there were 114 patients treated with IFL chemotherapy, 299 patients treated with FOLFOX-4 regimen and 107 patients who received IrOX chemotherapy<xref ref-type="bibr" rid="b6">6</xref>. Therefore, despite the 520 initial patients assessed for pharmacogenetic analyses, this remarkable study population was diluted among three treatments arms, with a small number of patients assessable for an oxaliplatin-based regimen. In this study, ≥grade 3 neutropenia, neurotoxicity and diarrhea occurred in the 27%, 13% and 13% of patients respectively. In the FOLFOX-4 regimen analysis, the <italic>GST-P1 TT</italic> genotype carriers were more likely to suffer from febrile neutropenia and to discontinue the treatment because of neurotoxicity, carriers of the <italic>GST-M1 null</italic> genotype were at increased risk of neutropenia<xref ref-type="bibr" rid="b6">6</xref>. In the Fédération Francophone de Cancérologie Digestive 2000-05 trial, metastatic colorectal cancer patients were randomized to receive 5-FU plus leucovorin followed by FOLFOX-6, followed by FOLFIRI (arm A), or FOLFOX-6 followed by FOLFIRI (arm B). The pharmacogenetic analysis included 346 patients who received more regimens in a different sequence<xref ref-type="bibr" rid="b7">7</xref>. There was a remarkable frequency of ≥grade 2 neurotoxicity (about half of the patients) and ≥grade 3 myelotoxicity in about one-third of the patients. The <italic>XPD</italic> C allele (rs13181) was significantly associated with an increased risk of FOLFOX-induced hematologic toxicity (p = 0.01). In the pharmacogenetic analysis associated with the randomized FOCUS UK trial, 1.188 patients were assessed<xref ref-type="bibr" rid="b8">8</xref>. In this study, metastatic colorectal cancer patients were randomized to receive three treatment strategies according to a different sequence of the following regimens: 5-FU alone, irinotecan alone, 5-FU with irinotecan and 5-FU with oxaliplatin. Only 280 patients were assessable for first- or second-line oxaliplatin-based chemotherapy. No significant pharmacogenetic association was found in this study<xref ref-type="bibr" rid="b8">8</xref>. The most recently published analysis in metastatic colorectal cancer patients depicts the results of a large panel of genetic variants in a robust sample of more than 2,000 patients enrolled in the COIN trials in UK<xref ref-type="bibr" rid="b9">9</xref>. Again, this study ruled out clinically relevant associations between pharmacogenetics and clinical outcomes of patients treated with fluoropyrimidine/oxaliplatin with or without cetuximab<xref ref-type="bibr" rid="b9">9</xref>.</p><p>As far as the adjuvant setting is concerned, the recently published pharmacogenetic study from the QUASAR2 trial has investigated the role of fluoropyrimidine-related polymorphisms in 927 patients who were randomized between capecitabine and capecitabine with bevacizumab. Of the 36 assessed polymorphisms only four TS and DPYD genetic variants were associated with grade ≥ 3 global toxicity, but with modest predictive power<xref ref-type="bibr" rid="b14">14</xref>.</p><p>Considering the characteristics of the above mentioned studies, we would emphasize the remarkable sample size in the adjuvant setting of our oxaliplatin-based study population, as well as the quality of pharmacogenetic analyses in a prospective and controlled collection of clinical data<xref ref-type="bibr" rid="b10">10</xref>. It seems that we recorded a lower frequency of grade ≥ 2 neurotoxicity and grade > 3 neutropenia than previously reported in the literature<xref ref-type="bibr" rid="b2">2</xref>. Generally, we observed a global lower incidence of toxicity events than expected. This finding is likely related to the accuracy of physicians in the monitoring of patients with early detection of signs of side-effects and consequently, their conservative attitudes towards treatment delays and dose-reductions.</p><p>However, this did not jeopardize the study plan of the ancillary pharmacogenetic study and a sufficient number of events for neurotoxicity and neutropenia was observed. Unfortunately, given the low rate of other severe toxicities, we cannot rule out the risk of observing false-negative associations in these cases.</p><p>A number of drug- and host-related variables contribute to pharmacodynamic and pharmacokinetic changes of chemotherapy drugs. Therefore, because of the moderate functional effects of polymorphism in the enzyme/target activity, their clinical impact may be masked according study populations and clinical settings. This may also explain the heterogeneity of results across pharmacogenetic studies. On the whole, we highlight the necessity for large-scale validation trials before pharmacogenetic findings from small studies are incorporated into clinical practice<xref ref-type="bibr" rid="b12">12</xref><xref ref-type="bibr" rid="b13">13</xref><xref ref-type="bibr" rid="b14">14</xref><xref ref-type="bibr" rid="b15">15</xref>. In fact, our findings, together with the results of the analyses in metastatic colorectal cancer<xref ref-type="bibr" rid="b6">6</xref><xref ref-type="bibr" rid="b7">7</xref><xref ref-type="bibr" rid="b8">8</xref><xref ref-type="bibr" rid="b9">9</xref> and other malignancies<xref ref-type="bibr" rid="b3">3</xref>, mitigate the positive expectations for the routine use of pharmacogenetics. It is a matter of fact that in spite of the growing burden of small, retrospective published studies on the predictive/prognostic role of polymorphisms in colorectal cancer patients, only UDP glucuronosyltransferase 1 family, polypeptide A1 (<italic>UGT1A1</italic>) and dihydrophyrimidine dehydrogenase (<italic>DPYD</italic>) genetic variants have shown a promising level of evidence for clinical practice<xref ref-type="bibr" rid="b16">16</xref>. However, we did not study the <italic>UGT1A1*28</italic> genotype analysis since it is typically associated with Irinotecan pharmacokinetic and toxicity<xref ref-type="bibr" rid="b16">16</xref>. As far as the <italic>DPYD IVS14 + 1G > A</italic> splice mutation is concerned, we did not include this variant for 5-fluorouracil toxicity analysis because of its very low frequency<xref ref-type="bibr" rid="b16">16</xref>. In fact, there were 2 heterozygous carriers in the 346 patients (0.5%) of the French trial<xref ref-type="bibr" rid="b6">6</xref>, 4 heterozygous carriers in the 520 patients (0.7%) of US trial<xref ref-type="bibr" rid="b7">7</xref> and 12 heterozygous carriers in the 1088 patients (1.1%) of FOCUS trial<xref ref-type="bibr" rid="b8">8</xref>.</p><p>Pharmacogenetics may still offer a unique opportunity for tailoring the administration of chemotherapy and novel biologic agents to cancer patients. Hopefully, new sophisticated techniques such as SNP arrays and genome-wide association studies (GWAS) will identify new and more promising genetic variants to be tested in future studies<xref ref-type="bibr" rid="b17">17</xref><xref ref-type="bibr" rid="b18">18</xref>.</p></sec><sec disp-level="1"><title>Author Contributions</title><p>A.R., Francesco G., E.G. and M.M. conceived and performed the study design, performed the manuscript preparation and data interpretation. Fabio G. performed coordination study. Francesca G., I.F., Fabio G. and E.R. performed statistical analysis, data interpretation and manuscript preparation. S.L., M.R., B.M., V.Z., N.P., C.M., R.L., M.T.I., E.V., P.S., S.B., V.R., L.F., M.N., E.B., A.B., D.T., S.L., C.V., F.B., A.S. and L.F., collected samples and patients' data, and commented the manuscript. R.L., L.F. and A.S. participated in the study design and data interpretation, and helped to draft the manuscript. All authors reviewed the manuscript.</p></sec><sec sec-type="supplementary-material" id="s1"><title>Supplementary Material</title><supplementary-material id="d33e18" content-type="local-data"><caption><title>Supplementary Information</title></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="srep06828-s1.doc"/></supplementary-material></sec> |
Efficient bi-allelic gene knockout and site-specific knock-in mediated by TALENs in pigs | <p>Pigs are ideal organ donors for xenotransplantation and an excellent model for studying human diseases, such as neurodegenerative disease. Transcription activator-like effector nucleases (TALENs) are used widely for gene targeting in various model animals. Here, we developed a strategy using TALENs to target the <italic>GGTA1</italic>, <italic>Parkin</italic> and <italic>DJ-1</italic> genes in the porcine genome using Large White porcine fibroblast cells without any foreign gene integration. In total, 5% (2/40), 2.5% (2/80), and 22% (11/50) of the obtained colonies of fibroblast cells were mutated for <italic>GGTA1</italic>, <italic>Parkin</italic>, and <italic>DJ-1</italic>, respectively. Among these mutant colonies, over 1/3 were bi-allelic knockouts (KO), and no off-target cleavage was detected. We also successfully used single-strand oligodeoxynucleotides to introduce a short sequence into the <italic>DJ-1</italic> locus. Mixed <italic>DJ-1</italic> mutant colonies were used as donor cells for somatic cell nuclear transfer (SCNT), and three female piglets were obtained (two were bi-allelically mutated, and one was mono-allelically mutated). Western blot analysis showed that the expression of the DJ-1 protein was disrupted in KO piglets. These results imply that a combination of TALENs technology with SCNT can efficiently generate bi-allelic KO pigs without the integration of exogenous DNA. These <italic>DJ-1</italic> KO pigs will provide valuable information for studying Parkinson's disease.</p> | <contrib contrib-type="author"><name><surname>Yao</surname><given-names>Jing</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Huang</surname><given-names>Jiaojiao</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Hai</surname><given-names>Tang</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Wang</surname><given-names>Xianlong</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Qin</surname><given-names>Guosong</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Zhang</surname><given-names>Hongyong</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Wu</surname><given-names>Rong</given-names></name><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Cao</surname><given-names>Chunwei</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Xi</surname><given-names>Jianzhong Jeff</given-names></name><xref ref-type="aff" rid="a4">4</xref></contrib><contrib contrib-type="author"><name><surname>Yuan</surname><given-names>Zengqiang</given-names></name><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Zhao</surname><given-names>Jianguo</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a2">2</xref></contrib><aff id="a1"><label>1</label><institution>State Key laboratory of Reproductive Biology, Institute of Zoology</institution>, Chinese Academy of Sciences, P. R. China, 100101</aff><aff id="a2"><label>2</label><institution>Beijing Farm Animal Research Center, Institute of Zoology</institution>, Chinese Academy of Sciences, P. R. China, 100101</aff><aff id="a3"><label>3</label><institution>State Key Laboratory of Brain and Cognitive Sciences, Institute of Biophysics</institution>, Chinese Academy of Sciences, Beijing, P. R. China, <country>100101</country></aff><aff id="a4"><label>4</label><institution>Biomedical Engineering Department, College of Engineering, Peking University</institution>, Beijing, P. R. China, 100871</aff> | Scientific Reports | <p>Pigs have been extensively used in biomedical research and are an excellent models for human diseases, especially for cardiovascular disease, atherosclerosis, cutaneous pharmacology, wound repair, cancer, diabetes, ophthalmological studies, toxicology research, lipoprotein metabolism, lung disease, and neurodegenerative diseases, and being considered as a potential source of organs for xenotransplantation<xref ref-type="bibr" rid="b1">1</xref>. The similarities of organ size, anatomic and physiologic characteristics as well as genome sequence between pigs and humans have stimulated a wide range of application of pigs in biomedical research. While pigs offer many similarities to human anatomy, metabolism, neurobiology and physiology<xref ref-type="bibr" rid="b2">2</xref><xref ref-type="bibr" rid="b3">3</xref><xref ref-type="bibr" rid="b4">4</xref><xref ref-type="bibr" rid="b5">5</xref>, furthermore, pigs have a longer life span, are easily bred, reach puberty at 6–8 months, and generate less ethical concerns when compared with non-human primates.</p><p>The ability to make genetic modifications in pigs facilitates the establishment of a human genetic disease model for the exploration and development of new therapies. Because no characterized embryonic stem cells (ESCs) have been generated in pigs that can be used for gene targeting, homologous recombination (HR) in somatic cells followed by somatic cell nuclear transfer (SCNT) is the primary strategy for creating knockout (KO) animals<xref ref-type="bibr" rid="b6">6</xref>. However, the very low rate of HR in somatic cells has hindered the establishment of KO animals. Therefore, highly efficient targeting in somatic cells is imperative and will facilitate the ability to genetically modify pigs. The emergence of zinc finger nucleases (ZFNs) has improved the efficiency of gene targeting greatly<xref ref-type="bibr" rid="b7">7</xref>, and ZFNs have been used to make various KO animals, such as pigs<xref ref-type="bibr" rid="b8">8</xref><xref ref-type="bibr" rid="b9">9</xref><xref ref-type="bibr" rid="b10">10</xref>, human cells<xref ref-type="bibr" rid="b11">11</xref>, and rats<xref ref-type="bibr" rid="b12">12</xref>. Although ZFN-mediated gene targeting has progressed in large animals, some disadvantages of ZFNs, such as off-target cleavage, cytotoxicity, and limited target sites<xref ref-type="bibr" rid="b13">13</xref><xref ref-type="bibr" rid="b14">14</xref><xref ref-type="bibr" rid="b15">15</xref>, have limited their applications.</p><p>Transcription activator-like effector nucleases (TALENs) were first reported in 2010<xref ref-type="bibr" rid="b16">16</xref>. Similar to ZFNs, TALENs are composed of a TALE DNA binding domain and a <italic>FokI</italic> endonuclease. TALENs have been shown to possess comparable or higher efficiency for gene targeting, are more straightforward, and can theoretically target any sequence. TALEN-mediated targeting has been used successfully in different organisms, such as zebrafish<xref ref-type="bibr" rid="b17">17</xref><xref ref-type="bibr" rid="b18">18</xref>, rat<xref ref-type="bibr" rid="b19">19</xref>, and pig<xref ref-type="bibr" rid="b6">6</xref>.</p><p>Normally, antibiotic selection was used to screen the targeted cells that were successfully transfected with targeting vectors. The integration of antibiotic genes may cause concerns about food safety and unexpected biological effects. Therefore, highly efficient gene targeting without exogenous DNA integration is worth studying. Additionally, for large animals, making precise genetic modifications in donor cells and then using these donor cells to generate gene KO animals through SCNT technology ensure that all the offspring carry the mutations, thus reducing the cost of maintaining large animals. Few studies related to TALEN-mediated knock-in animals have been reported.</p><p>Parkinson's disease (PD) is a common neurodegenerative movement disorder. PD is a progressive disease that is characterized by the degeneration of dopamine neurons in the substantia nigra and the intracytoplasmic inclusion of <italic>Lewy</italic> bodies. To date, 11 genes from 16 PD loci have been identified in familial PD patients, and 5 of these genes have been studied extensively, namely <italic>α-synuclein</italic> (also known as <italic>SNCA</italic>), <italic>parkin</italic> (<italic>PARK2</italic>), <italic>PINK1</italic> (<italic>PARK6</italic>), <italic>DJ-1</italic> (<italic>PARK7</italic>), and <italic>leucine-rich repeat kinase</italic> (also known as <italic>LRRK2</italic>, <italic>PARK8</italic>)<xref ref-type="bibr" rid="b20">20</xref>. To elucidate the mechanisms of PD, many PD models have been generated by genetic manipulating these 5 genes in different organisms, such as mice, <italic>Drosophila melanogaster</italic>, and <italic>Caenorhabditis elegans</italic>. Although many mouse models with different fragment loss of the <italic>Parkin</italic> and <italic>DJ-1</italic> genes have been generated<xref ref-type="bibr" rid="b21">21</xref><xref ref-type="bibr" rid="b22">22</xref><xref ref-type="bibr" rid="b23">23</xref><xref ref-type="bibr" rid="b24">24</xref><xref ref-type="bibr" rid="b25">25</xref><xref ref-type="bibr" rid="b26">26</xref><xref ref-type="bibr" rid="b27">27</xref><xref ref-type="bibr" rid="b28">28</xref><xref ref-type="bibr" rid="b29">29</xref>, few of these models recapitulate the human disease process well, especially the most important phenotype: dopaminergic loss in the substantia nigra. Considering the high level of similarity between pigs and humans, we hypothesized that pigs are a more suitable model for studying Parkinson's disease.</p><p>To overcome the shortage of human organs for organ transplantation, the use of organs from other species is an alternative. Considering the high level of similarity between humans and pigs, pigs are suggested to be most suitable for supplying organs to humans. Gal epitopes encoded by the α-1,3-galactosyltransferase gene exist on the cell surfaces of all porcine tissues; this gene product is the major antigen in pig-to-human transplantation and causes hyperacute rejection. The knockout of this gene may repress hyperacute rejection and GGTA1 disrupted pigs have been made by Lai <italic>et al.</italic> through homologous recombination method in 2002<xref ref-type="bibr" rid="b30">30</xref>.</p><p>In the current study, we aimed to develop highly efficient gene targeting strategies without the integration of exogenous DNA and site-specific knock-in mediated by TALENs in pigs. Bi-allelic deletions were generated via single transfection of somatic cell with TALENs targeting <italic>GGTA1</italic> and <italic>DJ-1</italic>. Furthermore, TALEN-mediated introduction of a short sequence containing an enzymatic restriction site into the <italic>DJ-1</italic> locus was achieved by single-strand oligodeoxynucleotides. Using SCNT and embryo transfer, <italic>DJ-1</italic> KO piglets were generated. Western blot analysis verified the disruption of DJ-1 in these KO piglets. These <italic>DJ-1</italic> KO piglets can provide valuable information on PD in humans.</p><sec disp-level="1" sec-type="results"><title>Results</title><sec disp-level="2"><title>TALENs mediated highly efficient gene targeting without the integration of exogenous DNA</title><p>TALEN pair targeting <italic>GGTA1</italic> or <italic>Parkin</italic> was co-transfected with the pCAG-GFP plasmid into male pig fetal fibroblast cells (FFCs), respectively, and single cells were sorted into 96 well plates by FACS. For the <italic>GGTA1</italic> TALENs, a total of 40 colonies were obtained, and 2 of them (the G3-16 and G3-28 colony) were shown to be mutated by the T7 endonuclease I (T7EI) assay (<xref ref-type="fig" rid="f1">Fig. 1A and 1B</xref>). Sequencing results showed that both of the colonies were bi-allelically mutated. One allele of G3-16 harbored a 17-bp deletion, and another allele harbored a 226-bp deletion and a 12-bp replacement (<xref ref-type="fig" rid="f1">Fig. 1C</xref>). There was a 1-bp insertion in one allele of the G3-28 colony and a 4-bp deletion in another allele (<xref ref-type="fig" rid="f1">Fig. 1C</xref>). For the <italic>Parkin</italic> TALENs, 2 of 80 colonies were shown to be modified with mono-allelic mutations (<xref ref-type="fig" rid="f2">Fig. 2A and 2B</xref>). Sequencing results showed that one allele of the P3 colony had a 17-bp deletion, and the P40 colony had a 14-bp deletion (<xref ref-type="fig" rid="f2">Fig. 2C</xref>). The <italic>DJ-1</italic> TALENs were transfected into female pig FFCs; 11 of 50 colonies were mutated, and 4 of them were bi-allelically mutated (the D10, D21, D27 and D37 colonies), as shown in <xref ref-type="fig" rid="f3">Fig. 3</xref>. The targeting efficiency is summarized in <xref ref-type="table" rid="t1">Table 1</xref>. The results showed that the TALEN-mediated targeting efficiency ranged from 2.5% to 22%, and more than 1/3 of the mutant colonies were bi-allelically modified.</p><p>PCR amplification was used to determine the integration of the GFP fragment and TALEN DNA. The mutant cell colonies were negative for GFP fluorescence under a fluorescence microscope. The PCR results confirmed the loss of GFP in all the mutant cell colonies (<xref ref-type="fig" rid="f4">Fig. 4A</xref>). Furthermore, the integration of TALEN plasmids was not detected in any of the mutant cell colonies by PCR amplification of the <italic>FokI</italic> endonuclease domain (<xref ref-type="fig" rid="f4">Fig. 4B</xref>).</p></sec><sec disp-level="2"><title>Off-target analysis</title><p>To assess the specificity of the 3 pairs of TALEN plasmids used in this study, we used two approaches to scan the porcine genome to predict potential off-target sites. For E-PCR<xref ref-type="bibr" rid="b31">31</xref><xref ref-type="bibr" rid="b32">32</xref>, no off-target sites were predicted for the 3 TALEN pairs. Based on the TALENoffer score<xref ref-type="bibr" rid="b33">33</xref>, 5-7 potential off-target sites were verified by PCR amplification and sequencing (<xref ref-type="supplementary-material" rid="s1">Supplementary Tables S1–S3</xref> online). No mutations were observed at these sites in the G3-16 and G3-28 colonies for the <italic>GGTA1</italic> TALENs, the P3 and P40 colonies for the <italic>Parkin</italic> TALENs, and the D9 and D27 colonies for the <italic>DJ-1</italic> TALENs. These data suggest that the 3 pairs of TALENs used in the current study have high specificity for their target sequences.</p></sec><sec disp-level="2"><title>Site-specific insertion via TALENs</title><p>We next investigated the possibility of TALEN-mediated site-specific insertion by co-transfection of TALENs with single-strand oligodeoxynucleotides (ssODN). ssODN was first used in homology-directed repair (HDR) in ZFN-induced gene editing, and at least 20–25 bp homology arms on each side are recommended<xref ref-type="bibr" rid="b34">34</xref>. Considering the high targeting efficiency of the <italic>DJ-1</italic> TALENs, it was chosen for subsequent investigation in female FFCs. An ssODN was designed to contain a <italic>HindIII</italic> restriction enzyme site (<xref ref-type="fig" rid="f5">Fig. 5A</xref>) that would facilitate the subsequent detection of an insertion. The ssODN was co-transfected into fibroblasts with the <italic>DJ-1</italic> TALENs and the pCAG-GFP plasmid, and single colonies were screened as described above. Ten of 49 colonies were mutated in the <italic>DJ-1</italic> gene, and one (the DS21 colony) had the <italic>HindIII</italic> site insertion (<xref ref-type="fig" rid="f5">Fig. 5B</xref>). Sequencing results confirmed the successful site-specific insertion of ssODN that was mediated by the <italic>DJ-1</italic> TALENs (<xref ref-type="fig" rid="f5">Fig. 5C</xref>). However, results of the restriction digest map and sequencing showed that the DS21 colony was not truly mono-colony. It may be mixed with wild type cells, which would be caused by the method we used for cell plating. The insertion rate was 10% among edited.</p></sec><sec disp-level="2"><title>Production of TALEN-modified piglets by somatic cloning</title><p>The <italic>DJ-1</italic> mutant female FFCs (a mix of mono-allelic and bi-allelic mutant colonies) were used as donor cells for SCNT and embryo transfer to examine TALEN-mediated KO cells for producing KO animals. A total of 687 embryos were delivered to 5 surrogates, and one pregnancy was established. Three live-born piglets and one stillborn piglet were obtained, and the piglets were genotyped as 1 mono-allelic mutation and 3 bi-allelic mutations (<xref ref-type="fig" rid="f6">Fig. 6A</xref>). The sequencing results showed that the mono-allelic KO piglet was derived from the D9 colony (<xref ref-type="fig" rid="f6">Fig. 6B</xref>), which harbored an insertion-induced frame-shift in one allele, resulting in a prematurely truncated DJ-1 ORF. The two bi-allelic KO live-born piglets and the one stillborn piglet were derived from the D27 colony (<xref ref-type="fig" rid="f6">Fig. 6B</xref>), which harbored an 8-bp deletion in one allele and a 10-bp deletion in another allele, also resulting in a prematurely truncated DJ-1 ORF. Unfortunately, the 2 bi-allelic mutant live-born piglets died within two days after birth, and the mono-allelic mutant piglet died at day 8. No obvious defect in the pig organs was observed by autopsy, and the deaths may have been caused by cloning defects. Tissues were dissected from the dead piglets, and Western blot analysis was performed to confirm the disruption of DJ-1 in these pigs. Compared with wild-type piglets, DJ-1 was partially repressed in the mono-allelic mutant piglet and completely absent in the bi-allelic mutant piglet (<xref ref-type="fig" rid="f6">Fig. 6C</xref>), suggesting the successful generation of <italic>DJ-1</italic> KO piglets.</p></sec></sec><sec disp-level="1" sec-type="discussion"><title>Discussion</title><p>In the current study, three pairs of TALEN expression plasmids were designed to target the <italic>GGTA1, DJ-1</italic> and <italic>Parkin</italic> genes in pigs. First, the efficiency of TALEN-mediated gene targeting was detected for the <italic>GGTA1</italic> gene. Exon 6 was chosen for gene targeting because there is a catalytic domain of the α1, 3-galactosyltransferase enzyme. The targeting efficiency was 5% (2/40), and both colonies were bi-allelically modified, demonstrating the establishment of TALEN-mediated gene targeting in the lab. Then, two more TALEN pairs were designed to target the <italic>Parkin</italic> and <italic>DJ-1</italic> genes, respectively. The efficiency of TALEN pairs designed against the <italic>DJ-1</italic> gene was 22% (11/50). Various mutation patterns at the target site were observed, including small deletion (-1 base), large deletions (-226 bases), and small insertions (+1 base), which are characteristic of non-homologous end joining (NHEJ)-induced DNA damage response. The vast majority of mutations were deletions, which were also reported previously in mice and pigs<xref ref-type="bibr" rid="b32">32</xref><xref ref-type="bibr" rid="b35">35</xref>. Among all the targeted cell colonies, more than 1/3 were bi-allelic mutations, and this amount was consistent with previous reports in pigs<xref ref-type="bibr" rid="b6">6</xref><xref ref-type="bibr" rid="b32">32</xref>.</p><p>To induce mutations into the pig genome without the integration of exogenous DNA, circular pCAG-GFP plasmid was co-transfected with each TALEN pairs into pig FFCs and then sorted based on GFP expression by FACS to screen the mutant cell colonies instead of the usage of antibiotic markers to screen positive colonies. PCR results confirmed that there was no integration of the GFP or TALEN plasmids in the targeted cell colonies.</p><p>Off-target activity is another concern regarding nucleases mediated genome editing. In the current study, no off-target cleavages were identified for all the 3 pairs of TALENs. This lack of off-target cleavage is an advantage of TALENs over ZFNs and CRISPR/Cas9 system, which was reported to cause high off-target mutagenesis<xref ref-type="bibr" rid="b13">13</xref><xref ref-type="bibr" rid="b14">14</xref><xref ref-type="bibr" rid="b36">36</xref>. Take these data described above, TALENs are a reliable gene targeting tool for high efficient genetic modification in porcine somatic cells.</p><p>Site-specific knock-in is another advantage of nuclease-mediated gene targeting in large animals. TALEN-mediated site-specific insertion using ssODN with a <italic>HindIII</italic> site was successfully introduced to the somatic cell genome, however, a base substitution just before the <italic>HindIII</italic> site was identified. This substitution may be the result of re-cleavage of TALEN pairs after insertion of <italic>HindIII</italic><xref ref-type="bibr" rid="b37">37</xref> because the length of the spacer region between the TALEN pairs was the same as the wild-type sequence, which could be easily re-cleaved in cells cultured for extended periods. ssODN combined with ZFNs<xref ref-type="bibr" rid="b34">34</xref><xref ref-type="bibr" rid="b38">38</xref><xref ref-type="bibr" rid="b39">39</xref>, TALENs<xref ref-type="bibr" rid="b18">18</xref><xref ref-type="bibr" rid="b40">40</xref> and Cripsr/Cas9<xref ref-type="bibr" rid="b41">41</xref> was used widely for introducing small indels, point mutation, and even deletion of large fragment (up to 100 kb) because of its easy design, rapid synthesis, and comparable efficiency as classical targeting vector<xref ref-type="bibr" rid="b34">34</xref><xref ref-type="bibr" rid="b39">39</xref>. However, since ssODN is very short (tens of nucleotides), it's difficult to detect the random integration of ssODN into genome. Deep sequencing might be necessary to evaluate the random integration of ssDNA. Now, we are trying to obtain longer donor template (>2 kb) to mediate site-specific insertion of long fragments, which will complement the future advances of gene therapy for repairing disease-related genes in humans.</p><p>Previous studies have constructed many mouse models with different fragments deleted from the <italic>Parkin</italic> and <italic>DJ-1</italic> genes<xref ref-type="bibr" rid="b21">21</xref><xref ref-type="bibr" rid="b22">22</xref><xref ref-type="bibr" rid="b23">23</xref><xref ref-type="bibr" rid="b24">24</xref><xref ref-type="bibr" rid="b25">25</xref><xref ref-type="bibr" rid="b26">26</xref><xref ref-type="bibr" rid="b27">27</xref><xref ref-type="bibr" rid="b28">28</xref><xref ref-type="bibr" rid="b29">29</xref>, but few of them recapitulate human PD very well. One of the reasons may be that mice have a short life span, and PD is age-dependent, often occurring in aged people. Considering that pigs have a longer life span than mice and the other similarities between pigs and humans, pigs are ideal for constructing models of PD. Therefore, we used the <italic>DJ-1</italic> KO cell colonies combined with SCNT and embryo transfer to generate KO piglets. One <italic>DJ-1</italic>
<sup>+/−</sup> and two <italic>DJ-1</italic>
<sup>−/−</sup> female piglets were obtained. Western blot analysis showed that the DJ-1 protein was repressed in all the detected tissues of <italic>DJ-1</italic> KO piglets, indicating the successful construction of a <italic>DJ-1</italic> KO pig model. Unfortunately, the piglets all died due to cloning defect.</p><p>In conclusion, TALEN mediated efficiently generation of bi-allelic <italic>DJ-1</italic> KO pigs without the integration of exogenous DNA was successfully developed. The highly efficient gene targeting was also verified in the targeting of <italic>GGTA1</italic> and <italic>Parkin</italic>. The results in the current study further confirmed that TALENs can be used conveniently and efficiently for site-specific knock-in for precise gene modification in large animals. The bi-allelic <italic>DJ-1</italic> KO pigs could provide valuable information for understanding the mechanisms underlying PD.</p></sec><sec disp-level="1" sec-type="methods"><title>Methods</title><sec disp-level="2"><title>Animals</title><p>Pigs were raised at the Beijing Farm Animal Research Center and had <italic>ad libitum</italic> access to a commercial pig diet (nutrient levels according to the NRC) and water during the entire experimental period. All experiments involving animals were performed according to procedures approved by the Institutional Animal Care and Use Committee of the Institute of Zoology, Chinese Academy of Sciences, China.</p></sec><sec disp-level="2"><title>Generation of TALENs</title><p>TALENs expression constructs were assembled by Viewsolid Biotech Company (Beijing, China). Three pairs of TALENs were designed to target exon 6, exon 1, and exon 2 in the porcine <italic>GGTA1</italic>, <italic>Parkin</italic>, and <italic>DJ-1</italic> genes (ensembl ID: ENSSSCG00000005518, ENSSSCG00000004032 and ENSSSCG00000003385), respectively. The <italic>GGTA1</italic> TALENs recognition sequences were as follows: left TALEN 5′- CTTATCCCCAGAATACT-3′ and right TALEN 5′- ATCCACAGACATGCCT -3′. The <italic>Parkin</italic> TALENs recognition sequences were as follows: left TALEN 5′- GTTTGTCAGGTTCAACT-3′ and right TALEN 5′- GTCAGAATCGACCTCCACT-3′. Between the two binding sites was a 16-bp spacer with a <italic>NcoI</italic> site (CCAG<underline>CCATGG</underline>TTTCCC, <italic>NcoI</italic> underlined). The <italic>DJ-1</italic> TALENs recognition sequences were as follows: left TALEN 5′- GGAGACGGTTATTCCT-3′ and right TALEN 5′- GGAGGACTTACTCCAGCT-3′. Between the two binding sites was a 17-bp spacer with a <italic>BspHI</italic> sequence (GTAGATG<underline>TCATGA</underline>GACG, <italic>BspHI</italic> underlined). The TALEN pairs were transfected into porcine fetal fibroblast cells to detect the targeting efficiency.</p></sec><sec disp-level="2"><title>Cell culture and transfection</title><p>Primary fetal fibroblast cells (FFCs) were isolated from 42-day-old Large White porcine fetuses (male and female) and digested by collagenase-DNaseI in cell culture medium (DMEM high glucose (Gibco BRL, Grand Island, NY) supplemented with 15% fetal bovine serum (FBS, Hyclone, Logan, UT), 0.032% collagenase (Sigma, St. Louis, MO), 25 Kunitz units/ml DNaseI (Sigma, St. Louis, MO) and 40 μg/ml gentamicin (Sigma, St. Louis, MO) for 4–6 h at 37°C. Digested cells were washed and plated in 25-cm<sup>2</sup> flasks in cell culture medium (DMEM high glucose supplemented with 15% FBS). FFCs were frozen when they became confluent. Two days before transfection, FFCs were thawed and cultured in 75-cm<sup>2</sup> flasks. When the cells become 80% confluent, transfection was performed by electroporation as described previously<xref ref-type="bibr" rid="b42">42</xref>. To avoid antibiotic gene insertion, the pCAG-GFP plasmid was co-transfected with TALENs to be used as an indicator for FACS sorting and was eliminated after several passages. Forty-eight to seventy-two hours after transfection, cells were subjected to FACS sorting based on the expression of GFP fluorescence. Single cells were plated in each well of 96-well plates and cultured for approximately 10 days in cell culture medium supplemented with 2.5 ng/mL basic fibroblast growth factor<xref ref-type="bibr" rid="b42">42</xref><xref ref-type="bibr" rid="b43">43</xref><xref ref-type="bibr" rid="b44">44</xref> (Sigma, St. Louis, MO). The medium was replaced every 4 days. Confluent cell colonies were propagated and genotyped by PCR, restriction enzyme digestion and sequencing. Cell colonies harboring mutations were cryopreserved for SCNT.</p><p>For TALEN/ssODN-induced insertion, 0.5 μg of each of the TALEN pairs was co-transfected into female FFCs with 2 nmol ssDNA (a 20-bp 5′ homologous arm and a 20-bp 3′ homologous arm with a <italic>HindIII</italic> recognition site in the center, synthesized by Invitrogen). The pCAG-GFP plasmid was also included in the transfection system to provide an indicator for FACS sorting. A similar strategy was used to screen single-cell colonies and for genotyping.</p></sec><sec disp-level="2"><title>Detection of gene mutations</title><p>The genomic DNA of each cell colony was extracted using the TIANamp genomic DNA kit (Tiangen, Beijing, China). Genotyping was conducted using PCR followed by restriction enzyme digestion. For <italic>GGTA1</italic>, the genotyping primers were as follows: 5′-AGCACGAGGTGGACTTC-3′ and 5′-CAGATGTTATTTCTAACCAAAT-3′. The primers for <italic>Parkin</italic> genotyping were as follows: 5′-AAGGGCAGTCCCACAGGGAAACTC-3′ and 5′- TGGAGTAAAGTTCAAGGAATCCCC-3′. The primers for <italic>DJ-1</italic> genotyping were as follows: 5′- GTGCTTCTTCCTATTCATTTGTGC-3′ and 5′- CAGGTCTGTAAGAAATATGGGATG-3′. Mutations in <italic>GGTA1</italic> were assessed by the T7 endonuclease I (T7EI) assay. Briefly, the purified PCR products from colonies and wild-type cells (200 ng each) were denatured at 95°C for 5 min and re-annealed at room temperature for 10 min, then digested by T7EI (New England Biolabs) at 37°C for 0.5 h. Different bands were expected for mutant colonies. Mutations in <italic>Parkin</italic> and <italic>DJ-1</italic> were assessed by the loss of restriction enzyme sites harbored in the spacer region. PCR products of colonies were cut by <italic>NcoI</italic> or <italic>BspHI</italic>. Uncut bands were expected for mutant colonies. To verify the mutations, the gel-purified PCR products were cloned into the pEASY-T3 vector using the pEASY-T3 cloning kit (Transgen, Beijing, China) and then sequenced. To detect whether there was pCAG-GFP or TALEN plasmid integration in the positive colonies, PCR was conducted using the primers 5′-CAAGCTTCGAATTCTGCAGTCG-3′ and 5′-TGATGAGTTTGGACAAACCACAAC-3′ for GFP detection and the primers 5′-ATTTGGGTGGATCAAGGA-3′ and 5′-GTTTATCTCGCCGTTATT-3′ for <italic>FokI</italic> detection. One hundred and fifty ng of genomic DNA was used for PCR analysis. Conditions for PCR amplification were one cycle of pre-denaturation at 94°C for 5 min followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 62.7°C (for GFP primers) or 52°C (for <italic>FokI</italic> primers) for 30 seconds, and elongation at 72°C for 30 seconds, and one cycle of post-elongation at 72°C for 7 min. To assess the <italic>HindIII</italic> insertion, the PCR products of each cell colony were digested by the <italic>HindIII</italic> restriction enzyme, and colonies that could be cut by <italic>HindIII</italic> were then confirmed by sequencing.</p></sec><sec disp-level="2"><title>Oocyte maturation</title><p>Pig ovaries were collected from a local slaughterhouse and transported to the laboratory within 1 h in 0.9% saline maintained at 37°C. Oocytes were aspirated from antral follicles (3–6 mm in diameter) with an 18-gauge needle fixed to a vacuum pump. Compact cumulus oocyte complexes (COCs) were selected under a stereomicroscope, washed and cultured in maturation medium at 39°C under 5% CO<sub>2</sub>. The maturation medium was composed of tissue culture medium (TCM) 199 (Hyclone, Logan, UT) supplemented with 0.1% (w/v) polyvinyl alcohol, 3.05 mM D-glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 0.5 μg/ml of luteinizing hormone, 0.5 μg/ml of follicle-stimulating hormone, 10 ng/ml of epidermal growth factor, 10% (v:v) of porcine follicular fluid, and 10 μg/ml of gentamicin. After 42–44 h of <italic>in vitro</italic> maturation culture, COCs were denuded by vigorous vortexing for 5 min in TL-HEPES supplemented with 0.1% polyvinyl alcohol and 0.1% hyaluronidase. Oocytes with polar bodies were selected under a stereomicroscope and used for somatic cell nuclear transfer (SCNT).</p></sec><sec disp-level="2"><title>Somatic cell nuclear transfer and embryo transfer</title><p>Mature MII oocytes were enucleated by aspirating the first polar body and adjacent cytoplasm with a glass pipette (20 μm in diameter) in manipulation medium supplemented with cytochalasin B (5 mg/ml stock, 4 μl stock per 3 ml manipulation medium). A single <italic>DJ-1</italic> KO fibroblast cell (donor cell) was injected into the perivitelline space of an enucleated oocyte. The donor cell membrane should be in contact with the oocyte cytoplasmic membrane. Oocyte cytoplasm-cell complexes were then fused and activated by electric pulse. Reconstructed embryos were cultured in porcine zygote medium (PZM3) in 5% CO<sub>2</sub> at 39°C for 14–16 h until embryo transfer. Embryos in good condition (120–200 embryos) were surgically transferred into the oviduct of a surrogate the day after observed estrus. Gestation was detected by ultrasound at day 28 after embryo transfer. If the surrogate was pregnant, the gestation was monitored every 2 weeks. On the day of birth, an ear skin biopsy was isolated from each piglet for genomic DNA extraction and genotyping.</p></sec><sec disp-level="2"><title>Western blot analysis</title><p>Different tissues were dissected from dead piglets, frozen immediately in liquid nitrogen and stored at −80°C until use. Twenty to thirty milligrams of different tissues was lysed in a buffer containing 50 mM HEPES, pH 7.4, 150 mM NaCl, 100 mM NaF, 0.05% SDS, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 2 g/ml aprotinin and leupeptin, 2 mM benzamidine, 1 mM sodium vanadate, 1 mM EGTA and 0.1% deoxycholate for 15 min on ice, and 50 μg of protein was subjected to Western blot analysis. The <italic>DJ-1</italic> antibody (DJ-1 (D29E5) XP® Rabbit mAb #5933) was purchased from Cell Signaling Technology (Danvers, MA, USA), and a concentration of 1:1000 was used to detect the expression of DJ-1 in different tissues. β-actin and β-tubulin were used as housekeeping genes to confirm equal sample loading.</p></sec><sec disp-level="2"><title>Off-target analysis</title><p>To examine whether these three pairs of TALENs have off-target effects, we explored two approaches to predict potential off-target sites in the porcine genome, namely E-PCR<xref ref-type="bibr" rid="b31">31</xref><xref ref-type="bibr" rid="b32">32</xref> and TALENoffer<xref ref-type="bibr" rid="b33">33</xref>. For E-PCR (<ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/tools/epcr/">http://www.ncbi.nlm.nih.gov/tools/epcr/</ext-link>), we used the same criteria as Yong Lei and Xiaogang Guo (2012) described. Briefly, up to three mismatches and a 1-bp gap for each binding site was allowed, and the spacer between the two TALEN binding sites was <100 bp because longer spacers could interfere with <italic>FokI</italic> dimerization<xref ref-type="bibr" rid="b45">45</xref>. For TALENoffer, the default parameters were used to perform the predictions. The regions around potential off-target sites were amplified in mutant colonies and sequenced to confirm off-target cleavage.</p></sec></sec><sec disp-level="1"><title>Author Contributions</title><p>Z.Y. and J.Z. conceived and designed the study. J.Y., J.H., T.H., X.W., G.Q., H.Z. and R.W. performed the study. C.C. predicted the off-target sites. J.X. designed the TALEN plasmid pairs. J.Y. and J.Z. analyzed the data and wrote the manuscript. All authors reviewed the manuscript.</p></sec><sec sec-type="supplementary-material" id="s1"><title>Supplementary Material</title><supplementary-material id="d33e25" content-type="local-data"><caption><title>Supplementary Information</title><p>Full length gel of Fig. 6A</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="srep06926-s1.doc"/></supplementary-material><supplementary-material id="d33e31" content-type="local-data"><caption><title>Supplementary Information</title><p>S1-S3</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="srep06926-s2.xls"/></supplementary-material></sec> |
Anatomy and efficiency of urban multimodal mobility | <p>The growth of transportation networks and their increasing interconnections, although positive, has the downside effect of an increasing complexity which make them difficult to use, to assess, and limits their efficiency. On average in the UK, 23% of travel time is lost in connections for trips with more than one mode, and the lack of synchronization decreases very slowly with population size. This lack of synchronization between modes induces differences between the theoretical quickest trip and the ‘time-respecting' path, which takes into account waiting times at interconnection nodes. We analyse here the statistics of these paths on the multilayer, temporal network of the entire, multimodal british public transportation system. We propose a statistical decomposition – the ‘anatomy' – of trips in urban areas, in terms of riding, waiting and walking times, and which shows how the temporal structure of trips varies with distance and allows us to compare different cities. Weaknesses in systems can be either insufficient transportation speed or service frequency, but the key parameter controlling their global efficiency is the total number of stop events per hour for all modes. This analysis suggests the need for better optimization strategies, adapted to short, long unimodal or multimodal trips.</p> | <contrib contrib-type="author"><name><surname>Gallotti</surname><given-names>Riccardo</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Barthelemy</surname><given-names>Marc</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a1">1</xref></contrib><aff id="a1"><label>1</label><institution>Institut de Physique Théorique, CEA, CNRS-URA 2306</institution>, F-91191, Gif-sur-Yvette, <country>France</country></aff> | Scientific Reports | <p>Although the coupling between different transportation networks is fundamental<xref ref-type="bibr" rid="b1">1</xref>, most of the studies on Public Transport Networks have been performed considering only one single transportation mode: private cars<xref ref-type="bibr" rid="b2">2</xref><xref ref-type="bibr" rid="b3">3</xref><xref ref-type="bibr" rid="b4">4</xref>, taxis<xref ref-type="bibr" rid="b5">5</xref><xref ref-type="bibr" rid="b6">6</xref><xref ref-type="bibr" rid="b7">7</xref><xref ref-type="bibr" rid="b8">8</xref>, Subway<xref ref-type="bibr" rid="b9">9</xref><xref ref-type="bibr" rid="b10">10</xref><xref ref-type="bibr" rid="b11">11</xref><xref ref-type="bibr" rid="b12">12</xref><xref ref-type="bibr" rid="b13">13</xref>, Train<xref ref-type="bibr" rid="b15">15</xref><xref ref-type="bibr" rid="b16">16</xref><xref ref-type="bibr" rid="b17">17</xref><xref ref-type="bibr" rid="b18">18</xref><xref ref-type="bibr" rid="b19">19</xref><xref ref-type="bibr" rid="b20">20</xref>, Bus and Trams<xref ref-type="bibr" rid="b18">18</xref><xref ref-type="bibr" rid="b21">21</xref><xref ref-type="bibr" rid="b22">22</xref><xref ref-type="bibr" rid="b23">23</xref>, and at a worldwide scale, airline networks (see<xref ref-type="bibr" rid="b24">24</xref> and references therein). However, most transportation systems are coupled to each other and as it was recently shown in<xref ref-type="bibr" rid="b25">25</xref>, interconnections can have dramatic consequences on the behavior of the whole system. This finding triggered a wealth of studies<xref ref-type="bibr" rid="b26">26</xref><xref ref-type="bibr" rid="b27">27</xref><xref ref-type="bibr" rid="b28">28</xref><xref ref-type="bibr" rid="b29">29</xref><xref ref-type="bibr" rid="b30">30</xref><xref ref-type="bibr" rid="b31">31</xref> on multilayer networks — also coined multiplex networks — providing a new paradigm for studying these coupled systems. Public Transport Networks belong to this class and provides a paradigmatically example of spatial<xref ref-type="bibr" rid="b32">32</xref>, temporal<xref ref-type="bibr" rid="b33">33</xref>, and multilayer Network<xref ref-type="bibr" rid="b30">30</xref> where each layer corresponds to a single transportation mode.</p><p>A few studies only considered many modes merged in an unique network<xref ref-type="bibr" rid="b34">34</xref>, but this aggregation might hide important structural features due to the intrinsical multilayer nature of the network<xref ref-type="bibr" rid="b35">35</xref>. In particular, in the case of urban transport, not considering the connection times can lead to unprecise estimates for the network's navigability<xref ref-type="bibr" rid="b36">36</xref>. We note also that interchanges are not symmetrical: rail-to-bus and bus-to-rail waiting time are different and are independent from the actual traffic volume<xref ref-type="bibr" rid="b37">37</xref> (at least as long as capacity limits are not taken into account<xref ref-type="bibr" rid="b14">14</xref>). In addition, the existence of alternative trajectories on differerent transportation modes enhance the system resilience<xref ref-type="bibr" rid="b36">36</xref>.</p><p>Inter- and intra-modal connections can be intensively optimized just through modifications and offsetting of the existing timetables, allowing to reduce waiting times at transfer points of a city like Washington D.C. of about 26%<xref ref-type="bibr" rid="b38">38</xref>. A better knowledge of the structure and layout of the Public Transport System would impact a wide range of areas. Indeed, mode choice is one of the fundamental steps in transportation forecasting<xref ref-type="bibr" rid="b39">39</xref> and has represented in the past a perfect experimental field for the study of individual choice behavior<xref ref-type="bibr" rid="b40">40</xref>. Developments in the availability of urban public transport has the potential to improve significantly the air quality in metropolitan areas<xref ref-type="bibr" rid="b41">41</xref> and directly influences the social geography of a city<xref ref-type="bibr" rid="b42">42</xref>. However, multi-modality not only means the existence of more and better alternative options, but having to deal with all these alternatives at the same time. From the users point of view, the difficulty of dealing with the enormous amount of information needed for describing and taking advantage of the public transport of a city is such that it is no more managed by personal experience and habits, but by services offered by major information technology companies. From the transport agencies point of view, the managing task becomes significantly harder because: i) different modes are run by separate agencies and both data handling and optimization tasks have to cross high organizational barriers; ii) it is not trivial to identify aspects of the system that are relevant for service optimization.</p><p>Therefore, in order to help decision makers, new quantitative approaches are needed to highlight the limits of the system and to assess the impact of new infrastructural development. Our capacity to understand transfer behavior and to evaluate transfer improvements are indeed limited by the lack of proper analytical tools, as we have to take into account many important aspects simultaneously<xref ref-type="bibr" rid="b43">43</xref>. If we want the public transit system to become a viable alternative to automobile, it is crucial to design cost-competitive and reliable public transportation systems that guarantees both short travel times and a travel experience comparable to those of car trips<xref ref-type="bibr" rid="b44">44</xref>.</p><p>Another important difficulty in the study of transportation systems is the data availability. In particular, it is usually very difficult to obtain traffic related data, and we take advantage here of the availability of another type of data which will enable us to assess the structural efficiency of the system. This open-data information consists in the set of timetables for all transportation modes in the United Kingdom, except for Northern Ireland (see Methods and the <xref ref-type="supplementary-material" rid="s1">Supplementary Information</xref> for more details). We will focus on the urban scale and identify key quantities characterizing the efficiency of the system, providing directions to improve urban Public Transport Systems. More precisely, our goal is to determine:<list id="l1" list-type="bullet"><list-item><p>how far is an urban, multimodal public transportation system from optimum, </p></list-item><list-item><p>how the temporal aspects impact the structure of quickest paths, </p></list-item><list-item><p>how important are the multilayer aspects, </p></list-item><list-item><p>the key differences between transportation systems in different cities. </p></list-item></list></p><p>Our study is based on the statistical analysis of the quickest paths on the multimodal transportation networks. We assume that origins and destinations are uniformly and independently distributed on the location served by the transport system and we do not take into account access time at the departure location. This uniform demand does not take into account how flows are actually allocated over the network (a piece of information that is usually not directly available) but allows us to focus directly on the structural features of the network, and not on its actual use and on the qualities perceived by the average user. In this sense, the weaknesses and optimization that we discuss here, concern an ideal optimum where all possible routes with all possible origins and destinations would be improved. The methodology developped here can however be very easily adapted to the case where origin-destination matrices are known.</p><sec disp-level="1" sec-type="results"><title>Results</title><p>We first define different types of paths in these systems. In particular, in order to understand how far are urban transportation systems from an ideal optimum, we compare the quickest time-respecting paths with the minimal path. The minimal path is the quickest one, computed by using the largest speed observed on each link and by neglecting waiting times, and represents an unreachable condition, equivalent to having all the existing transportation systems perfectly synchronized for the specific trip under consideration. In contrast, the time-respecting path is the quickest path but where we use the real timetable and where walking and waiting times are taken into account. The time-respecting path is by definition longer than the minimal path and as we can see on an example shown in <xref ref-type="fig" rid="f1">figure 1</xref>, they can be extremely different from each other. In addition, real trips are bound to the transportation system and do not follow a straight line of euclidean distance <italic>d</italic>(<italic>a</italic>, <italic>b</italic>) from the origin <italic>a</italic> to the destination <italic>b</italic>. The topographical and infrastructural constraints induce differences of the transportation network topology between cities. A consequence of this is that the length <inline-formula id="m23"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e247" xlink:href="srep06911-m23.jpg"/></inline-formula> (<italic>a</italic>, <italic>b</italic>) of the quickest (time-respecting and minimal) trips on the network might be very different from <italic>d</italic>(<italic>a</italic>, <italic>b</italic>), and this difference can be measured by the detour <inline-formula id="m9"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e266" xlink:href="srep06911-m9.jpg"/></inline-formula> that can be interpreted as a cost-benefit ratio<xref ref-type="bibr" rid="b45">45</xref>. In order to compare the availability of routes in different networks, we use the quantity <italic>R</italic> = max<italic><sub>d</sub></italic><sub>>1</sub>
<italic>r</italic>(<italic>d</italic>) for a fixed <italic>d</italic> subset (see <xref ref-type="supplementary-material" rid="s1">Supplementary Information</xref>). The values of <italic>R</italic> in different cities are strongly anti-correlated (−0.95) with the static network normalized cyclomatic number<xref ref-type="bibr" rid="b46">46</xref>
<italic>M<sub>N</sub></italic> = (<italic>E</italic> − <italic>N</italic> − 1)/<italic>N</italic>, reflecting the fact that the more loops are present in the network and the less the detour. In the following, we would like to exclude this topological influence and in order to compare various cities, we will use as a spatial metric the effective length <inline-formula id="m24"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e313" xlink:href="srep06911-m24.jpg"/></inline-formula> on the network.</p><p>Only large cities can afford significant rail-based elements (trains, metro, tram) in their public transport systems and therefore can have a high propensity to interchange<xref ref-type="bibr" rid="b47">47</xref>. Other transportation modes such as ferries and coaches, play a secondary role at an urban level (and air transportation is naturally out of the game). Coaches emerge for minimal paths in certain cities, but their low frequencies are completely excluding them from time-respecting paths. Other forms of road transportation are usually more accessible and, for this reason, bus is the dominant layer for short distances. If cities have enough suitable street space dedicated to Bus and Bus Rapid Transit systems, they are even able to outperform metro and rail systems<xref ref-type="bibr" rid="b44">44</xref>.</p><p>Each transportation mode is characterized by its cruise speed, departure frequencies and accessibility. A consequence of these peculiarities is that, depending on the length of the trip, the public transport system offers different optimal time-respecting solutions. At the national scale (<xref ref-type="fig" rid="f2">Figure 2</xref>, Left), different strategies emerge at different spatial scales. We will not consider very short trips for which the origin and destination are closer than 1 km, because distances so short could be easily covered by walking and usually do not rely on transportation systems. Above this scale, the vast majority of short trips are made within the bus layer, and the rail system becomes dominant for inter-urban trips of length larger than approximately 40 kms. Air transportation emerges naturally for longer distances above 200 kms, and its importance increases significantly for distances of order 400 kms (e.g. Glasgow-Birmingham) and <italic>d</italic> ≈ 500 kms (e.g. Glasgow-London Luton), and becomes finally dominant for trips longer than 700 kms, connecting for example the southern part of England with the northern part of Scotland.</p><p>At the urban level, transportation modes that capture a significant fraction of the time-respecting paths are Bus, Railways and, when available, the Metro and Tramway layer (<xref ref-type="fig" rid="f3">Figure 3</xref>). The bus stops represent the vast majority of the possible origins and destinations and are almost always used in our paths. This bus layer contains in general the largest part of both minimal and time-respecting paths. The use of the fast transportation modes emerges progressively with increasing <inline-formula id="m25"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e335" xlink:href="srep06911-m25.jpg"/></inline-formula> (<xref ref-type="fig" rid="f2">Figure 2</xref>, right), with a higher rate in larger cities such as London, Manchester and Birmingham, where the Metro-Tramway systems are present. As this transportation mode has a high frequency and a fast speed, and is not affected by congestion, it is naturally used as a quickest alternative to buses across city centres. Nevertheless, due to its limited accessibility, the largest fraction of short trips are done in the bus layer, also in cities where the system has an extended offer of metro lines (<xref ref-type="fig" rid="f3">Figure 3</xref>). The metro layer is in competition with the rail layer, which has higher speed but lower departure frequencies. In cities with high multi-modality (i.e. high average number of modes used per trip), the rail network attracts the largest part of the mobility at distances much lower than at the national level. Indeed, for London (<xref ref-type="fig" rid="f3">Figure 3</xref>, left) and Manchester (<xref ref-type="fig" rid="f3">Figure 3</xref>, right), the length done by train overcomes the one by buses at <inline-formula id="m26"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e351" xlink:href="srep06911-m26.jpg"/></inline-formula> and <inline-formula id="m27"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e354" xlink:href="srep06911-m27.jpg"/></inline-formula>.</p><sec disp-level="2"><title>Comparing minimal with time-respecting paths</title><p>We first analyze the multi-modal aspect of trips, quantified by the numbers Λ<italic><sub>m</sub></italic><sub>(<italic>t</italic>)</sub> which represent the number of different modes for the minimal (m) or time-respecting (t) paths. For some cities, the time-respecting paths display a larger Λ<italic><sub>t</sub></italic> than for minimal paths, while for others, it is the opposite (<xref ref-type="fig" rid="f4">Fig. 4a–b</xref>). The relative loss in multi-modality due to synchronization can be measured by <disp-formula id="m1"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e378" xlink:href="srep06911-m1.jpg"/></disp-formula>The larger Δ and the larger the difference between minimal and time-respecting paths. We see in <xref ref-type="fig" rid="f4">Fig. 4c</xref> that Δ is positive when the average speed of the alternative (ie. non-bus) layers <italic>V<sub>nb</sub></italic> is sensibly larger (>2.5 times) than the average speed <italic>V<sub>b</sub></italic> of the bus layer (see <xref ref-type="supplementary-material" rid="s1">Supplementary Information</xref> for more details). The quicker the rail and metro layers are, the more multimodal the minimal path would tend to be. Indeed, for minimal path, the use of fast non-bus layers is only limited by their accessibility, i.e. by the extra-time needed for reaching the inter-layer connection point. For time-respecting paths, multi-modality also implies the importance of synchronization, and it appears that in cities where metro or rail are sensibly faster, their frequency is also lower (<xref ref-type="fig" rid="f4">Fig. 4d</xref>). In other words, in cities where the fast layers are extremely advantageous in term of speed, the system suffers from synchronization problems. This empirical finding suggests the existence of a structural limit to transportation systems' possibilities that policy makers should take into account in the search of a efficient optimization strategy.</p><p>As a consequence, if the rail and metro layers are relatively quick, they are used for minimal paths while additional waiting times due to mode change can be too costly for the time-respecting paths. On the other hand, in cities where the bus layer is fast but with a frequency as low as for faster layers (eg. London, Liverpool, Cardiff), minimal paths tend to use buses only, while the time-respecting paths face the synchronization limits of the bus layer itself (see for example <xref ref-type="fig" rid="f1">figure 1</xref>). More generally, the factors responsible for the time difference between time-respecting and minimal paths are: (i) waiting times (both intra- and inter-layer); (ii) the fact that the optimal riding times used to compute minimal paths may differ from the riding times at a particular hour; (iii) a long walking time for connecting different modes in a wide stop area. In order to quantify the differences between minimal and time-respecting paths, we introduce the synchronization inefficiency <italic>δ</italic>, computed as the ratio of time-respecting travel time <italic>τ<sub>t</sub></italic> and minimal travel time <italic>τ<sub>m</sub></italic>
<disp-formula id="m2"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e419" xlink:href="srep06911-m2.jpg"/></disp-formula>For all cities, <italic>δ</italic> reaches its maximum <italic>δ<sub>max</sub></italic> for short trips, where waiting times are long compared to the travel time, and then decreases with the distance <inline-formula id="m28"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e431" xlink:href="srep06911-m28.jpg"/></inline-formula> according to the following function, valid for all cities (see <xref ref-type="fig" rid="f5">Fig. 5</xref>, left) <disp-formula id="m3"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e437" xlink:href="srep06911-m3.jpg"/></disp-formula>where <italic>ν</italic> ≈ 0.5. The collapse observed for <italic>δ</italic> for all cities suggests that there is an underlying process describing the accumulation of waiting and walking times along time-respecting paths. The specifics of the different cities appear in the system efficiency in both the worst <italic>δ<sub>max</sub></italic> and best <italic>δ<sub>min</sub></italic> limits. We note here that this <xref ref-type="disp-formula" rid="m3">Eq. 3</xref> is consistent with a simple argument based on the central limit theorem leading to <italic>ν</italic> = 1/2.</p><p>Time-respecting paths are however not completely different from the minimal ones and we can measure the similarity of two paths by using their spatial overlap <italic>q</italic>, defined as the fraction of length of edges they have in common. The overlap is <italic>q</italic> = 1 for extremely short trips (if a single edge is used, waiting times are not playing any role), and then decreases with <inline-formula id="m29"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e471" xlink:href="srep06911-m29.jpg"/></inline-formula> for all cities as (<xref ref-type="fig" rid="f5">Fig. 5</xref>, right) <disp-formula id="m4"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e477" xlink:href="srep06911-m4.jpg"/></disp-formula>where <inline-formula id="m30"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e481" xlink:href="srep06911-m30.jpg"/></inline-formula> is the scale parameter for each city. The function <inline-formula id="m31"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e484" xlink:href="srep06911-m31.jpg"/></inline-formula> converges to a limiting value <italic>q<sub>min</sub></italic> in the range [0.15, 0.33]. This minimal overlap is due to the limited number of good options available, especially close to the origin and the destination. The constraints due to the local connectivity and optimal cruise speeds make the minimal path the best option also when time causality starts playing a role. The exponential decay of the overlap with <inline-formula id="m32"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e492" xlink:href="srep06911-m32.jpg"/></inline-formula> suggests that there is a typical ‘branching' length <inline-formula id="m33"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e495" xlink:href="srep06911-m33.jpg"/></inline-formula> for each city, which sets the probability of having alternate routes. In other words, the probability for the time-respecting path to deviate at each <inline-formula id="m34"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e498" xlink:href="srep06911-m34.jpg"/></inline-formula> from the minimal path is proportional to <inline-formula id="m10"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e502" xlink:href="srep06911-m10.jpg"/></inline-formula>.</p></sec><sec disp-level="2"><title>Anatomy of a trip</title><p>We have seen so far that the length <inline-formula id="m35"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e510" xlink:href="srep06911-m35.jpg"/></inline-formula> governs the behavior of most quantities characterizing a trip. In order to identify the role of the temporal and multilayer aspects of the network in the structure of the time-respecting paths, we detail how the total travel time can be decomposed into different components: the riding time (with any mode), and waiting and walking times at interchanges. In addition, in order to take into account the multi-modal aspect of the network, we discriminate riding times per layer and we separate intra-layer from inter-layer waiting time. This wide spectrum of temporal quantities forms what we call the ‘anatomy' of a trip, and is represented in <xref ref-type="fig" rid="f6">Figure 6 a–c</xref> for different cities. This figure allows for a quick understanding of how the temporal structure of trips varies with distance.</p><p>We first note that the travel speed grows with <inline-formula id="m36"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e518" xlink:href="srep06911-m36.jpg"/></inline-formula> (see <xref ref-type="supplementary-material" rid="s1">Supplementary Information</xref>) which implies that travel time for time-respecting paths grows sub-linearly with the distance covered. Another important contribution in trips is due to walking between modes in the multilayer network, which represents a fixed cost of multi-modality (in addition to inter-layer waiting times). We naturally expect walking times to grow with <inline-formula id="m37"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e524" xlink:href="srep06911-m37.jpg"/></inline-formula> as the number Λ<italic><sub>t</sub></italic> of layers used in time-respecting paths (see <xref ref-type="supplementary-material" rid="s1">Supplementary Information</xref> for more details). Finally, waiting times are one of the two main contributions to the synchronization inefficiency <italic>δ</italic>, the other being the difference between optimal and actual edges' riding times. These times will play a relatively minor role for long distances, as their relative importance compared to riding times decreases.</p><p>The analysis of these anatomy plots shows the following. At short distance, in all cities but London, most of the travel time is spent in intra-layer waiting time. Most trips start at the bus layer, and the first connections within this layer are those that make the system extremely inefficient. We therefore define for each city a distance <inline-formula id="m38"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e540" xlink:href="srep06911-m38.jpg"/></inline-formula> such that for trips shorter than <inline-formula id="m39"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e543" xlink:href="srep06911-m39.jpg"/></inline-formula> the waiting time represents more than 50% of the travel time. For <inline-formula id="m40"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e546" xlink:href="srep06911-m40.jpg"/></inline-formula>, the lack of synchronization is dominant and the temporal network is far from being optimal. This distance interval corresponds to values of <inline-formula id="m41"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e549" xlink:href="srep06911-m41.jpg"/></inline-formula> where the overlap <italic>q</italic> is larger than 50% (<xref ref-type="fig" rid="f6">Fig. 6d</xref>). For short trips, we have few alternative paths and cannot avoid waiting times due to synchronization problems. Time-respecting paths are thus very similar to minimal paths, and (large) waiting times are directly added to optimal riding times.</p><p>For long distances, we already saw that the multi-modal nature of the systems becomes important. The use of fast transportation modes becomes advantageous only when the difference in speed compensates for the time necessary to reach the rail or metro network. In order to measure this effect, we define the distance <inline-formula id="m42"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e561" xlink:href="srep06911-m42.jpg"/></inline-formula> such that for <inline-formula id="m43"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e564" xlink:href="srep06911-m43.jpg"/></inline-formula> the largest part of the trip is done with a transportation mode different from the bus. Trips with <inline-formula id="m44"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e567" xlink:href="srep06911-m44.jpg"/></inline-formula> are then essentially made within the bus layer, and most of the travel time is due to actual transfer (walking or riding). We observe for large cities like London, Manchester, or Birmingham, a finite value of <inline-formula id="m45"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e570" xlink:href="srep06911-m45.jpg"/></inline-formula> indicating that at a certain point the bus layer loses its dominant role. This is in contrast with smaller cities where <inline-formula id="m46"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e573" xlink:href="srep06911-m46.jpg"/></inline-formula> is larger than the city radius, implying that fast layers always play a marginal role in these cases.</p><p>If we take into account time respecting paths with at least one inter-layer connections only, we find that on average for all cities considered in this work, the time spent in connections (walking and inter-layer waiting times) represents a significant fraction (23 ± 6%, see <xref ref-type="supplementary-material" rid="s1">Supplementary Information</xref>) of the total travel time. The different regimes identified in <xref ref-type="fig" rid="f6">figure 6d</xref> suggest that different strategies might have the better impact for each city for optimizing the transport time of trips of specific distances. Short trips are indeed dominated by intra-layer waiting times, while long trips by riding times. In the cases where the multimodality becomes dominant, inter-layer waiting and walking times, together with the fast layers' cruise speed, become instead the most relevant quantities for the optimization task.</p><sec disp-level="3"><title>The role of the total number of stop events</title><p>An interesting question concerns the characterization of a multilayer, temporal network such as the transportation systems that we consider here. Obviously, the number of modes and their frequency play important roles in their efficiency. A simple, natural quantity is then given by the average number of stop events per unit time <disp-formula id="m5"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e589" xlink:href="srep06911-m5.jpg"/></disp-formula>where <italic>C<sub>α</sub></italic> is the number of stop events in the layer <italic>α</italic> and Δ<italic>t</italic> the duration of the time interval considered in the analysis of the temporal network (see Methods). In this study, we considered a starting time of 8:00 am (monday) and a duration Δ<italic>t</italic> = 16 <italic>h</italic> which covers a whole day of mobility. The quantity Ω represents a global measure of the transportation service offered in a city, of the infrastructural cost of the transport network, and is indeed proportional to the cities population (<xref ref-type="fig" rid="f7">Fig. 7a</xref>). In order to improve the transportation system and to serve more people, one may add new lines, new connections, increase the frequency of a line, or even introduce a new transportation mode in the network, and the quantity Ω integrates all these modifications.</p><p>As we will see, it is actually remarkable and unexpected that a single network indicator such as Ω is enough to explain the behavior of many key quantities characterizing the public transport network of different cities. For example, the interplay between temporal and multilayer aspect of the public transport network is highlighted in <xref ref-type="fig" rid="f7">Fig. 7b</xref>, showing that the fraction <italic>λ<sub>t</sub></italic> of time-respecting paths using more than one mode<xref ref-type="bibr" rid="b1">1</xref> is larger for cities with a larger number of stop events. If we assume that the average number of possible alternative to bus layer path (which is always an available option) is <inline-formula id="m11"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e625" xlink:href="srep06911-m11.jpg"/></inline-formula>, the expected fraction of unimodal trips is <inline-formula id="m12"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e628" xlink:href="srep06911-m12.jpg"/></inline-formula>. The average interdependency of the time-respecting paths is then 1 − <italic>ω</italic>
<disp-formula id="m6"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e635" xlink:href="srep06911-m6.jpg"/></disp-formula>Using this form to fit the data shown in <xref ref-type="fig" rid="f7">Fig. 7b</xref>, we obtain <inline-formula id="m13"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e641" xlink:href="srep06911-m13.jpg"/></inline-formula> hour/stops.</p><p>Similarly, Ω is related to the cruise speed <inline-formula id="m47"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e646" xlink:href="srep06911-m47.jpg"/></inline-formula> (where <italic>τ<sub>cruise</sub></italic> is the time spent in a moving vehicle) and therefore also to the time respecting paths travel speed <inline-formula id="m48"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e654" xlink:href="srep06911-m48.jpg"/></inline-formula> (<xref ref-type="fig" rid="f7">Fig. 7c</xref>). Indeed, we can assume that the fraction <italic>ω</italic> of unimodal paths is traveled at the average bus layer speed <inline-formula id="m14"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e664" xlink:href="srep06911-m14.jpg"/></inline-formula>, while the fraction 1 − <italic>ω</italic> of multi-modal paths is traveled at a speed <inline-formula id="m15"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e670" xlink:href="srep06911-m15.jpg"/></inline-formula>, where <inline-formula id="m49"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e673" xlink:href="srep06911-m49.jpg"/></inline-formula> represents the ratio of lengths on the non-bus and bus layers (<inline-formula id="m16"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e676" xlink:href="srep06911-m16.jpg"/></inline-formula> is the average speed for the fast layers — see <xref ref-type="supplementary-material" rid="s1">Supplementary Information</xref>). The average travel speed <inline-formula id="m17"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e683" xlink:href="srep06911-m17.jpg"/></inline-formula> grows then with Ω as <disp-formula id="m7"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e686" xlink:href="srep06911-m7.jpg"/></disp-formula>Using the value for <inline-formula id="m18"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e689" xlink:href="srep06911-m18.jpg"/></inline-formula> obtained above, we minimize the variance between the estimated and empirical values of <inline-formula id="m19"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e692" xlink:href="srep06911-m19.jpg"/></inline-formula> and we find the optimal value: <italic>k</italic> ≈ 0.8 (see <xref ref-type="fig" rid="f7">Fig. 7c</xref>). This result shows that for all cities considered here (and under uniform demand), approximately 45% of time-respecting paths are on non-bus transportation modes.</p><p>In addition, the quantity Ω also characterizes the efficiency of a public transportation network in terms of synchronization. Indeed, we observe that the average synchronization inefficiency measure <inline-formula id="m20"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e703" xlink:href="srep06911-m20.jpg"/></inline-formula> decreases with Ω as a power law (see <xref ref-type="fig" rid="f7">Fig. 7d</xref>) <disp-formula id="m8"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e709" xlink:href="srep06911-m8.jpg"/></disp-formula>where <italic>μ</italic> ≈ 0.3 ± 0.1. The expected decrease is naturally due to the fact that larger values of Ω implies larger frequency and thus a better synchronization between modes. The small value of <italic>μ</italic> is however bad news in terms of efficiency: in order to divide <inline-formula id="m21"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e719" xlink:href="srep06911-m21.jpg"/></inline-formula> by a factor 2 we need to multiply Ω by a factor of almost 10. We can however hope that when exact origin-destination matrices are known, a better optimization of the system can be obtained through targeted improvements. It is not unusual to observe power law behavior in urban systems<xref ref-type="bibr" rid="b49">49</xref><xref ref-type="bibr" rid="b50">50</xref>, and although the fits are not perfect (essentially due to the small number of available decades), this result <xref ref-type="disp-formula" rid="m8">Eq. 8</xref> could be useful for constructing coarse-grained models of transportation in cities. Besides this, we note here that the city of Edinburgh is an outlier in all <xref ref-type="fig" rid="f7">figures 7(a–d</xref>) and, for this reason, has been excluded from the best-fit of <xref ref-type="fig" rid="f7">figures 7b and 7c</xref>. Indeed, even if Ω is relatively high for this city, Edinburgh's public transport system seems to use a significantly different strategy in managing the mobility demand, characterized by an extremely high bus-frequency. The network is therefore extremely efficient in terms of synchronization but not performant in terms of cruise speed (see <xref ref-type="fig" rid="f4">fig. 4d</xref>), as can be seen with time-respecting paths that are mostly composed of slow unimodal bus trajectories (see <xref ref-type="fig" rid="f7">figures 7b and 7c</xref>).</p></sec></sec></sec><sec disp-level="1" sec-type="discussion"><title>Discussion</title><p>We identified the total number of stop events per hour Ω as the key quantity which characterizes the efficiency of a transportation system and its efficiency in terms of speed, multimodality and synchronization. Naturally, Ω is not the only parameter at play: multi-modality depends also on the different cruise speed and departure frequencies in the different layer. In the UK transportation system, these two quantities are anti-correlated, as if a city system might try to optimize rail and metro systems, with respect of the bus system, either making them faster or more frequent. This relationship has important practical applications, as it constitute a limit that policy makers need to take into account in their system optimization, and can serve as a support for evaluating alternative Public Transport Systems' designs.</p><p>The temporal aspect of the Public Transport Networks appears to be influential for trips covering all distances. Short time-respecting paths tend to be mostly similar to the minimal ones, and waiting times are directly added to the riding times of the associated minimal paths. Waiting times then represent the largest fraction of the total travel times, and at this scale an increase of bus departure frequency, or methods like timetables offsetting<xref ref-type="bibr" rid="b37">37</xref><xref ref-type="bibr" rid="b38">38</xref> of the bus service may represent a good optimization strategy. Longer time-respecting paths tend instead to diverge from minimal ones, and very large waiting times can be avoided thanks to the availability of alternative routes, and when it is possible, longer trips are progressively taking advantage of the multi-modality of the system. For cities with a large level of multi-modality, as it is the case for London, Birmingham Manchester, it becomes hard to disentangle the temporal and multilayer aspects of the system. Waiting time (together with walking time) does not represent a simple cost to minimize, but a price to pay to access to fast transportation.</p><p>The value of waiting and walking times are perceived as higher than the time spent travelling<xref ref-type="bibr" rid="b52">52</xref>, in particular because walking demands a greater physical effort<xref ref-type="bibr" rid="b51">51</xref>. Waiting time has an higher perceived cost because of the frustration due to the sheer inconvenience of waiting<xref ref-type="bibr" rid="b52">52</xref>. All these costs have to be integrated with those related to the time needed for accessing the network<xref ref-type="bibr" rid="b44">44</xref>, the stress of the transfer experience<xref ref-type="bibr" rid="b43">43</xref>, breaking personal habits<xref ref-type="bibr" rid="b53">53</xref>, scheduling costs and those caused by the unreliability of arrival times<xref ref-type="bibr" rid="b52">52</xref>. In order to optimize the travel experience and to minimize the perceived mobility cost, it is then necessary to consider the full anatomy of trips and to distinguish between transportation modes and between the nature of time spent (riding, waiting, walking). In this respect, we believe that the tools and the methodology developed here will allow for an integrated view of these systems and will be helpful for testing and finding specific optimization strategies.</p></sec><sec disp-level="1" sec-type="methods"><title>Methods</title><sec disp-level="2"><title>Data</title><p>The land transport timetables used in these papers are provided by the National Public Transport Data Repository<xref ref-type="bibr" rid="b54">54</xref> under Open Government licence. A snapshot of every public transport journey is recorded for all services running in Great Britain (England, Scotland, Wales) during a full week in October 2010. The raw files contain the information available in the travel-lines web sites and call-centres during the selected week. For road transport, transportation agencies take into account the average traffic conditions at different hours and days for the design of timetables, so that they implicitely contain congestion effects.</p><p>The modes covered and identified are bus, coach, train (national rail), ferry and metro (including Underground, tram, light rail and non-national rail trains). All routes are referenced to stops coded using the NaPTAN scheme (National Public Transport Access Nodes) data<xref ref-type="bibr" rid="b55">55</xref>. In the NaPTAN scheme, every UK rail or metro station, coach terminus, airport, ferry terminal, bus stop or taxi rank is associated to at least one Stop Point. Not all Stop Points are actually used, so only those that were present in the timetables are considered active and have been taken into account. Stop point are then organized in Stop Areas representing facilities (Airports, Bus/Metro/Coach/Railway Stations) or possible interchange points. The definition of these Stop areas has been taken as a basis for defining a multilayer network from the timetable data. A further process of data cleaning and aggregation has been performed to have a consistent definition of inter-modal exchange points (see <xref ref-type="supplementary-material" rid="s1">Supplementary Information</xref>). To complete the spectrum of transportation modes, we use detailed schedules of all non-stop UK domestic flights, provided by Innovata LLC<xref ref-type="bibr" rid="b56">56</xref> for the week of 18–24 October 2010. Each of these flights has been associated to the Stop Points of the arrival and departure airport (and eventually to a specific terminal). The multilayer temporal network dataset derived from these data is publicly available at <ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="http://www.quanturb.com/data.html">http://www.quanturb.com/data.html</ext-link>.</p></sec><sec disp-level="2"><title>Multilayer temporal network</title><p>The inter-modal exchange points are identified by (i) original NaPTAN Stop Areas, (ii) new Stop Areas obtained by a spatial aggregation of Stop Points (see <xref ref-type="supplementary-material" rid="s1">Supplementary Information</xref>). To be an exchange point, journeys of different transportation modes should stop in that Area and to correctly define a Multilayer network<xref ref-type="bibr" rid="b30">30</xref>, we associate all Stop Points to a layer <italic>α</italic>, representing a specific transportation mode. If a Stop Point belongs to a Stop Area, the point is not represented in the network and all vehicle stops in that point are associated to the area. Both Areas and Points are identified by an id <italic>i</italic>. As buses and coaches may stop in the same location, a copy of the same Stop Point can be defined in two different layers, and thus associated to two different vertices <italic>v<sub>iα</sub></italic> and <italic>v<sub>iβ</sub></italic> in the multilayer network. Similarly, if an Area <italic>i</italic> has associated points belonging to a set of layers <italic>α</italic>, <italic>β</italic>, <italic>γ</italic>, …, a vertex representing that Area is defined in each of those layers (<italic>v<sub>iα</sub></italic>, <italic>v<sub>iβ</sub></italic>, <italic>v<sub>iγ</sub></italic>, …). Inter-layer edges connects all couples of vertices associated to the same Point or Area in different layers in both directions (<italic>i<sub>α</sub></italic>, <italic>i<sub>β</sub></italic>) and (<italic>i<sub>β</sub></italic>, <italic>i<sub>α</sub></italic>). If the connection from a layer <italic>α</italic> to a layer <italic>β</italic> is performed by walking, a walking distance is assigned to each of these edges (<italic>i<sub>α</sub></italic>, <italic>i<sub>β</sub></italic>) which is calculated as the average distance between all couples of active Stops Points in <italic>i</italic> belonging to the two different layers <italic>α</italic> and <italic>β</italic>. The travel time has been then computed using a standard walking speed of 5 km/h<xref ref-type="bibr" rid="b44">44</xref>. In addition to the walking times, additional 30 minutes are added to the inter-links from the air-flights layer to all the others, in order to take into account the characteristic waiting times in airports. Similarly, two hours of check-in and security control times are added to the inter-links towards the airline layer (which corresponds to the time suggested by airlines to be at the airport before departure time).</p><p>In each layer <italic>α</italic>, we thus have a set of <italic>N<sub>α</sub></italic> vertices, representing stops locations. The timetables define a set of events occurring in these vertices. Each vehicle departure can be associated to a directed connection between two vertices <italic>v<sub>iα</sub></italic> and <italic>v<sub>jα</sub></italic> that occurs at a certain time. These events can be represented as <italic>C<sub>α</sub></italic> quadruplets (<italic>i</italic>, <italic>j</italic>, <italic>t</italic>, <italic>δt</italic>), where <italic>i</italic>, <italic>j</italic> ∈ <italic>V<sub>α</sub></italic>, <italic>t</italic> denotes the departure time and <italic>δt</italic> the riding time for that specific trip<xref ref-type="bibr" rid="b57">57</xref>. Besides the temporal network, we can also study the static topology of the public transport network by defining a set of <italic>E</italic> edges, where the edge (<italic>i<sub>α</sub></italic>, <italic>j<sub>α</sub></italic>) exists if there is at any time at least a connection between <italic>v<sub>iα</sub></italic> and <italic>v<sub>jα</sub></italic>. For each of these edges, we compute the minimal riding time observed at any time <italic>δt<sub>min</sub></italic>. We define the minimal path as the shortest path on this static network, where the cost associated to each link is the minimal riding times. We use these minimal paths as a benchmark which represents the optimal mobility though the multi layer network. All the measures performed in this paper are limited to the largest strongly connected component<xref ref-type="bibr" rid="b46">46</xref> of the static network associated to the corresponding area.</p></sec><sec disp-level="2"><title>Time-respecting paths</title><p>Paths performed through the network must respect the time-ordered sequences of contacts. For this reason, a journey has to follow causal temporal paths defined as a sequence of connections with non-decreasing times<xref ref-type="bibr" rid="b33">33</xref>. We define the travel duration <italic>τ<sub>ab</sub></italic>(<italic>t</italic>) as the shortest time needed to reach <italic>b</italic> starting from a connection from <italic>a</italic> departing at a time <italic>t</italic>′ ≥ <italic>t</italic>. The duration is not static but depends upon <italic>t</italic>. In this paper, we focus on the morning rush hour, and thus we chose <italic>t</italic><sub>0</sub> = Monday, 8:00am. The temporal distance is measured starting from the actual beginning of the trip, without taking into account the first waiting time <italic>t</italic>′ − <italic>t</italic>. Furthermore, to limit the contribution of a small number of location from where connections are extremely rare, we introduce a waiting time cutoff Δ<italic><sub>c</sub></italic> = 2 h limiting the maximum delay allowed for a single connection<xref ref-type="bibr" rid="b57">57</xref>. Even while working on a static connected component, this cutoff limits the number of allowed paths. At a national scale, approximately 16% of the trips in the largest strongly connected component of the static network have been excluded because unreachable with this choice of <italic>t</italic><sub>0</sub> and Δ<italic><sub>c</sub></italic>.</p></sec></sec><sec disp-level="1"><title>Author Contributions</title><p>R.G. and M.B. designed, performed research and wrote the paper.</p></sec><sec sec-type="supplementary-material" id="s1"><title>Supplementary Material</title><supplementary-material id="d33e87" content-type="local-data"><caption><title>Supplementary Information</title><p>Supplementary information</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="srep06911-s1.pdf"/></supplementary-material></sec> |
Incident light adjustable solar cell by periodic nanolens architecture | <p>Could nanostructures act as lenses to focus incident light for efficient utilization of photovoltaics? Is it possible, in order to avoid serious recombination loss, to realize periodic nanostructures in solar cells without direct etching in a light absorbing semiconductor? Here we propose and demonstrate a promising architecture to shape nanolenses on a planar semiconductor. Optically transparent and electrically conductive nanolenses simultaneously provide the optical benefit of modulating the incident light and the electrical advantage of supporting carrier transportation. A transparent indium-tin-oxide (ITO) nanolens was designed to focus the incident light-spectrum in focal lengths overlapping to a strong electric field region for high carrier collection efficiency. The ITO nanolens effectively broadens near-zero reflection and provides high tolerance to the incident light angles. We present a record high light-conversion efficiency of 16.0% for a periodic nanostructured Si solar cell.</p> | <contrib contrib-type="author"><name><surname>Yun</surname><given-names>Ju-Hyung</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Lee</surname><given-names>Eunsongyi</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Park</surname><given-names>Hyeong-Ho</given-names></name><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Kim</surname><given-names>Dong-Wook</given-names></name><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Anderson</surname><given-names>Wayne A.</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Kim</surname><given-names>Joondong</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a4">4</xref></contrib><contrib contrib-type="author"><name><surname>Litchinitser</surname><given-names>Natalia M.</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Zeng</surname><given-names>Jinwei</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Yi</surname><given-names>Junsin</given-names></name><xref ref-type="aff" rid="a5">5</xref></contrib><contrib contrib-type="author"><name><surname>Kumar</surname><given-names>M. Melvin David</given-names></name><xref ref-type="aff" rid="a4">4</xref></contrib><contrib contrib-type="author"><name><surname>Sun</surname><given-names>Jingbo</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><aff id="a1"><label>1</label><institution>Department of Electrical Engineering, University at Buffalo, State University of New York</institution>, Buffalo, New York 14260, <country>USA</country></aff><aff id="a2"><label>2</label><institution>Department of Physics, Ewha Womans University</institution>, Seoul 120750, <country>Korea</country></aff><aff id="a3"><label>3</label><institution>Patterning Process Department, Nano Process Division, Korea Advanced Nano Fab Center</institution>, Suwon 443270, <country>Korea</country></aff><aff id="a4"><label>4</label><institution>Department of Electrical Engineering, Incheon National University</institution>, Incheon 406772, Korea</aff><aff id="a5"><label>5</label><institution>College of Information and Communication Engineering, Sungkyunkwan University</institution>, Suwon 440746, <country>Korea</country></aff> | Scientific Reports | <p>Many approaches have been reported to achieve low-cost<xref ref-type="bibr" rid="b1">1</xref><xref ref-type="bibr" rid="b2">2</xref><xref ref-type="bibr" rid="b3">3</xref><xref ref-type="bibr" rid="b4">4</xref><xref ref-type="bibr" rid="b5">5</xref><xref ref-type="bibr" rid="b6">6</xref><xref ref-type="bibr" rid="b7">7</xref> and high-performing photovoltaics (PVs) using designs for light-incident surficial structures<xref ref-type="bibr" rid="b3">3</xref><xref ref-type="bibr" rid="b8">8</xref><xref ref-type="bibr" rid="b9">9</xref><xref ref-type="bibr" rid="b10">10</xref><xref ref-type="bibr" rid="b11">11</xref><xref ref-type="bibr" rid="b12">12</xref><xref ref-type="bibr" rid="b13">13</xref>, and/or optical behaviors<xref ref-type="bibr" rid="b14">14</xref><xref ref-type="bibr" rid="b15">15</xref><xref ref-type="bibr" rid="b16">16</xref><xref ref-type="bibr" rid="b17">17</xref><xref ref-type="bibr" rid="b18">18</xref>. Recently, great interest has been focused on the periodic structure of light-active materials for optical enhancement<xref ref-type="bibr" rid="b19">19</xref><xref ref-type="bibr" rid="b20">20</xref><xref ref-type="bibr" rid="b21">21</xref><xref ref-type="bibr" rid="b22">22</xref>. Lambertian light trapping is proposed for an ideally random surface, which can extend the optical path length by 4n<sup>2</sup> or higher<xref ref-type="bibr" rid="b21">21</xref><xref ref-type="bibr" rid="b23">23</xref><xref ref-type="bibr" rid="b24">24</xref>, where n is the refractive index. A strong candidate for extraordinary optical enhancement by 14.5n<sup>2</sup> has been proposed<xref ref-type="bibr" rid="b25">25</xref><xref ref-type="bibr" rid="b26">26</xref> using a periodic nanoscale grating structure<xref ref-type="bibr" rid="b27">27</xref>.</p><p>Microscale designs are attractive for fabricating structures using a commercial method for large scale application<xref ref-type="bibr" rid="b11">11</xref><xref ref-type="bibr" rid="b12">12</xref><xref ref-type="bibr" rid="b13">13</xref>. Nanoscale architectures are definitely promising in attempts to reduce light-reflection with an enormous enlargement of the light-active surface area; however, it is extremely difficult to establish a neutral region<xref ref-type="bibr" rid="b28">28</xref><xref ref-type="bibr" rid="b29">29</xref> or a space charge region (SCR)<xref ref-type="bibr" rid="b11">11</xref><xref ref-type="bibr" rid="b13">13</xref><xref ref-type="bibr" rid="b30">30</xref> for nanoscale entities due to their tiny geometry. The SCR has the highest carrier collection efficiency, benefitting from a strong electric field (<italic>E</italic>) existing inside the region. Recent investigations have revealed that the position of an SCR is peculiarly crucial inside light-absorbing entities<xref ref-type="bibr" rid="b11">11</xref><xref ref-type="bibr" rid="b13">13</xref>.</p><p>Though the ITO nanowire and nanorod structures have been employed in photovoltaic and LED applications<xref ref-type="bibr" rid="b31">31</xref><xref ref-type="bibr" rid="b32">32</xref><xref ref-type="bibr" rid="b33">33</xref>, there are limitations in shaping the junctions of the nanoscale devices<xref ref-type="bibr" rid="b9">9</xref><xref ref-type="bibr" rid="b34">34</xref>. A direct etching method has been typically used to pattern semiconducting material. However, the direct etching of a semiconductor almost always leads to surface defects<xref ref-type="bibr" rid="b3">3</xref><xref ref-type="bibr" rid="b24">24</xref><xref ref-type="bibr" rid="b27">27</xref><xref ref-type="bibr" rid="b28">28</xref><xref ref-type="bibr" rid="b30">30</xref><xref ref-type="bibr" rid="b35">35</xref>. Such defects in the semiconducting material readily cause low carrier collection efficiency<xref ref-type="bibr" rid="b34">34</xref>, resulting in degraded solar cell performance of ~10% or less for Si and 13.8% for compound semiconductors; those achievements<xref ref-type="bibr" rid="b5">5</xref><xref ref-type="bibr" rid="b27">27</xref><xref ref-type="bibr" rid="b28">28</xref><xref ref-type="bibr" rid="b30">30</xref><xref ref-type="bibr" rid="b36">36</xref> are far behind from the pursued efficiency of over 20% of Si solar cells<xref ref-type="bibr" rid="b6">6</xref><xref ref-type="bibr" rid="b22">22</xref><xref ref-type="bibr" rid="b35">35</xref><xref ref-type="bibr" rid="b37">37</xref>.</p><p>The defect-induced recombination loss is a main reason to cause a discrepancy between the optical benefit and the electrical degradation of the direct nanoscale patterned semiconductor PVs. And thus, an urgent issue is assigned to the nanostructured PVs active light management<xref ref-type="bibr" rid="b23">23</xref><xref ref-type="bibr" rid="b38">38</xref>. Is it possible to form the optically beneficial nanostructures without electrical recombination loss by surface defects of the direct patterned semiconductor?</p><p>One promising scheme can be a periodically nanoscale patterned transparent conductor. In this design approach, it is possible to assign nanostructures for a planar semiconductor without a need of the direct etching in semiconducting material.</p><p>In this paper, we propose and demonstrate a periodic nanoscale-patterned high performing Si solar cell, without direct etching in a light-active semiconductor. This design is realized by an imprint method to form nanoscale transparent patterns on a bare 4-inch Si wafer.</p><p>In this paper the following challenges are addressed:</p><p>-  Fabrication of the periodically nanostructured PV device without a direct etching of the semiconducting material.Optically transparent indium-tin-oxide (ITO) nanolenses were periodically formed on a planar Si, which enables to fabricate the nanostructure PV device without damage to the semiconductor. And thus, the light-absorbing semiconductor (Si) is free from the surface defects, which are readily caused by the direct etching of the semiconductor. This approach ideally provides no etching-induced recombination loss from the semiconductor.</p><p>-  Optical and electrical functions of the ITO nanolensThe incident light has a form of plane wave and passes a planar semiconductor without an optical modulation. We have optimized the ITO nanolens to work as an optical lens by tuning the propagation length of the incident light. The nanolens geometries were designed to focus the various photon wavelengths at a designated spot to maximize the collection efficiency of photo-generated carriers. In terms of an electrical aspect, the electrical conducting ITO nanolens-arrays support the photo-generated carrier transport.</p><p>-  Reflection minimization for the incident photon wavelengthsTo improve solar cell performances, it is essential to drive the incident light into a light-absorber with little reflection at the surface. The ITO material has an intermediate refractive index value between air and Si. And thus, an ITO layer insertion spontaneously reduces the light reflection. We demonstrated an effective scheme of near-zero reflection for a broad range of wavelengths by using the ITO nanolenses.</p><p>-  High tolerance to the incident light anglesNot only is the solar cell efficiency important, but also it is a crucial issue to extend the solar utilization time, which directly increases the solar power generation. Unfortunately, the incident light angle to a solar cell varies with time during the day and throughout the year. This limits the power generation time for a conventional solar cell. The ITO nanolenses-embedded solar cell shows a strong potential of the high tolerance to broad incident light angles.</p><p>-  A spatial overlapping of the photo-generated carrier region and the SCR location to enhance the carrier collection efficiency.The incident light generates the photo-generated carriers in the light-absorbing Si material and establishes the light-induced <italic>E</italic>. For a solar cell, there exists a built-in <italic>E</italic> in the SCR, which is a driving force to collect photo-generated carriers. It is reported that few of the nanostructure systems such as metallic nanospheres<xref ref-type="bibr" rid="b39">39</xref>, silver nanorods<xref ref-type="bibr" rid="b40">40</xref> have been used to focus and magnify the broadband color images through localized surface plasmon (LSP) resonance. In the present work, a nanolens system is applied in renewable energy generation. Hence, the nanolens was designed to a focus broadband solar spectrum over the SCR rather than transfer the specific color and magnitude information for imaging. The more important geometric factor of the nanolens is the radius, which determines the focal length of the light. The spatial overlapping of the light-induced <italic>E</italic> and the built-in <italic>E</italic> due to focusing the photon wavelengths over SCR enhances the carrier collection efficiency<xref ref-type="bibr" rid="b41">41</xref>.</p><sec disp-level="1" sec-type="results"><title>Results</title><sec disp-level="2"><title>Formation of nanolens-arrays</title><p><xref ref-type="fig" rid="f1">Figure 1</xref> shows the schematic of the proposed structure. Nanostructure features were embedded in an optically transparent ITO layer to fabricate nanolenses, instead of using a Si substrate. This approach properly formed the periodically nanostructured PV device without a direct etching process to Si.</p><p>In order to fabricate the proposed ITO nanolens structure, we used a nanoimprint method to fabricate nanoscale patterns, which is suitable for patterning of large-scale periodic structures with low fabrication cost and high throughput<xref ref-type="bibr" rid="b42">42</xref>. Our nanosize hole-array was patterned on a 4-inch Si wafer as a target substrate. A polymer mold (polyurethane acrylate, PUA) was used to inversely replicate the structure from a master stamp (<xref ref-type="fig" rid="f1">Fig. 1a</xref>). The UV imprinting process was performed to pattern hole-arrays on the target Si substrate (<xref ref-type="fig" rid="f1">Fig. 1b</xref>). The inversely replicated PUA mold was then pressed against the UV-curable resin. By detaching the PUA mold from the UV-irradiated resin, the hole-arrays of the imprinted resin layer were produced on a 200 nm-thick polymethyl methacrylate (PMMA) layer. Subsequently, an O<sub>2</sub>-etching process was used to pattern identical hole-arrays on a PMMA layer (<xref ref-type="fig" rid="f1">Fig. 1c, d, e</xref>).</p><p>We deposited a 200 nm-thick ITO film on the PMMA hole-array Si substrate. A lift-off process was performed to remove the PMMA layer in an acetone solution under 15 min of sonication; there remained a 200 nm-height ITO-nanolens (with a radius of 180 nm) array on the Si substrate (See Methods), as presented in the top-view image (<xref ref-type="fig" rid="f1">Fig. 1f</xref>) and in the cross-sectional view image (<xref ref-type="fig" rid="f1">Fig. 1g</xref>). Each ITO-nanolens spontaneously contacts to the Si substrate; however, it is electrically isolated from the neighboring nanolenses. An additional an ITO film (80 nm) was coated on the previously formed 200 nm-height ITO-nanolens-arrays on the Si substrate (<xref ref-type="fig" rid="f1">Fig. 1h</xref>). A cross sectional image (<xref ref-type="fig" rid="f1">Fig. 1i</xref>) clearly depicts the ITO-film coating layer on the ITO nanolenses and the Si substrate, which ensures the electrical connection through the entire nanolens-arrays.</p><p>As a result, the front surface has nanoscale patterns comprised of an 80 nm-thick ITO film layer with 200 nm-height ITO-nanolens-arrays. Electrical conduction was obtained from this front surface; the device was found to have a low sheet resistance of 29.4 Ω/sq, which will ensure good electrical contact<xref ref-type="bibr" rid="b12">12</xref><xref ref-type="bibr" rid="b43">43</xref><xref ref-type="bibr" rid="b44">44</xref>. The optical transmittance was measured and found to be 90.95%. Photographic images are presented for the ITO nanolens-array patterned on the 4-inch Si wafer (<xref ref-type="fig" rid="f1">Fig. 1j</xref>) and the glass substrate (<xref ref-type="fig" rid="f1">Fig. 1k</xref>).</p></sec><sec disp-level="2"><title>Reflection profiles for the flat ITO film, nanolens-arrays, and nanolens-arrays with ITO film-coating on a Si substrate</title><p>In order to characterize the ITO-nanolens structure, we performed the following optical and electrical measurements. A reduction in reflection is crucial to drive more photons into a light-absorber. We measured reflectance profiles from various ITO structures on a Si substrate (<xref ref-type="fig" rid="f2">Fig. 2a</xref>). A flat 280 nm ITO film was prepared to compare the reflection features of a 200 nm-height ITO nanolens-array (Nanolens) and a 200 nm-height ITO-nanolens-array with a coating of 80 nm-thick ITO film (Nanolens + 80 nm-ITO film). The thickness of the additional 80 nm ITO coating film was chosen using an optical interfacial design for bare Si (See Methods), in order to minimize the reflectance<xref ref-type="bibr" rid="b28">28</xref><xref ref-type="bibr" rid="b44">44</xref>.</p><p>Average reflectance was found in the range of 400–1100 nm. A bare Si substrate gave a high reflection value of 38.90%. Meanwhile, a 280 nm-ITO film embedded Si substrate moderately reduced the reflection value to 18.63%. A nanolens-embedded surface more effectively reduced the reflection value (13.85%). A minimum reflection point (5.39%) was found at λ = 510 nm, without touching the zero reflection point.</p><p>A further reduction of the reflection of 4.70% was achieved from a nanolens with an ITO film-coating surface. The additional ITO film-coating fully covered the surface without leaving behind a bare Si region. It is worthwhile to note that this structure provides broad near-zero reflection (R < 1%) for wavelengths between 608 nm and 751 nm.</p><p>A practical solar cell has a peak electric power generation condition, in which the light incident angle is normal to the solar cell, having a zero angle value. However, the incident angle is time-varying and therefore a sustainably high tolerance of the independence to the incident light angle is an important factor<xref ref-type="bibr" rid="b8">8</xref><xref ref-type="bibr" rid="b10">10</xref><xref ref-type="bibr" rid="b18">18</xref><xref ref-type="bibr" rid="b21">21</xref><xref ref-type="bibr" rid="b45">45</xref> to improve the solar cell power-generation amount and the utilization time as well.</p><p>An ellipsometric system was used to measure the angle-dependent reflectance profiles for the flat ITO film, nanolens-arrays, and nanolens-arrays with ITO film-coating on a Si substrate with incident angle variations from 30° to 75°; data are presented as color maps (<xref ref-type="fig" rid="f2">Fig. 2b–d</xref>). The flat ITO film has very limited wavelength range for zero-reflection and showed substantial reflectance profiles for short-wavelengths, indicating crucial angle dependencies. Meanwhile, the nanolens surface seems to be efficient at reducing reflectance values for wavelength variations. In addition, this structure sufficiently relieves the incident angle dependency. A further improvement has clearly been shown with the nanolens-arrays with ITO film. This structure significantly expands the near-zero reflection regions with a high tolerance to incident-light angles for broader wavelengths (<xref ref-type="supplementary-material" rid="s1">Supplementary section</xref> ‘Weighted- reflectance').</p></sec><sec disp-level="2"><title>Light distribution according to wavelengths</title><p>The transparent nanolens structure has a convex feature, and thus can work as a lens. In conventional optics, focusing size and depth are limited by diffraction phenomenon<xref ref-type="bibr" rid="b46">46</xref><xref ref-type="bibr" rid="b47">47</xref>. A solid immersion lens has been shown to have the potential to overcome the diffraction limit by filling an object space with a solid material, that has a high-refractive index<xref ref-type="bibr" rid="b47">47</xref><xref ref-type="bibr" rid="b48">48</xref><xref ref-type="bibr" rid="b49">49</xref><xref ref-type="bibr" rid="b50">50</xref>.</p><p>The ITO-nanolens can provide an active solution for efficient light management; it can focus the incident light into a light-absorber. When a plane wave is incident on the ITO-nanolens, the light is refracted by the interface curvature and establishes a length that can be focused. We have investigated the focal length (F<sub>lens</sub>) formation for short, medium, and long wavelengths, as shown in <xref ref-type="table" rid="t1">Table 1</xref> (also see Methods).</p><p>The behaviors of refraction tuning are directly related to the F<sub>lens</sub> formation, which is determined according to wavelength variations (<xref ref-type="supplementary-material" rid="s1">Supplementary section</xref> ‘Refraction tuning'). In the visible range, the ITO-nanolens effectively modulates the light propagation, focusing the shorter wavelength light into a deeper Si absorber and the longer wavelength light into a thinner Si absorber (<xref ref-type="fig" rid="f3">Fig. 3a</xref>).</p><p>The light-induced electric field (<italic>E<sub>light</sub></italic>) intensity distribution in a Si absorber is controlled by two factors. First, the absorption coefficient of Si (α<sub>si</sub>) is high at short-wavelengths<xref ref-type="bibr" rid="b51">51</xref>, resulting in fast decay of the field intensity in Si. Second, the F<sub>lens</sub> of the nanolens is gradually decreased, with increased wavelengths in the visible range (<xref ref-type="table" rid="t1">Table 1</xref>), due to the reduction of the refractive index of Si (n<sub>Si</sub>).</p><p>In order to investigate the optical interaction and the <italic>E<sub>light</sub></italic> distribution of the ITO-nanolens-embedded Si, a numerical simulation based on a finite-difference time-domain (FDTD) calculation (<xref ref-type="supplementary-material" rid="s1">Supplementary section</xref> ‘FDTD simulation') was performed for the ITO-nanolens/Si unit cell according to various wavelength photons (<xref ref-type="fig" rid="f3">Fig. 3b–e</xref>). Comparison results are also presented for the flat-ITO film on the Si substrate (<xref ref-type="fig" rid="f3">Fig. 3f–i</xref>). For the flat ITO film, the field magnitude is readily exponentially decayed in the Si absorber due to the increasing distance from the surface (<xref ref-type="fig" rid="f3">Fig. 3l</xref>). In contrast, the plot of <italic>E<sub>x</sub></italic>/<italic>E</italic><sub>0</sub> of the ITO-nanolens array has characteristic patterns by modulating of the incident light, demonstrating the tendency of a strong <italic>E<sub>light</sub></italic> to appear along the z-direction of the Si depth (<xref ref-type="fig" rid="f3">Fig. 3j,k</xref>).</p><p>The ITO front surface consists of the nanolens-arrays. Thus, the light focused by the nanolens will meet the light from the neighboring nanolenses, which can cause interference. As a result, the lens array will generate second and third peaks (<xref ref-type="fig" rid="f3">Fig. 3a</xref>). The FDTD calculations show the field pattern, consistent with expectations for the intermediate wavelengths. For light wavelengths (<italic>λ</italic>) of 600 nm and 710 nm, the Si absorber holds a moderate value of α<sub>si</sub>. The first focusing peak appears close to the surface of the Si absorber. Neighboring nanolenses adjust the further light behavior, forming the second and third peak generations to the Si depth, (<xref ref-type="fig" rid="f3">Fig. 3c,d</xref>). The second peak generation is distinctively found along the ‘b-line', which is the center-point of neighboring nanolenses in the z-direction (<xref ref-type="fig" rid="f3">Fig. 3l</xref>). The third peak is formed along the ‘a-line', which is a direction of a single nanolens in the z-direction (<xref ref-type="fig" rid="f3">Fig. 3j</xref>). This light redirection phenomenon of incident light is uniquely observed for the ITO-nanolens structure, different from the multiple peak appearances of the tailored Si structure<xref ref-type="bibr" rid="b21">21</xref> or the light propagation by scattering<xref ref-type="bibr" rid="b38">38</xref>.</p><p>At a short-wavelength of 500 nm, the <italic>E<sub>light</sub></italic> intensity distribution shows a dominant first peak, close to an ITO-Si interface (<xref ref-type="fig" rid="f3">Fig. 3j</xref>). No second or third peak can be clearly found at λ = 500 nm due to the higher α<sub>si</sub> value at short-wavelengths, which results in fast decay of the field intensity (<xref ref-type="fig" rid="f3">Fig. 3b</xref>).</p><p>For long-wavelengths (e.g., λ = 1,100 nm), no significant peak can be clearly seen. The low refractive index of Si is the main physical origin that results in the weak lens effect. The incident photons are almost uniformly distributed in the Si absorber through the flat ITO-film without redirection of the incident light<xref ref-type="bibr" rid="b16">16</xref>. The span of the <italic>E</italic> is directly proportional to the wavelengths of the incident plane waves (<xref ref-type="fig" rid="f3">Fig. 3f–i</xref>). The flat ITO-film surface does not have an effect of focusing the incident light and thus, the light absorption of Si is a function of the distance from the surface, at a fixed wavelength. For a long-wavelength, the period of the peak <italic>E<sub>light</sub></italic> is extended compared to that of a short-wavelength. This is attributed to the increased span of the <italic>E<sub>light</sub></italic> distribution according to the increases of wavelength. The long-wavelength photons induce relatively little contribution due to the low α<sub>si</sub> value for plane wave propagation. Meanwhile, the nanolens seems to be an appropriate solution to improve the utilization of long-wavelength photons for Si solar cells.</p><p>The generation rate of photo-carriers is proportional to the field intensity in a Si absorber. Thus, a spatial distribution of the photo-carriers can be readily inferred from the <italic>E<sub>light</sub></italic> intensity distributions. Practically, some of the photo-generated carriers are recombined, limiting the energy conversion efficiency of the solar cell. The SCR holds a strong built-in potential (0.786 V, <xref ref-type="supplementary-material" rid="s1">Supplementary section</xref> ‘Built-in potential'), which is a driving force to sweep the photo-generated minority carriers (electrons in p-Si and holes in n-Si) in opposite directions<xref ref-type="bibr" rid="b11">11</xref>.</p><p>A strong built-in <italic>E</italic> develops inside the SCR (<italic>E<sub>SCR</sub></italic>) and the probability of photo-generated carrier collection is ideally unity in the SCR. Otherwise, a significant decay of the collection probability occurs in the neutral region (p-Si) with an increasing distance from the SCR due to the exponential decrease of photo-generated electrons. In addition, the carriers should be diffusively collected without assistance of the <italic>E<sub>SCR</sub></italic>. Meanwhile, a heavily doped emitter layer (n-Si) is an easy spot for serious recombination of photo-generated carriers<xref ref-type="bibr" rid="b11">11</xref><xref ref-type="bibr" rid="b29">29</xref>. This suggests an important clue. An enhanced performance solar cell may be achieved using a spatial overlapping of the high carrier generation region (<italic>E<sub>light</sub></italic>) and the space of the high carrier collection probability of SCR (<italic>E<sub>SCR</sub></italic>).</p></sec><sec disp-level="2"><title>Solar cell performances</title><p>According to the doping profile, we have measured the emitter thickness and found it to be 394 nm (<xref ref-type="fig" rid="f4">Fig. 4a</xref>, see Methods). Considering the heavy dopant concentrations (10<sup>18</sup>/cm<sup>3</sup> or higher), it is desirable to form a long F<sub>lens</sub> beyond the emitter thickness to minimize the serious recombination loss at the surface. The SCR width of our cells was 572 nm (<xref ref-type="supplementary-material" rid="s1">Supplementary section</xref> ‘SCR analysis'). Therefore, an effective <italic>E</italic> distribution should be positioned at a depth of 394–966 nm, where the SCR is located from the surface. The nanolens feature was designed with this consideration and fully satisfies the focal lengths in order to be positioned in the SCR for broad-band wavelengths (<xref ref-type="table" rid="t1">Table 1</xref>).</p><p>The geometric layout of the ITO-nanolens solar cell is presented as a schematic diagram (<xref ref-type="fig" rid="f4">Fig. 4b</xref>). A relatively large-size nanolens-embedded solar cell was prepared (<xref ref-type="fig" rid="f4">Fig. 4c</xref>) and we characterized light-performance, under one-sun illumination (see Methods). The nanolens device provided substantially improved open circuit voltage (590 mV), short circuit current (35.82 mA/cm<sup>2</sup>), and a conversion efficiency (16.0%) from 520 mV, 27.80 mA/cm<sup>2</sup> and 10.9% efficiency of a flat ITO-film solar cell (<xref ref-type="fig" rid="f4">Fig. 4d</xref>).</p><p>In what follows we discuss the main optical benefits of the ITO-nanolens solar cell performance. Overall, the ITO-nanolens device has higher internal quantum efficiency (IQE) values for the broad wavelengths than did the planar device (<xref ref-type="fig" rid="f4">Fig. 4e</xref>); this situation was different from that of the direct Si-etched structure<xref ref-type="bibr" rid="b12">12</xref><xref ref-type="bibr" rid="b52">52</xref>. A clear comparison of the two devices according to wavelength can be seen by plotting the relative IQE values of the ITO-nanolens over those of the ITO film (<xref ref-type="fig" rid="f4">Fig. 4f</xref>). The ITO nanolens collimates light into the SCR and also is effective to reduce reflection.</p><p>The ITO film has an intrinsic free carrier loss for short and long-wavelengths, without creating electron-hole pairs<xref ref-type="bibr" rid="b53">53</xref><xref ref-type="bibr" rid="b54">54</xref>, causing low IQE values for the flat ITO film device. Otherwise, the ITO-nanolens provides improved performance according to the manipulation of the incident light for short, long, and visible wavelength light by propagation, concentration, and tuning effect, respectively.</p><p>For short wavelengths, serious ITO loss can be relieved by passing the incident light through the nanolens to extend the short wavelength photon propagation into a deeper area of the Si. This significantly relieves the fast decay of the short-wavelength photons at the interface between the air and the ITO medium. Substantially enhanced IQE values were achieved at short-wavelengths. At λ = 300 nm, the IQE value of the ITO-nanolens was improved by 313% compared to that of the planar device. This is a distinctive advantage of the ITO-nanolens in that it can reduce the inherent ITO reflection loss for short-wavelengths<xref ref-type="bibr" rid="b21">21</xref>. The nanolens is effective in modulating the light propagation at short wavelengths, driving more photons to head towards the Si light-absorber.</p><p>For the visible range, the ITO-nanolens device showed generally improved IQE values. A 10% enhanced IQE value was achieved for the ITO-nanolens device compared to that of the planar device at λ = 600 nm, which is the most important incident light for a Si light-absorber<xref ref-type="bibr" rid="b11">11</xref>. The nanolens effectively modulates the visible wavelengths. Relatively long-wavelengths of visible light (red region, λ = 600–700 nm) have shorter F<sub>lens</sub> values (830.5–832.2 nm) than that (882.3 nm) of blue light (λ = 500 nm). The nanolens is efficient in tuning the visible-wavelength photons so that they are positioned in the SCR.</p><p>For long-wavelengths, the ITO-nanolens device shows prominently improved IQE performance, demonstrating an active scheme for low photon-energy utilization<xref ref-type="bibr" rid="b49">49</xref>. Light trapping of 800 nm–1100 nm wavelengths is crucial for Si<xref ref-type="bibr" rid="b2">2</xref>. At λ = 1100 nm, the IQE value of the ITO-nanolens was enhanced by 193.8%. Incident light moves as a plane wave into the Si absorber through the ITO medium. For long-wavelengths, the Si absorber has insufficient absorption of long wave photons. Thus, a planar ITO film device identically bears a critical problem against a long collection length for minority carriers (electrons) in p-type Si. This typically causes an extremely low QE performance for infrared regimes<xref ref-type="bibr" rid="b55">55</xref>. Meanwhile, the ITO-nanolens efficiently drives long-wavelength photons to be focused near the SCR. This light-adjustable effect spontaneously resolves the low light absorption limit of Si at long- wavelengths and simultaneously establishes a short collection length. Due to an existing strong <italic>E</italic> in the SCR, the photo-generated carriers can be effectively collected, resulting in improved QE values. The nanolens has a distinctive benefit in being able to concentrate the long-wavelengths into Si.</p><p>In summary, we propose a promising approach to nanostructure PVs by using a transparent ITO-nanolens without a direct etching of the semiconductor material and demonstrated a record high light-conversion efficiency of 16.0% among the periodic Si solar cells. A periodic structure of an ITO-nanolens was formed using a commercially viable printing method for large-scale devices. The ITO-nanolens provides optical and electrical benefits for nanostructured PVs. Due to the electrical conducting property, the ITO-nanolens supports the photo-generated carrier transport. For the optical aspects, the ITO-nanolens effectively adjusts the focal lengths for various light wavelengths, resulting in the overlapping of <italic>E<sub>light</sub></italic> and <italic>E<sub>SCR</sub></italic> to give a higher carrier collection efficiency. In previous work<xref ref-type="bibr" rid="b12">12</xref>, a microscale ITO lens was used. However, the efficiency improvement was not significant due to the off-positioned focal length location from SCR. Additionally, the ITO-nanolens can broaden near-zero reflection and provide high tolerance to the incident light angles. Ultimately, these benefits may enhance solar power-generation and utilization time. This optically transparent and electrically conductive nanolens architecture would be a promising scheme for the high-efficient nanostructure PVs.</p></sec></sec><sec disp-level="1" sec-type="methods"><title>Methods</title><sec disp-level="2"><title>The p-n junction formation</title><p>An emitter layer (n-Si) was formed on a Czochralski (CZ) grown 4-inch p-type (100) Si wafer, having a resistivity of 1–10 Ωcm. Phosphoryl chloride (POCl<sub>3</sub>) was used as a doping agent and was flowed into a doping furnace at 800°C for 40 min. The phosphosilicate glass (PSG) formed during the doping step was removed using buffered hydrofluoric acid (10 wt% HF).</p></sec><sec disp-level="2"><title>ITO-coating and design</title><p>Nanolens-arrays were deployed on a Si substrate. An additional ITO-coating layer (2<sup>nd</sup> ITO) was deposited to induce an electrical connection through all the nanolens entities. This is also an important detail that is used to control the high reflection of the bare Si region. A nanolens sitting on a Si region is optically affected by the ITO nanolens; however, a region not covered by an ITO nanolens displays the optical behavior of bare Si. Due to the intermediate refractive index of ITO film for air (n = 1) and Si (n = 3.54 ~ 5.57) systems, the insertion of an ITO film (n = 1.76 ~ 2.12) relieves the sudden changes of refractive indexes and therefore effectively diminishes the reflection. The optimum thickness of the 2<sup>nd</sup> ITO film was determined to be ~80 nm, after considering a quarter wavelength anti-reflection scheme (<italic>d = λ/4n</italic>), in which <italic>n</italic> is the refractive index of ITO (1.85 at λ = 600 nm). ITO depositions were performed in a dc-sputtering system using a 4-inch target composed of In<sub>2</sub>O<sub>2</sub> containing 10 wt% SnO<sub>2</sub> at a working pressure of 5 mTorr under Ar/O<sub>2</sub> (50/1) ambient condition.</p></sec><sec disp-level="2"><title>Calculation of focal lengths</title><p>The radius (R) of the ITO nanolens curvature can be achieved as in the following equation: <disp-formula id="m1"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e694" xlink:href="srep06879-m1.jpg"/></disp-formula>where h is the height of the ITO nanolens (200 nm) and r is the radius (180 nm). Considering these values, we have achieved an R value of 181 nm. In air conditions, the focal length (F<sub>air</sub>) of the ITO nanolens can be found using the following equation: <disp-formula id="m2"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e700" xlink:href="srep06879-m2.jpg"/></disp-formula>When we consider a Si medium, the focal length is multiplied by the refractive index of Si (n<sub>si</sub>). And thus, the effective focal length of the ITO nanolens (F<sub>lens</sub>) on Si can be extended according to the following sequence<xref ref-type="bibr" rid="b46">46</xref>: <disp-formula id="m3"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e712" xlink:href="srep06879-m3.jpg"/></disp-formula></p></sec><sec disp-level="2"><title>Emitter layer determination</title><p>The emitter depth was measured by secondary ion mass spectroscopy (SIMS, Cameca, magnetic sector ims7f). Regarding the p-Si doping level (~10<sup>16</sup>/cm<sup>3</sup>), a two order higher level (10<sup>18</sup>/cm<sup>3</sup>) was chosen for the n-type doping region in order to give a 394 nm-thickness to the emitter (<xref ref-type="fig" rid="f4">Fig. 4a</xref>).</p></sec><sec disp-level="2"><title>Device fabrication and light responses</title><p>A flat ITO device and a nanolens device were tailored to a size of 3.2 × 3.2 cm<sup>2</sup>. Solar cell performances were obtained under one-sun illumination using a simulator (McScience, K3000). The profiles of the carrier collection, according to wavelength variations, were obtained using a quantum efficiency measurement system (McScience, K3100).</p></sec></sec><sec disp-level="1"><title>Author Contributions</title><p>J.K. conceived this research. J.H.Y. designed the ITO-nanolens geometry and fabricated solar cell devices. J.Y. analyzed device performances. H.H.P. performed the nanoimprint method to fabricate the ITO-nanolens-arrays. E.L. performed FDTD simulation. J.Z. and J.S. performed the ellipsometric measurement. D.W.K. supervised E.L. W.A.A. supervised J.-H. N.M.L. supervised J.Z. J.K., D.W.K., W.A.A. and N.M.L. participated in discussion throughout the work and intensively cooperated to analyze results. M.M.D. Kumar analyzed QE performances. All the authors contributed to prepare this manuscript.</p></sec><sec sec-type="supplementary-material" id="s1"><title>Supplementary Material</title><supplementary-material id="d33e26" content-type="local-data"><caption><title>Supplementary Information</title><p>Incident light adjustable solar cell by periodic nanolens architecture</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="srep06879-s1.pdf"/></supplementary-material></sec> |
The effect of fluorescent nanodiamonds on neuronal survival and morphogenesis | <p>Nanodiamond (ND) has emerged as a promising carbon nanomaterial for therapeutic applications. In previous studies, ND has been reported to have outstanding biocompatibility and high uptake rate in various cell types. ND containing nitrogen-vacancy centers exhibit fluorescence property is called fluorescent nanodiamond (FND), and has been applied for bio-labeling agent. However, the influence and application of FND on the nervous system remain elusive. In order to study the compatibility of FND on the nervous system, neurons treated with FNDs <italic>in vitro</italic> and <italic>in vivo</italic> were examined. FND did not induce cytotoxicity in primary neurons from either central (CNS) or peripheral nervous system (PNS); neither did intracranial injection of FND affect animal behavior. The neuronal uptake of FNDs was confirmed using flow cytometry and confocal microscopy. However, FND caused a concentration-dependent decrease in neurite length in both CNS and PNS neurons. Time-lapse live cell imaging showed that the reduction of neurite length was due to the spatial hindrance of FND on advancing axonal growth cone. These findings demonstrate that FNDs exhibit low neuronal toxicity but interfere with neuronal morphogenesis, and should be taken into consideration when applications involve actively growing neurites (e.g. nerve regeneration).</p> | <contrib contrib-type="author"><name><surname>Huang</surname><given-names>Yung-An</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a2">2</xref><xref ref-type="author-notes" rid="n1">*</xref></contrib><contrib contrib-type="author"><name><surname>Kao</surname><given-names>Chun-Wei</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a3">3</xref><xref ref-type="author-notes" rid="n1">*</xref></contrib><contrib contrib-type="author"><name><surname>Liu</surname><given-names>Kuang-Kai</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Huang</surname><given-names>Hou-Syun</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a4">4</xref></contrib><contrib contrib-type="author"><name><surname>Chiang</surname><given-names>Ming-Han</given-names></name><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Soo</surname><given-names>Ching-Ren</given-names></name><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Chang</surname><given-names>Huan-Cheng</given-names></name><xref ref-type="aff" rid="a4">4</xref></contrib><contrib contrib-type="author"><name><surname>Chiu</surname><given-names>Tzai-Wen</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Chao</surname><given-names>Jui-I</given-names></name><xref ref-type="corresp" rid="c2">b</xref><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Hwang</surname><given-names>Eric</given-names></name><xref ref-type="corresp" rid="c3">c</xref><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a2">2</xref><xref ref-type="aff" rid="a3">3</xref></contrib><aff id="a1"><label>1</label><institution>Department of Biological Science and Technology, National Chiao Tung University</institution>, Hsinchu 30068, Taiwan</aff><aff id="a2"><label>2</label><institution>Institute of Bioinformatics and Systems Biology, National Chiao Tung University</institution>, Hsinchu 30068, Taiwan</aff><aff id="a3"><label>3</label><institution>Institute of Molecular Medicine and Bioengineering, National Chiao Tung University</institution>, Hsinchu 30068, Taiwan</aff><aff id="a4"><label>4</label><institution>Institute of Atomic and Molecular Sciences</institution>, Academia Sinica, Taipei 10672, Taiwan</aff> | Scientific Reports | <p>Nanodiamond (ND), a carbon derivative nanomaterial has become a promising candidate for biomedical applications<xref ref-type="bibr" rid="b1">1</xref><xref ref-type="bibr" rid="b2">2</xref><xref ref-type="bibr" rid="b3">3</xref><xref ref-type="bibr" rid="b4">4</xref><xref ref-type="bibr" rid="b5">5</xref><xref ref-type="bibr" rid="b6">6</xref><xref ref-type="bibr" rid="b7">7</xref><xref ref-type="bibr" rid="b8">8</xref><xref ref-type="bibr" rid="b9">9</xref>. NDs have been evaluated as a new class of nanocarrier for chemotherapeutic drugs by covalent or non-covalent linkages<xref ref-type="bibr" rid="b4">4</xref><xref ref-type="bibr" rid="b8">8</xref>. Moreover, ND containing nitrogen-vacancy centers can emit fluorescence property without photobleaching that is called fluorescent nanodiamond (FND), which has been applied for bio-labeling agent<xref ref-type="bibr" rid="b10">10</xref><xref ref-type="bibr" rid="b11">11</xref>. Although FNDs did not induce significant toxicity in various cellular and animal systems<xref ref-type="bibr" rid="b12">12</xref><xref ref-type="bibr" rid="b13">13</xref>, the effects of FNDs on the nervous system are still unclear.</p><p>The excellent biocompatibility of nanodiamond (ND) in various biological systems has made it a promising carbon nanomaterial for therapeutic applications, especially in cancer therapy. It has recently been demonstrated that doxorubicin-complexed ND markedly reduced the brain tumor via convection-enhanced intracranial delivery<xref ref-type="bibr" rid="b14">14</xref>. This result opens up the possibility of using ND to treat nervous system-related diseases and injuries. Previous researches have demonstrated the low cytotoxicity of ND in primary neurons or neurons derived from embryonal carcinoma cells<xref ref-type="bibr" rid="b15">15</xref><xref ref-type="bibr" rid="b16">16</xref><xref ref-type="bibr" rid="b17">17</xref>. Thalhammer <italic>et al</italic>. first demonstrated that culturing mouse hippocampal neurons on ND monolayer did not affect the neuronal attachment, neurite outgrowth, or cell-autonomous neuronal excitability compared to the traditional ECM coating<xref ref-type="bibr" rid="b15">15</xref>. The same group recently showed that the size and curvature of the NDs exhibited a significant correlation with neurite extension, the smaller the nanodiamond the longer the neurites extend<xref ref-type="bibr" rid="b16">16</xref>. Other diamond or diamond-like carbon surfaces have also been tested and exhibited excellent biocompatibility with neurons<xref ref-type="bibr" rid="b18">18</xref><xref ref-type="bibr" rid="b19">19</xref>. These researches suggested ND as a potential material for drug delivery in the nervous system. However, none of the experiments mentioned above examined the effect of ND after neurons have been seeded and developed, which is the state of the damaged neurons to be treated.</p><p>It has been demonstrated that microtubule stabilization with taxol can induce axon regeneration and improve functional outcome<xref ref-type="bibr" rid="b20">20</xref><xref ref-type="bibr" rid="b21">21</xref>. We have previously shown that taxol can be covalently linked to NDs and this ND-taxol blocked the tumor growth <italic>in vivo</italic><xref ref-type="bibr" rid="b8">8</xref>. These observations suggested the possibility of using ND to deliver taxol into injured nervous system in order to promote nerve regeneration. We selected FND in this study for its fluorescent property and high photostability. To minimize diffusion once FND has been injected into the nervous system, we decided to use FND with the size of 100 nm.</p><p>We first examined the biocompatibility of FND <italic>in vitro</italic> and discovered that FND did not induce cytotoxicity in CNS or PNS neurons. Intracranial injection of FND into the hippocampi of live rats did not cause gross behavioral differences compared to saline-injected rats. In addition, we demonstrated that FND could be internalized by neurons <italic>in vitro</italic> using flow cytometry and confocal microscopy. Surprisingly, we also discovered that FND caused a dosage-dependent decrease on neurite length in both CNS and PNS neurons <italic>in vitro</italic>. Finally, we performed live cell imaging on neurons treated with FND <italic>in vitro</italic> and found that the neurite length reduction effect was most likely the result of FND aggregates serving as spatial hindrance instead of as growth cone poison.</p><sec disp-level="1" sec-type="results"><title>Results</title><sec disp-level="2"><title>Fluorescence nanodiamonds did not induce cytotoxicity in CNS or PNS neurons</title><p>To test whether FNDs exhibit any cytotoxic effect on neurons, we utilized two primary neuron cultures: mouse hippocampal neurons (represent CNS neurons) and mouse dorsal root ganglion (DRG) neurons (represent PNS neurons). Both neurons were isolated from embryonic mouse pups and seeded into optical bottom 96-well plates to facilitate image acquisition and analysis. Various dosages of FNDs (1, 5, 10, 25, 50, 100, 250 µg/mL) were then applied to the hippocampal neurons DRG neurons (<xref ref-type="fig" rid="f1">Figure 1</xref>). After fixation, neurons were immunofluorescence stained with antibody against neuron-specific β-III-tubulin and the DNA stain DAPI. Images of hippocampal or DRG neurons were acquired using an automated fluorescence microscope. We quantified the number of neurons remain attached to the culture surface. This is a reliable indicator of viable neurons, because toxin-induced cell death caused neurons to detach from the culture surface. The neuron morphology quantification software NeurphologyJ<xref ref-type="bibr" rid="b22">22</xref> was used to quantify the number of neurons in hippocampal and DRG neuron cultures after FND treatment. We did not detect any significant change in hippocampal or DRG neuron number under various FND dosages (<xref ref-type="fig" rid="f2">Figure 2</xref>). In addition, we analyzed the fragmentation of the nuclear DNA after FND treatment. This is because cells undergoing apoptosis often exhibit fragmented nuclear DNA<xref ref-type="bibr" rid="b23">23</xref>. The circularity of the nuclear DNA was measured in FND treated neurons to deduce the morphology of the nuclear fragmentation. Nuclear DNA in healthy cells displays high circularity (>0.7), whereas those undergoing apoptosis display low circularity (<0.5). None of the FND dosage we examined altered the circularity of the nuclear DNA in dissociated hippocampal neurons (<xref ref-type="fig" rid="f3">Figure 3</xref>). Finally, we examined the level of activated caspase-3 in dissociated hippocampal neurons after FND treatment. Caspase-3 exists as inactive a proenzyme that needs to undergo proteolytic cleavage to generate the activated caspase-3, and this is a well-known apoptotic marker in various cell types including neurons<xref ref-type="bibr" rid="b24">24</xref><xref ref-type="bibr" rid="b25">25</xref><xref ref-type="bibr" rid="b26">26</xref>. None of the FND dosages we examined significantly increased the level of activated caspase-3 in hippocampal neurons (<xref ref-type="supplementary-material" rid="s1">Figure S1</xref>). Taken together, these results indicate that FNDs did not induce cytotoxicity at concentration up to 250 µg/mL.</p></sec><sec disp-level="2"><title>Intracranial injection of fluorescence nanodiamonds did not alter gross animal behavior</title><p>To further examine the effect of FNDs on the nerves system <italic>in vivo</italic>, we intracranially injected 10 μL of FNDs at a concentration of 100 μg/mL into the hippocampi of post-weaned juvenile rats. After the injection, the body weight, fodder and water consumption were assessed on a daily basis for one week. There were no significant differences in the daily changes of body weight or the daily consumption of fodder or water between FND- and saline-injected rats (<xref ref-type="fig" rid="f4">Figure 4A–C</xref>). We also performed a behavioral test to determine whether there were more subtle effects of FNDs on the hippocampus. The novel object recognition test (NORT), developed by Ennaceur and Delacour<xref ref-type="bibr" rid="b27">27</xref>, is based on the natural preference of rats to explore novel objects more than familiar ones. It has been shown that drugs which can damage the hippocampus lowered the discriminating index of NORT<xref ref-type="bibr" rid="b28">28</xref><xref ref-type="bibr" rid="b29">29</xref>. Therefore, we used NORT to assess the effects of FNDs on hippocampal neurons in behaving animals. Neither the procedure of intracranial injection nor the injection of FNDs to the hippocampus altered the discriminating index of NORT (<xref ref-type="fig" rid="f4">Figure 4D</xref>). These results suggested that FNDs didn't interfere with the general function of the hippocampus in live animals.</p></sec><sec disp-level="2"><title>Fluorescence nanodiamonds can be internalized by neurons</title><p>It has been well documented that FNDs can be taken up by cancer cells<xref ref-type="bibr" rid="b9">9</xref><xref ref-type="bibr" rid="b30">30</xref>. And it has recently been documented that FNDs can also be taken up by neurons derived from embryonal carcinoma cells <italic>in vitro</italic><xref ref-type="bibr" rid="b17">17</xref>. To confirm whether FNDs can be internalized by primary neurons, we treated dissociated mouse cortical neurons with 0, 50, 250 μg/mL of FNDs for 3 days and examined using flow cytometry (<xref ref-type="fig" rid="f5">Figure 5</xref>). The red fluorescence intensity from FNDs was excited with a 488 nm laser and the emission was collected at 650 ~ 750 nm wavelength range with flow cytometry. We detected a FND dosage-dependent increase in FND fluorescence signal, suggesting FNDs remain associated with dissociated neurons after they were released from the culture surface. However, flow cytometry cannot distinguish whether FND localized on the surface or the inside of neurons. Therefore, confocal microscopy was utilized to determine whether FND was indeed internalized by neurons. We treated dissociated mouse cortical neurons with 20 μg/mL of FNDs for 2 days, fixed, and stained with concanavalin-A and DAPI to detect plasma membrane and nuclear DNA. Clusters of FNDs can be detected inside the cell bodies of the cortical neurons (<xref ref-type="fig" rid="f6">Figure 6</xref>). Interestingly, internalized FND clusters were surrounded by concanavalin-A-containing organelles (<xref ref-type="fig" rid="f6">Figure 6C–E</xref>). To assure the red fluorescence came from FNDs, we repeated scanned the soma region in an attempt to photobleach all fluorophores except the unbleachable FNDs. Indeed, the only remaining fluorescence after the photobleaching process was the red emission from FNDs (data not shown). These results demonstrated that FNDs can be internalized by neurons, and internalized FNDs tend to aggregated together inside membrane-surrounded organelles.</p></sec><sec disp-level="2"><title>Fluorescence nanodiamonds affected neuronal morphogenesis in CNS and PNS neurons</title><p>Even though FNDs did not have any cytotoxic effect for neurons <italic>in vitro</italic> and <italic>in vivo</italic>, there remains a possibility that they can interfere with normal neuronal morphogenesis, a process requires intricate interaction between neurons and their extracellular environment. One of the most prominent morphological features of neurons is the elongated neurites. We therefore examined the effect of FNDs on neurite length. Various dosages of FND (1, 5, 10, 25, 50, 100, 250 µg/mL) were applied to the cultured neurons (hippocampal neurons for 3 days and DRG neurons for 2 days). Due to the excessive growth of axons after 48 hours (which interfered with our image analysis), DRG neurons were treated with FNDs for only 2 days. After fixation and immunofluorescence staining, images of neurons were acquired using the automated fluorescence microscopy and the neurite length of hippocampal or DRG neurons was quantified using NeurphologyJ<xref ref-type="bibr" rid="b22">22</xref> (<xref ref-type="fig" rid="f1">Figure 1</xref>). Surprisingly, FNDs provoked a dosage-dependent reduction in neurite length in both CNS hippocampal neurons (<xref ref-type="fig" rid="f7">Figure 7</xref>) and PNS DRG neurons (<xref ref-type="fig" rid="f8">Figure 8</xref>). Furthermore, we discovered a striking morphological alteration in DRG neurons under high dosage FND treatment (100 μg/mL and above). Almost all DRG neurons generated numerous terminal branches under this condition (<xref ref-type="fig" rid="f8">Figure 8A</xref>). To better understand the neurite length reduction effect of FNDs, we performed live cell imaging on DRG neurons after treatment with low dosage of FND (1 μg/mL) for 24 hours. We observed that the advancing growth cone of the DRG neuron stopped and retracted whenever it encountered FND clusters (<xref ref-type="fig" rid="f9">Figure 9</xref>). However, this growth cone remained motile and regrew after selecting a different advancing route (<xref ref-type="fig" rid="f9">Figure 9</xref> and <xref ref-type="supplementary-material" rid="s1">supplemental movies</xref>). This result suggested that the reduction of neurite length was due to the FND acting as a spatial hindrance of the advancing neuronal growth cone instead of acting as a poison that compromised the motility of the growth cone.</p></sec></sec><sec disp-level="1" sec-type="discussion"><title>Discussion</title><p>Consistent with previous researches<xref ref-type="bibr" rid="b15">15</xref><xref ref-type="bibr" rid="b16">16</xref><xref ref-type="bibr" rid="b17">17</xref><xref ref-type="bibr" rid="b31">31</xref>, we did not observe any cytotoxic effect of FND on primary CNS or PNS neurons. Furthermore, intracranial injection of FND into the hippocampus of the rat did not alter the animal behavior. We did notice a slight increase of GFAP-positive astrocytes in FND-injected hippocampus compared to saline-injected hippocampus (data not shown). However, the sample size of this observation was rather small and requires further examination. While FND uptake has been observed in neurons derived from embryonal carcinoma cells<xref ref-type="bibr" rid="b17">17</xref>, neurons have very different membrane protein composition and whether other types of neurons can uptake FND remained unclear. Here we demonstrated that FNDs could be internalized by primary cortical neurons <italic>in vitro</italic>. The fact that FNDs can be internalized by neurons hints at their applications as neurite tracer. We do not yet know whether internalized FNDs possess anterograde and/or retrograde motility.</p><p>We observed a dosage-dependent decrease of neurite length in both CNS and PNS neuron cultures in this study. However, a recently research showed that ND-coated surface enhanced neurite outgrowth in CNS neuron culture<xref ref-type="bibr" rid="b16">16</xref>. These seemingly contradicting results may be explained by the particle size of the NDs used and the attachment of NDs to the culture surface. While FNDs utilized in our study have an average size of 114.7 nm, the previous study used NDs with the size ranging from 20 nm to 35 nm<xref ref-type="bibr" rid="b16">16</xref>. The same research also showed that the particle size of the ND is negatively correlated with the neurite length, with the smallest ND particles promoting the best neurite outgrowth<xref ref-type="bibr" rid="b16">16</xref>. FNDs utilized in our study were not pre-coated on the culture surface and might form even larger aggregates. It is therefore possible that when the particle size exceedes a certain threshold, the neurite promoting effect may switch to a neurite inhibitory effect. Another origin of the discrepancy may come from the attachment of ND to the culture surface. FNDs we applied to our neuronal cultures were not pre-attached on the culture surface, but Edgington <italic>et al</italic>. used various approaches to attach their NDs to the surface. Although we did not observe prominent movements for large FND aggregates (<xref ref-type="fig" rid="f9">Figure 9</xref> and <xref ref-type="supplementary-material" rid="s1">supplemental movies</xref>), it is a possibility that smaller FND particles invisible to phase contrast microscopy were moving on the culture surface and compromising neurite extension.</p><p>Interestingly, a recent paper showed that titanium dioxide, silicon dioxide, and hydroxyapatite nanoparticles could destabilize the microtubule cytoskeleton and induce focal adhesion remodeling<xref ref-type="bibr" rid="b32">32</xref>. We do not believe FNDs caused microtubule disruption as microtubule filaments can still be observed in neurons treated with 250 μg/mL of FNDs (data not shown). Furthermore, our live cell imaging experiment showed that axon extension, a process that requires intact microtubule cytoskeleton, was not impaired in FND-treated DRG neurons.</p><p>In addition to the reduced neurite length, we also observed a drastic alteration of neurite morphology in DRG neurons under high dosage of FND (<xref ref-type="fig" rid="f8">Figure 8A</xref>). Numerous terminal branches can be seen reminiscent of the synaptic terminals of unmyelinated DRG axons observed <italic>in vivo</italic><xref ref-type="bibr" rid="b33">33</xref>. Whether these terminal branches had really developed into synapses in our high-dosage FND cultures reminds to be tested. Nonetheless, the drastic morphological changes caution any ND application involves actively growing neurites (e.g. nerve regeneration). It is also worth noting that some nanomaterials have been shown to disrupt the homophilic interaction of VE–cadherin at the adherens junction of the endothelial cells<xref ref-type="bibr" rid="b34">34</xref> Whether FNDs affect the synaptic junction between neurons remains to be tested.</p></sec><sec disp-level="1" sec-type="methods"><title>Methods</title><sec disp-level="2"><title>FND preparation</title><p>NDs with an average size of 100 nm were purchased from Element Six (Micron+ MDA, Element Six, Ireland). ND powders were radiation-damaged by using either a 40-keV He<sup>+</sup> beam at a dose of ~1 × 10<sup>14</sup> ions/cm<sup>2</sup> or a 3-MeV H<sup>+</sup> beam at a dose of ~1 × 10<sup>16</sup> ions/cm<sup>2</sup> to create the optimum amount of vacancies in the diamond crystal lattice, as previously described<xref ref-type="bibr" rid="b10">10</xref>. ND particles were subsequently annealed in vacuum at 800°C for 2 hours to form FND. The nitrogen-vacancy-containing particles were extensively rinsed in distilled deionized water and stored at room temperature prior to use. The particle size and morphology of FNDs were examined by a scanning electron microscope (S6700, JEOL, Japan) (<xref ref-type="supplementary-material" rid="s1">Figure S2</xref>). The average size of FNDs was around 114.7 ± 8.4 nm when analyzed by dynamic light scattering (BI-200SM, Brookhaven Instruments, Holtsville, NY) (<xref ref-type="supplementary-material" rid="s1">Figure S3</xref>). We also observed that our FNDs carried negative charge around −20.65 ± 1.63 mV as determined by zeta potential analysis (ZetaPALS, Brookhaven Instruments, Holtsville, NY).</p></sec><sec disp-level="2"><title>Primary neuron cultures</title><p>All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of National Chiao Tung University (NCTU) and in accordance with the Guide for the Care and Use of Laboratory Animals.</p><p>Hippocampal neurons from E18 mouse embryos (C57BL/6) were prepared as described<xref ref-type="bibr" rid="b35">35</xref>. Dissociated hippocampal neurons were seeded into poly-L-lysine-coated 96-well optical bottom plates at the density of 2 × 10<sup>4</sup> cells per well in 200 μL serum-containing neuronal plating medium (minimum essential medium supplemented with 5% fetal bovine serum, 0.6% D-glucose, and 2 mM L-glutamine; Life Technologies, Carlsbad, CA). The serum-containing medium was replace with 100 μL serum-free neuronal maintenance medium (neurobasal medium with B27 supplement and 0.5 mM L-glutamine; Life Technologies) 3 hours after seeding. 100 μL of 2× FND-containing serum-free neuronal maintenance medium was added 4 hours after seeding. Neurons were treated with 1, 5, 10, 25, 50, 100, or 250 μg/mL of FND. FND stock solution was vortexed for 30 seconds and sonicated for 30 mins before diluted in serum-free neuronal maintenance medium to minimize aggregation.</p><p>DRG neurons from E14 mouse embryos (C57BL/6) were prepared as described<xref ref-type="bibr" rid="b36">36</xref>. Dissociated DRG neurons were seeded into poly-L-lysine- and laminin-coated 96-well optical bottom plates at the density of 3 × 10<sup>3</sup> cells per well in serum-free DRG growth medium (neurobasal medium with 25 ng/mL NGF, B27 supplement, 2 mM L-glutamine, Life Technologies). Neurons were treated with 1, 5, 10, 25, 50, 100, or 250 μg/mL of FND at the time of seeding. FND stock solution was vortexed for 30 seconds and sonicated for 30 mins before diluted in serum-free DRG growth medium to minimize aggregation.</p></sec><sec disp-level="2"><title>Immunofluorescence staining and microscopy</title><p>Hippocampal and DRG neurons were fixed with 3.7% formaldehyde in 1xPBS at 37°C for 15 mins and 30 mins, respectively. Fixed neurons were permeabilized with 0.25% triton X-100 for 5 mins at room temperature, and blocked for 30 mins at 37°C with 10% BSA. Neurons were incubated for 1 hour at 37°C with primary antibodies (TUJ1, 1:4000, Covence; cleaved caspase-3 antibody, 1:400, Cell Signaling) in 2% BSA. Alexa Fluor 488-labeled secondary antibodies (1:1000, Life Technologies) were incubated for 1 hour at 37°C in the dark. Fluorescence images were acquired with a Nikon Eclipse-Ti inverted microscope equipped with a Photometrics CoolSNAP HQ<sup>2</sup> CCD camera. A 10× 0.45 N.A. Plan Apochromat objective lens and the Nikon NIS-Element imaging software were used to automatically acquire fluorescence images for neuronal morphology quantification. A 20× 0.75 N.A. Plan Apochromat objective lens was used to automatically acquire fluorescence images for activated caspase-3 quantification.</p><p>Live cell imaging was performed on the same Nikon microscope equipped with a Tokai Hit TIZHB live cell chamber. Time-lapse series were acquired using a 20× 0.45 N.A. Ph1 Plan Fluor objective. Time-lapse series were acquired at 5 mins intervals. Dissociated DRG neurons were cultured with or without 1 μg/mL FNDs in a poly-L-lysine- and laminin-coated 12-well plate for 18 hours. Live cell imaging was conducted for the subsequent 22 hours.</p><p>For confocal microscopy of dissociated neurons, 5 × 10<sup>5</sup> mouse cortical neurons were seeded into a 35 mm optical bottomed dish (μ-dish, ibidi, Germany) in serum-containing neuronal plating medium. The serum-containing medium was replaced by freshly made serum-free neurobasal medium containing 20 μg/mL of FND 4 hours afterwards. Neurons were fixed 2 days after seeding with 3.7% formaldehyde in 1xPBS for 15 mins at 37°C. After washing with 1xPBS three times, neurons were incubated with Alexa Fluor 488-labeled concanavalin-A (50 μg/mL, Life Technologies) and DAPI (5 μg/mL, Life Technologies) for 1 hour at 37°C. FND particles were excited with wavelength centered at 580 nm and the emission was collected in the wavelength range of 610 ~ 750 nm using a Leica TCS-SP5-X microscopy system.</p></sec><sec disp-level="2"><title>Cleaved caspase-3 quantification</title><p>Dissociated hippocampal neurons treated with FNDs were fixed and immunofluorescence stained as previously described. DAPI stained images were used to identify the location of the nuclei, and the signal of the activated (cleaved) caspase-3 inside the nuclei was quantified using ImageJ. To eliminate the spectral bleed through of FND into the caspase-3 channel, signal before immunofluorescence staining was subtracted from the final caspase-3 signal.</p></sec><sec disp-level="2"><title>Intracranial injection</title><p>All experimental procedures were approved by the IACUC of NCTU and in accordance with the Guide for the Care and Use of Laboratory Animals. A total of 8 post-weaned juvenile rats (3 weeks old, 40–50 g, from BioLASCO, Taiwan) were used in this experiment. Each pair of control and experimental rats was selected from the same litter to minimize genetic variations. Both control and experimental rats were anesthetized with isofluorane (5% for induction and 2% for maintenance) and mounted on the standard stereotaxic instrument (Stoelting, Wood Dale, IL). A small holed was drilled according to landmarks in Paxinos and Watson's standard brain atlas<xref ref-type="bibr" rid="b37">37</xref>. 10 μL of FNDs (100 μg/mL) containing saline were injected into the experimental group hippocampi with a 31-gauge injection cannula that was connected to a 10 μL microsyringe (Hamilton, Reno, NV) (<xref ref-type="supplementary-material" rid="s1">Figure S4A</xref>). The solution was infused over a 10 mins period and the needle was left in hippocampus for 2 mins after the end of infusion. Control rats were injected with 10 μL saline. After the injection cannula was removed, Spongostan film (Ferrosan Medical Devices, Denmark) and bone wax (WPI Inc., Sarasota, FL) were covered on the opening of the skull and the resected skins were closed with fine suture (5/0; UNIK, Taiwan). After recovered from the anesthesia, rats were housed in individual chambers. The wounds, body weight, fodder and water consumptions were assessed on a daily basis. The daily changes of body weight, fodder and water consumptions were averaged and compared between experimental and control groups.</p></sec><sec disp-level="2"><title>The novel object recognition test (NORT)</title><p>The experimental environment (78 cm × 56 cm × 46 cm) was surrounded by white walls and the floor was covered with wood bedding. Objects were placed at the symmetrical positions 26 cm from the left or right sides, and 28 cm from the upper and lower sides of the box. At the end of each session, the 70% ethanol was used to clean the objects and box to remove any olfactory cues on the objects and in the box. To minimize the bias for the specific location, the familiar and non-familiar objects were randomly placed at the left or right position. To habituate to the perimental environment, rats were placed in the box and allowed for exploration for 15 mins an hour before the behavioral measurement. NORT consisted of the training and testing sessions and they were separated by a retention interval of 1 hour (<xref ref-type="supplementary-material" rid="s1">Figure S4B</xref>). NORT were conducted 1 week before and after the intracranial FNDs injection. During the training session, rats were placed in the box at the center of two identical objects and allowed to explore for 10 mins. At the end of the training session, the rats were return to the home cage and the two objects and the whole box were cleaned with 70% ethanol. After the retention interval, one familiar object was replaced by a novel object. In the testing session, rats were return to the box and exposed to the objects for 10 mins. Both the training and testing session were recorded by a digital video camera and analyzed. The object exploration was defined as the rat pointing its nose to the object within 1 cm. The difference in time exploring the novel versus familiar objects normalized by the total exploring time for both objects in the testing session was the discriminating index (DI). The population data of DI was expressed as mean ± SEM. The effects of intracranial injections of FNDs were assessed by the two-way ANOVA with repeated measurements.</p></sec><sec disp-level="2"><title>Flow cytometry</title><p>Mouse cortical neuron cells were plated at a density of 5 × 10<sup>6</sup> cells in a 6-well plate coated with poly-L-lysine in serum-containing neuronal plating medium for 4 hours. The serum-containing medium was replaced by freshly made serum-free neurobasal medium containing various concentrations of FND 4 hours afterwards. Cells were treated with 0, 50, 250 μg/mL FNDs and incubated for 72 hours. At the end of the incubation, cells were collected by treating with 0.25% trypsin at 37°C for 30 mins, centrifuged at 1200 rpm, and fixed with ice-cold 70% ethanol overnight at −20°C. Thereafter, cells were centrifuged at 1200 rpm and cell pellets were re-dissolved with 1 mL 1xPBS. To avoid aggregation, fixed cells were filtered through a nylon membrane (BD Biosciences, San Jose, CA). A minimum of 1 × 10<sup>4</sup> cells in each samples were subjected to the fluorescence intensities analysis by CellQuest software in a flow cytometer (FACSCalibur, BD Biosciences). FNDs were analyzed by FL3 laser system (excitation: 488 nm; emission: 650 ~ 750 nm).</p></sec></sec><sec disp-level="1"><title>Author Contributions</title><p>Y.H., C.K., K.L., H.H., M.C. and C.S. conducted experiments. H.C. supplied materials and equipments. T.C., J.C. and E.H. designed experiments and wrote the manuscript. All authors discussed on the manuscript.</p></sec><sec sec-type="supplementary-material" id="s1"><title>Supplementary Material</title><supplementary-material id="d33e28" content-type="local-data"><caption><title>Supplementary Information</title><p>Supplementary Data</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="srep06919-s1.doc"/></supplementary-material></sec> |
Controllable Schottky Barriers between MoS<sub>2</sub> and Permalloy | <p>MoS<sub>2</sub> is a layered two-dimensional material with strong spin-orbit coupling and long spin lifetime, which is promising for electronic and spintronic applications. However, because of its large band gap and small electron affinity, a considerable Schottky barrier exists between MoS<sub>2</sub> and contact metal, hindering the further study of spin transport and spin injection in MoS<sub>2</sub>. Although substantial progress has been made in improving device performance, the existence of metal-semiconductor Schottky barrier has not yet been fully understood. Here, we investigate permalloy (Py) contacts to both multilayer and monolayer MoS<sub>2</sub>. Ohmic contact is developed between multilayer MoS<sub>2</sub> and Py electrodes with a negative Schottky barrier, which yields a high field-effect mobility exceeding 55 cm<sup>2</sup>V<sup>−1</sup>s<sup>−1</sup> at low temperature. Further, by applying back gate voltage and inserting different thickness of Al<sub>2</sub>O<sub>3</sub> layer between the metal and monolayer MoS<sub>2</sub>, we have achieved a good tunability of the Schottky barrier height (down to zero). These results are important in improving the performance of MoS<sub>2</sub> transistor devices; and it may pave the way to realize spin transport and spin injection in MoS<sub>2</sub>.</p> | <contrib contrib-type="author"><name><surname>Wang</surname><given-names>Weiyi</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Liu</surname><given-names>Yanwen</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Tang</surname><given-names>Lei</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Jin</surname><given-names>Yibo</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Zhao</surname><given-names>Tongtong</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Xiu</surname><given-names>Faxian</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a1">1</xref></contrib><aff id="a1"><label>1</label><institution>State Key Laboratory of Surface Physics and Department of Physics, Fudan University</institution>, Shanghai 200433, <country>China</country></aff> | Scientific Reports | <p>Molybdenum disulphide (MoS<sub>2</sub>) is a layered two-dimensional (2D) material, which draws intensive attention because of its unique electrical, optical and mechanical properties. It has been considered as a promising candidate for various future nanoelectronic and spintronic applications. It is a semiconductor with an indirect bandgap (1.2 eV) for its bulk material and a direct bandgap (1.8 eV) for monolayer MoS<sub>2</sub><xref ref-type="bibr" rid="b1">1</xref><xref ref-type="bibr" rid="b2">2</xref><xref ref-type="bibr" rid="b3">3</xref><xref ref-type="bibr" rid="b4">4</xref><xref ref-type="bibr" rid="b5">5</xref><xref ref-type="bibr" rid="b6">6</xref>. The presence of the direct bandgap in monolayer MoS<sub>2</sub> allows room-temperature FETs with an on/off current ratio exceeding 10<sup>8</sup>
<xref ref-type="bibr" rid="b7">7</xref>. Studies also point out that MoS<sub>2</sub> could be used in sensors<xref ref-type="bibr" rid="b8">8</xref> or for photoluminescence<xref ref-type="bibr" rid="b9">9</xref> applications. The spin transport properties were theoretically predicted with a spin diffusion length of 400 nm at room temperature, and it becomes longer than 800 nm at low temperature (<italic>T</italic> < 77 K)<xref ref-type="bibr" rid="b10">10</xref>. Because of the semiconducting nature, the contacts between MoS<sub>2</sub> and metal electrodes naturally forms Schottky barriers, which plays a crucial role in spin transport. To cope with this problem, theorists proposed that titanium is a suitable electrode material because of favorable geometry and large electronic density of state at the interface<xref ref-type="bibr" rid="b11">11</xref>. And experimentalists found that devices with scandium electrodes have very good performance because of low metal work function<xref ref-type="bibr" rid="b12">12</xref>. However, until now there is not much research on the ferromagnetic contact with MoS<sub>2</sub><xref ref-type="bibr" rid="b13">13</xref><xref ref-type="bibr" rid="b14">14</xref>, which may lead to the reduced conductance mismatch and the enhanced spin polarization.</p><p>In this letter, we study the contact between MoS<sub>2</sub> and ferromagnetic permalloy (Py) electrodes. The Schottky barrier height (SBH) is extracted by 2D thermionic emission theory<xref ref-type="bibr" rid="b13">13</xref><xref ref-type="bibr" rid="b15">15</xref>. For multilayer MoS<sub>2</sub>, we observed a barrier height of −5.7 mV. This negative Schottky barrier leads to a perfect Ohmic contact between MoS<sub>2</sub> and Py electrodes which dramatically enhances the field effect transistor (FET) performance of MoS<sub>2</sub>: the mobility of multilayer MoS<sub>2</sub> with a single back gate reaches 55 cm<sup>2</sup> V<sup>−1</sup>s<sup>−1</sup> at low temperature. For monolayer MoS<sub>2</sub>, the SBH has a positive value, which can be readily tuned by both back gate voltage and by the insertion of an Al<sub>2</sub>O<sub>3</sub> tunneling layer. When inserting 2.5 nm Al<sub>2</sub>O<sub>3</sub>, the SBH is reduced from 80.2 to 2.7 mV; by applying a back gate voltage of 30 V on the samples without Al<sub>2</sub>O<sub>3</sub>, the SBH is reduced to −8.3 mV. These two approaches of tuning the barrier height are important for realizing spin injection into monolayer MoS<sub>2</sub>.</p><sec disp-level="1" sec-type="results"><title>Results</title><sec disp-level="2"><title>Structure of the FET devices and method for extracting SBH</title><p>A schematic diagram of our devices is shown in <xref ref-type="fig" rid="f1">Figure 1a</xref>. Electrode on Si substrate is used as a back gate to tune Fermi level and carrier density. <xref ref-type="fig" rid="f1">Figure 1b</xref> shows a scanning electron microscopy (SEM) picture of the device. The channel length of the device is 2 μm and the width of exfoliated MoS<sub>2</sub> is about 4 μm. The channel material is verified to be tri-layer MoS<sub>2</sub> by a Raman spectrum<xref ref-type="bibr" rid="b16">16</xref> as shown in <xref ref-type="fig" rid="f1">Figure 1c</xref>. The devices were characterized by two-probe <italic>I-V</italic> measurement for Schottky barrier height and by <italic>I<sub>D</sub>-V<sub>G</sub></italic> measurement for mobility.</p><p>The forward <italic>I-V</italic> characteristics of an ideal Schottky diode can be described as follows<xref ref-type="bibr" rid="b17">17</xref>: <disp-formula id="m1"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e314" xlink:href="srep06928-m1.jpg"/></disp-formula>where <disp-formula id="m2"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e317" xlink:href="srep06928-m2.jpg"/></disp-formula><italic>I<sub>s</sub></italic> is the diode saturation current, <italic>A</italic> is the Richardson constant, <italic>S</italic> is the contact area of junction, <italic>q</italic> is the electron charge, Φ<italic><sub>B</sub></italic> is the Schottky barrier height, and <italic>k<sub>B</sub></italic> is the Boltzmann constant. As the device is thin enough to be treated as a 2D material, the drain-source current <italic>I<sub>DS</sub></italic> can be defined by 2D thermionic emission equation<xref ref-type="bibr" rid="b18">18</xref>, which employs the reduced power law <italic>T<sup>3/2</sup></italic> for a 2D transport channel: <disp-formula id="m3"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e356" xlink:href="srep06928-m3.jpg"/></disp-formula>where <italic>A*<sub>2D</sub></italic> is the 2D equivalent Richardson constant, <italic>n</italic> is the ideality factor, and <italic>V<sub>DS</sub></italic> is the drain-source bias voltage. To determine the Schottky barrier height Φ<italic><sub>B</sub></italic>, temperature dependent <italic>I-V</italic> measurements were carried out. <xref ref-type="fig" rid="f2">Figure 2a</xref> shows the <italic>I-V</italic> curves at several temperatures on a logarithmic scale. To investigate the barrier, it is common to use Arrhenius plot, <italic>i.e.</italic>, ln(<italic>I<sub>DS</sub></italic>/<italic>T</italic><sup>3/2</sup>) against 1000/<italic>T</italic> for various <italic>V<sub>DS</sub></italic> in <xref ref-type="fig" rid="f2">Figure 2b</xref>. By fitting the data to each <italic>V<sub>DS</sub></italic>, we obtained the slopes with <inline-formula id="m6"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e416" xlink:href="srep06928-m6.jpg"/></inline-formula>. Then by plotting the slopes as a function of <italic>V<sub>DS</sub></italic>, the SBH could be extracted from the y-intercept <inline-formula id="m7"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e425" xlink:href="srep06928-m7.jpg"/></inline-formula> (<xref ref-type="fig" rid="f2">Figure 2c</xref>).</p></sec><sec disp-level="2"><title>Schottky barrier height between Py and tri-layer MoS<sub>2</sub></title><p>For our tri-layer MoS<sub>2</sub> transistor device, Φ<italic><sub>B</sub></italic> is found to be −5.7 mV in the temperature regime of 100 ~ 200 K. Such a negative Schottky barrier produces a good Ohmic contact with a perfect linear <italic>I-V</italic> curve between MoS<sub>2</sub> and Py electrodes (<xref ref-type="supplementary-material" rid="s1">Figure S1</xref>). Similar results were also reported in <italic>p</italic>-type MoS<sub>2</sub> transistors with MoO<sub>x</sub> electrodes<xref ref-type="bibr" rid="b19">19</xref>. According to the Schottky-Mott model<xref ref-type="bibr" rid="b20">20</xref>, we could roughly estimate the SBH based on the work function of the metal Φ<italic><sub>metal</sub></italic> relative to the electron affinity (or vacuum ionization energy) of the semiconductor χ<sub>semi</sub>: <disp-formula id="m4"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e476" xlink:href="srep06928-m4.jpg"/></disp-formula>The negative Schottky barrier suggests that the work function of Py is slightly smaller than the affinity of MoS<sub>2</sub>, as shown in <xref ref-type="fig" rid="f1">Figure 1d</xref>.</p><p>To investigate the FET performance, we have performed temperature-dependent <italic>I<sub>D</sub>-V<sub>G</sub></italic> measurements. <xref ref-type="fig" rid="f3">Figure 3a</xref> shows typical gate-dependent conductance curves at different temperatures and <xref ref-type="fig" rid="f3">Figure 3b</xref> displays the temperature-dependent conductance under different gate voltage. It is noted that when applying a small back gate voltage (<italic>V<sub>G</sub></italic> < 35 V), the channel tri-layer MoS<sub>2</sub> shows an insulating behavior that the conductance decreases as temperature decreases. While <italic>V<sub>G</sub></italic> > 35 V the conductance increases as temperature decreases. This is a hallmark of metallic behavior which suggests that the tri-layer MoS<sub>2</sub> has entered a metallic state.</p><p>The field effect mobility can be calculated from the linear regime (40 ~ 50 V) of conductance curves using the following expression<xref ref-type="bibr" rid="b21">21</xref>: <disp-formula id="m5"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e522" xlink:href="srep06928-m5.jpg"/></disp-formula>where d<italic>G</italic>/d<italic>V<sub>G</sub></italic> is the slope of the conductance curve in the linear regime, <italic>L</italic> = 2 μm is the channel length, <italic>W</italic> = 4 μm is channel width and <italic>C<sub>ox</sub></italic> = 1.3 × 10<sup>−4</sup> F m<sup>−2</sup> is the capacitance between the channel and the back gate per unit area (<italic>C<sub>ox</sub></italic> = <italic>ε</italic><sub>0</sub><italic>ε<sub>r</sub></italic>/<italic>d</italic>; <italic>ε<sub>r</sub></italic> = 3.9; <italic>d</italic> = 270 nm). <xref ref-type="fig" rid="f3">Figure 3c</xref> shows the extracted mobility as a function of temperature under different drain-source voltage. The mobility is nearly independent of temperature when <italic>T</italic> < 20 K, indicating that the scattering of charged impurities is reduced by drain-source voltage<xref ref-type="bibr" rid="b22">22</xref><xref ref-type="bibr" rid="b23">23</xref>. At higher temperatures (<italic>T</italic> > 100 K), the mobility of the tri-layer MoS<sub>2</sub> is mainly influenced by the phonon scattering<xref ref-type="bibr" rid="b23">23</xref><xref ref-type="bibr" rid="b24">24</xref>. Fitting to the expression <italic>μ</italic> ~ <italic>T<sup>−r</sup></italic>, the range of exponent part <italic>γ</italic> can be obtained between 0.47 and 0.66. This value is much smaller than the theoretical prediction, <italic>i.e.</italic>, <italic>γ</italic> = 1.69 for single-layer MoS<sub>2</sub><xref ref-type="bibr" rid="b25">25</xref> or <italic>γ</italic> = 2.6 for bulk crystals<xref ref-type="bibr" rid="b26">26</xref>, indicative of a weak electron-phonon interaction<xref ref-type="bibr" rid="b20">20</xref>. The mobility of our tri-layer MoS<sub>2</sub> exceeds 55 cm<sup>2</sup>V<sup>−1</sup>s<sup>−1</sup>, which is a comparatively high value for single back gate MoS<sub>2</sub> transistor devices. As the Schottky barrier can significantly impact the electron mobility<xref ref-type="bibr" rid="b12">12</xref>, the high mobility of our device also provides a strong evidence of a low SBH between Py and tri-layer MoS<sub>2</sub>.</p></sec><sec disp-level="2"><title>Schottky barrier height between Py and monolayer MoS<sub>2</sub></title><p>Different from the tri-layer, the SBH between Py and the monolayer MoS<sub>2</sub> turns out to be 80.2 mV (<xref ref-type="fig" rid="f4">Figure 4d</xref>). This can be understood by the fact that the monolayer MoS<sub>2</sub> has a large bandgap of 1.8 eV and consequently it has a smaller electron affinity<xref ref-type="bibr" rid="b4">4</xref>. Thus, the increase of Schottky barrier height in the monolayer system is consistent with the Schottky-Mott model (<xref ref-type="disp-formula" rid="m4">Equation 4</xref>), <italic>i.e.</italic>, for the same metal work function, when the vacuum affinity decreases, the Schottky barrier height increases.</p><p>However, great caution must be exercised when the Arrhenius plot is used to extract SBH. The data in <xref ref-type="fig" rid="f4">Figure 4</xref>
<xref ref-type="fig" rid="f4">Figure 4a</xref> are not completely linear for the entire temperature range. In the high temperature regime (<italic>T</italic> > 130 K), the data show negative slopes, corresponding to the positive SBH. At low temperatures, however, ln(<italic>I<sub>DS</sub></italic>/<italic>T</italic><sup>3/2</sup>) versus 1000/<italic>T</italic> has positive correlations, suggesting negative SBH. These fitted results are contradictory to the observation of “S” shape <italic>I-V</italic> curves at low temperatures (<xref ref-type="supplementary-material" rid="s1">Figure S2</xref>), which are strong evidence of positive Schottky barriers. To explain this contradiction, one needs to take the semiconducting nature of MoS<sub>2</sub> into consideration. As the temperature decreases, the resistivity of monolayer MoS<sub>2</sub> increases and the device reaches an “off” state. It is well known that at the off state the channel resistance is too large that the current does not change much with temperature<xref ref-type="bibr" rid="b20">20</xref>. Thus, the thermionic emission equation is no longer suitable to describe the current. With this limitation, the SBH could only be extracted in the high temperature regime (above the turning points) using 2D thermionic emission equation, while the “V” shaped turning point in the Arrhenius plot represents the entrance of device “off” state.</p><p>To investigate the tunability of the Schottky barrier, we have performed the gate-dependent <italic>I-V</italic> measurements (<xref ref-type="fig" rid="f4">Figure 4a–c</xref>). When <italic>V<sub>G</sub></italic> = 0 V, the turning point is about 130 K. As the voltage is increased to 10 and 20 V, the turning point shifts to 20 and 8 K, respectively. Further increasing the gate voltage (up to 30 V) makes the turning point completely vanished (<xref ref-type="supplementary-material" rid="s1">Figure S3</xref>). For traditional field effect transistors, the channel conductance can be tuned by gate voltage. When <italic>V<sub>G</sub></italic> > 0 V, the conduction channel is broadened such that it needs a much lower temperature to turn the device off, which explains the systematic shift of the turning point. <xref ref-type="fig" rid="f4">Figure 4d</xref> summarizes the SBH as a function of <italic>V<sub>G</sub></italic>: the SBH is reduced to 20.4, 1.1 and −8.3 mV when <italic>V<sub>G</sub></italic> changes from 10, 20 to 30 V, respectively. This is attributed to the upwards shift of Py Fermi level by positive gate voltage (<xref ref-type="fig" rid="f4">Figure 4d</xref> inset)<xref ref-type="bibr" rid="b27">27</xref>.</p><p>For the spin injection into monolayer MoS<sub>2</sub>, the contact between ferromagnetic metal and semiconductor is crucial<xref ref-type="bibr" rid="b28">28</xref>. The spin polarization of injected carriers through Ohmic contact is extremely small due to the conductance mismatch<xref ref-type="bibr" rid="b13">13</xref>. In order to alleviate this issue, the resistance-area (RA) product should be designed properly to obtain a significant spin polarization and magnetoresistance<xref ref-type="bibr" rid="b29">29</xref>. In conventional 3D semiconductors, the Schottky barrier can provide the required resistance<xref ref-type="bibr" rid="b30">30</xref>, but the surface doping is needed to facilitate single step tunneling<xref ref-type="bibr" rid="b31">31</xref>. Another approach to obtain the proper RA product is to achieve a pinning-free FM/Oxide/SC interface by inserting a tunneling oxide layer, which could lower the SBH and the resistance could be tuned by the thickness of the oxide layer. Here we choose Al<sub>2</sub>O<sub>3</sub> as the tunneling oxide layer, which is well known for high spin injection efficiency and large tunneling magnetoresistance in magnetic tunnel junctions<xref ref-type="bibr" rid="b32">32</xref><xref ref-type="bibr" rid="b33">33</xref><xref ref-type="bibr" rid="b34">34</xref><xref ref-type="bibr" rid="b35">35</xref><xref ref-type="bibr" rid="b36">36</xref><xref ref-type="bibr" rid="b37">37</xref>.</p><p>Similar to the back gate voltage, the insertion of Al<sub>2</sub>O<sub>3</sub> layer also causes the “V” shaped turning point in the Arrhenius plot to shift towards lower temperatures (<xref ref-type="fig" rid="f5">Figure 5a–c</xref>). When the thickness of Al is 0.8, 1.7 and 2.5 nm, the turning point shifts to 40, 25 and 10 K respectively. This can be explained by the reduction of conductance mismatch<xref ref-type="bibr" rid="b38">38</xref> (or reduced SBH) between the electrodes and monolayer MoS<sub>2</sub> as Al<sub>2</sub>O<sub>3</sub> thickness increases. For that reason, the shift of the turning point could be regarded as an evidence of the alleviation of conductance mismatch with the inserted Al<sub>2</sub>O<sub>3</sub> layer. The corresponding SBH is extracted to be 32.1, 15.9 and 2.7 mV (<xref ref-type="fig" rid="f5">Figure 5d</xref>), respectively. Such a dramatic decrease shows an effective control of the SBH via changing the thickness of oxide layer. By the combination of applying a gate voltage and inserting an Al<sub>2</sub>O<sub>3</sub> layer, we could minimize the conductance mismatch and tune the Schottky barrier height down to zero, which may help to achieve the proper RA product.</p></sec></sec><sec disp-level="1" sec-type="discussion"><title>Discussion</title><p>In conclusion, we have investigated the properties of MoS<sub>2</sub> FET with ferromagnetic Py electrodes; the Schottky barrier height is extracted using 2D thermionic emission analysis of <italic>I-V</italic> curves. For the tri-layer MoS<sub>2</sub>, there is a negative Schottky barrier between Py and MoS<sub>2</sub> and this Ohmic contact yields a high mobility due to low contact resistance. For the monolayer MoS<sub>2</sub>, there is a positive Schottky barrier, which is dramatically reduced either by applying a gate voltage or inserting a tunneling Al<sub>2</sub>O<sub>3</sub> layer. To some extent, the insertion of Al<sub>2</sub>O<sub>3</sub> layer also alleviates the conductance mismatch. Such control of Schottky barrier paves the way of proper design of the RA product, which sheds light on the future research of spin transport and spin injections in MoS<sub>2</sub>.</p></sec><sec disp-level="1"><title>Experimental Section</title><p>Multilayer MoS<sub>2</sub> is obtained through mechanical exfoliation from bulk MoS<sub>2</sub> crystals onto pre-patterned SiO<sub>2</sub>/Si substrate (the thickness of SiO<sub>2</sub> is 270 nm). FET devices were fabricated by <italic>e</italic>-beam lithography (EBL) using PMMA/MMA bilayer polymer. Subsequently, Py electrodes are deposited by magnetron sputtering, followed by a deposition of gold layer to protect Py from oxidation. All the data are measured from electrode 1 and 2 in <xref ref-type="fig" rid="f1">Figure 1b</xref>. For the monolayer transistors, MoS<sub>2</sub> is obtained via chemical vapor deposition (CVD) using high purity molybdenum and sulphur as the source materials, similar to the previous report<xref ref-type="bibr" rid="b39">39</xref>. After growth, they were transferred onto clean SiO<sub>2</sub>/Si substrate for the following EBL process using PMMA stamping method<xref ref-type="bibr" rid="b40">40</xref>. The tunneling Al<sub>2</sub>O<sub>3</sub> layer was produced as follows: first a thin layer of Al was deposited by <italic>e</italic>-beam evaporation (the SEM picture in <xref ref-type="supplementary-material" rid="s1">Figure S4</xref> shows a high-quality Al layer without visible pinholes). Then the samples were placed in the air overnight for natural oxidation to develop Al<sub>2</sub>O<sub>3</sub>. The thickness of Al<sub>2</sub>O<sub>3</sub> can be estimated by <inline-formula id="m8"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e908" xlink:href="srep06928-m8.jpg"/></inline-formula><xref ref-type="bibr" rid="b41">41</xref>. Before measurement, the devices were annealed at 360 K for two hours in vacuum to remove polymer residues between the interface of Py and MoS<sub>2</sub><xref ref-type="bibr" rid="b42">42</xref><xref ref-type="bibr" rid="b43">43</xref>.</p></sec><sec disp-level="1"><title>Author Contributions</title><p>F.X. conceived the ideas and supervised the overall research. W.W. fabricated the devices and carried out the entire characterizations. Y.L. and T.Z. contributed to the sample preparation and measurement. L.T. and Y.J. provided the monolayer MoS<sub>2</sub> and took the Raman spectrum. W.W. and F.X. wrote the paper. All authors reviewed the manuscript.</p></sec><sec sec-type="supplementary-material" id="s1"><title>Supplementary Material</title><supplementary-material id="d33e33" content-type="local-data"><caption><title>Supplementary Information</title><p>Supplementary</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="srep06928-s1.pdf"/></supplementary-material></sec> |
Continuity of states between the cholesteric → line hexatic transition and the condensation transition in DNA solutions | <p>A new method of finely temperature-tuning osmotic pressure allows one to identify the cholesteric → line hexatic transition of oriented or unoriented long-fragment DNA bundles in monovalent salt solutions as first order, with a small but finite volume discontinuity. This transition is similar to the osmotic pressure-induced expanded → condensed DNA transition in polyvalent salt solutions at small enough polyvalent salt concentrations. Therefore there exists a continuity of states between the two. This finding, together with the corresponding empirical equation of state, effectively relates the phase diagram of DNA solutions for monovalent salts to that for polyvalent salts and sheds some light on the complicated interactions between DNA molecules at high densities.</p> | <contrib contrib-type="author"><name><surname>Yasar</surname><given-names>Selcuk</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Podgornik</surname><given-names>Rudolf</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a2">2</xref><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Valle-Orero</surname><given-names>Jessica</given-names></name><xref ref-type="aff" rid="a4">4</xref><xref ref-type="aff" rid="a5">5</xref><xref ref-type="author-notes" rid="n1">*</xref></contrib><contrib contrib-type="author"><name><surname>Johnson</surname><given-names>Mark R.</given-names></name><xref ref-type="aff" rid="a6">6</xref></contrib><contrib contrib-type="author"><name><surname>Parsegian</surname><given-names>V. Adrian</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a1">1</xref></contrib><aff id="a1"><label>1</label><institution>Department of Physics, University of Massachusetts</institution>, Amherst, MA 01003, <country>United States</country></aff><aff id="a2"><label>2</label><institution>Department of Theoretical Physics, J. Stefan Institute</institution>, SI-1000 Ljubljana, <country>Slovenia</country></aff><aff id="a3"><label>3</label><institution>Department of Physics, Faculty of Mathematics and Physics, University of Ljubljana</institution>, SI-1000 Ljubljana, <country>Slovenia</country></aff><aff id="a4"><label>4</label><institution>Institut Laue Langevin, BP 156</institution>, 6, rue Jules Horowitz 38042 Grenoble Cedex 9, <country>France</country></aff><aff id="a5"><label>5</label><institution>Laboratoire de Physique, Ecole Normale Superiéure de Lyon</institution>, 46 allée d'Italie, 69364 Lyon Cedex 07, <country>France</country></aff><aff id="a6"><label>6</label><institution>Institut Laue-Langevin, 6 rue Jules Horowitz</institution>, BP156 38042, Grenoble, <country>France</country></aff> | Scientific Reports | <p>DNA at elevated osmotic pressures, accessible in highly concentrated DNA solutions <italic>in vitro</italic>, exhibits a sequence of ordered liquid-crystalline mesophases<xref ref-type="bibr" rid="b1">1</xref><xref ref-type="bibr" rid="b2">2</xref><xref ref-type="bibr" rid="b3">3</xref> whose properties determine the nature of high density DNA compaction also in the biological milieu characterized by similar DNA densities, bathing solution conditions, and osmotic pressures. These biologically relevant examples of high density DNA compaction include most importantly DNA packing within virus capsids at osmotic pressures exceeding 60 atm and at densities within the regime of highly concentrated DNA solutions<xref ref-type="bibr" rid="b4">4</xref><xref ref-type="bibr" rid="b5">5</xref><xref ref-type="bibr" rid="b6">6</xref><xref ref-type="bibr" rid="b7">7</xref><xref ref-type="bibr" rid="b8">8</xref>. Moreover, in eukaryotic sperm cells, DNA is packaged by a variety of simple basic proteins with positively charged polypeptide chains<xref ref-type="bibr" rid="b9">9</xref><xref ref-type="bibr" rid="b10">10</xref><xref ref-type="bibr" rid="b11">11</xref><xref ref-type="bibr" rid="b12">12</xref> that condense DNA as condensing agents do in solution conditions<xref ref-type="bibr" rid="b13">13</xref><xref ref-type="bibr" rid="b14">14</xref>. While the general outlines of the long-fragment (few microns long) DNA phase diagram seem to be properly characterized, with a well established progressive ordering sequence, isotropic → cholesteric → line hexatic → orthorhombic phases, many details including the fragment length dependence<xref ref-type="bibr" rid="b15">15</xref> remain to be systematically investigated.</p><p>In this paper, we concentrate on the question of the organization and packing of genomic length DNA chains and parameterization of the forces governing their interactions at biologically relevant DNA densities and osmotic pressures. The two most important DNA liquid-crystalline phases at these densities are the line hexatic<xref ref-type="bibr" rid="b1">1</xref> (LH) and the cholesteric<xref ref-type="bibr" rid="b16">16</xref>. The more ordered LH phase is observed at higher DNA densities, i.e., from approximately 300 mg/ml to 700 mg/ml, while the cholesteric phase then extends all the way to the isotropic phase, in which DNA is not ordered. In both the cholesteric and LH phases, DNA is locally oriented and positionally organized in a lattice with hexagonal bond orientational order<xref ref-type="bibr" rid="b17">17</xref>. However, while the bond orientational order in the cholesteric phase is short-range, it becomes <italic>macroscopic</italic> in the LH phase<xref ref-type="bibr" rid="b18">18</xref>, leading to the appearance of a sixfold azimuthally modulated x-ray diffraction intensity of the first-order diffraction peak when the chains are aligned parallel to the x-ray beam<xref ref-type="bibr" rid="b1">1</xref>. Previous work could not definitively address the question whether this change in the nature of order is continuous or not.</p><p>While for long-fragment DNA the positional order in both phases remains liquid-like, the range of ordering changes quantitatively as the system is pushed through the transition between these two phases. In what follows, we present evidence that the range of the bond orientational order and the nature of the local positional ordering change abruptly at the transition between these two phases, leading to a discontinuous jump in the DNA density and thus exhibiting the characteristics of a first-order transition. Furthermore, we note that the abrupt change in the radial widths of the first-order diffraction peaks, indicating the range of the positional order, occurs concurrently with the appearance of the sixfold azimuthal modulation of the first-order diffraction peak. A sharper well-defined peak seen in the LH phase indicates strong suppression of conformational fluctuations and the interaxial separation between the neighboring DNA chains changes discontinuously at the cholesteric → LH transition, signaling a discontinuous change also in DNA density, controlled and varied by the osmotic stress of the bathing solution<xref ref-type="bibr" rid="b19">19</xref>. In order to observe this discontinuous change induced by the solution osmotic pressure, separating a first-order from a second-order transition between these two mesophases, a very precise means of controlling the DNA density is needed, implying also an accurate tuning of the osmotic pressure of the DNA solution. The latter is accomplished by fine temperature tuning of the bathing solution and its osmotic pressure, as described below.</p><p>We address two important unresolved issues pertaining to the phase diagram of long-fragment DNA: (<italic>i</italic>) the nature and the ionic strength dependence of the cholesteric → LH phase transition and (<italic>ii</italic>) the connection between this DNA ordering transition in monovalent salts and the DNA condensation transition in polyvalent salts<xref ref-type="bibr" rid="b20">20</xref>. By using a new method of finely temperature-tuning the osmotic pressure, we thus find that the cholesteric → LH transition in monovalent salts is first order, just like the cholesteric → columnar hexagonal transition in short-fragment (≈50 nm long) DNA<xref ref-type="bibr" rid="b15">15</xref>, with a small but finite volume change. We also find a <italic>continuity of states</italic>, as opposed to the qualitatively different behavior usually invoked when comparing monovalent and polyvalent salt DNA solutions<xref ref-type="bibr" rid="b20">20</xref>, between this ordering transition in monovalent salts and the condensation transition induced by osmotic pressure at subcritical <inline-formula id="m11"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e279" xlink:href="srep06877-m11.jpg"/></inline-formula> (i.e., CoHex) concentration. The elucidation of these features of the DNA phase diagram in various solution conditions is particularly important for the description of DNA packing in bacteriophage capsids<xref ref-type="bibr" rid="b4">4</xref><xref ref-type="bibr" rid="b5">5</xref><xref ref-type="bibr" rid="b6">6</xref><xref ref-type="bibr" rid="b7">7</xref><xref ref-type="bibr" rid="b8">8</xref>, occurring within the same range of DNA densities and osmotic pressures, as well as for understanding the long-range interactions that drive the DNA condensation in polyvalent salts. They have both been the subject of focused theoretical efforts<xref ref-type="bibr" rid="b21">21</xref><xref ref-type="bibr" rid="b22">22</xref><xref ref-type="bibr" rid="b23">23</xref>.</p><p>Ever since its discovery<xref ref-type="bibr" rid="b1">1</xref><xref ref-type="bibr" rid="b24">24</xref>, the nature of the cholesteric → LH transition has remained unresolved and was presumed to be either continuous (second order) or weak first-order with a small volume discontinuity. The implied <italic>caveat</italic> has always been that the available accuracy of osmotic pressure resolution does not allow for a definitive resolution of the order of the transition and that the absence of detectable density discontinuity in the equation of state should not be interpreted as a definitive evidence that the transition is second order. A distinct possibility would thus exist that there is an extremely narrow phase-coexistence window that can not be resolved by the osmotic stress method<xref ref-type="bibr" rid="b2">2</xref>. However, this phase-coexistence window, on the order of ~1–2Å interaxial spacing wide, has now been detected in monovalent salt DNA solutions through high resolution control of the osmotic pressure, based on its known temperature variation<xref ref-type="bibr" rid="b25">25</xref>. While not completely unexpected, the existence of this phase-coexistence window in monovalent salt solutions is nevertheless surprising. In fact, phase coexistence at finite osmotic pressures has been heretofore observed only in the case of DNA solutions with polyvalent counterions, e.g., with CoHex, or other condensing agents, when the polyvalent counterion concentration is below a critical value that would induce an immediate precipitation of DNA. In fact, at these subcritical polyvalent salt concentrations DNA condensation does not occur spontaneously but has to be induced by an additional solution osmotic pressure<xref ref-type="bibr" rid="b26">26</xref> that then pushes DNA through a clearly detectable first-order expanded → condensed transition. If the concentration of the polyvalent salt is then increased above a critical value that depends on other solution parameters, DNA condenses spontaneously without any need for an additional osmotic pressure push from the solution. The ensuing DNA condensation transition then becomes second order.</p><p>We now connect the existence of the finite density jump for subcritical polyvalent salt solutions with a similar first-order cholesteric → LH transition in the case of monovalent (NaCl) salts. This effectively unifies the phase diagrams of DNA for mono- and polyvalent salts and allows us to describe quantitatively the whole regime of DNA equation of state, i.e., the osmotic pressures vs. density dependence, including the volume discontinuity at the cholesteric → LH for monovalent salts or expanded → condensed DNA transitions for polyvalent salts, with a simple empirical equation of state. It identifies a universal attractive interaction between DNA molecules, even in a monovalent (NaCl) salt, that probably stems from a structural adaptation of DNA helices to the strong interaxial hydration and/or electrostatic interactions at high densities. A picture showing more intimate connection between the nature of the positional, orientational, and bond orientational order<xref ref-type="bibr" rid="b17">17</xref><xref ref-type="bibr" rid="b18">18</xref><xref ref-type="bibr" rid="b27">27</xref> and the ensuing interaxial interactions between DNA double helices is thus clearly emerging. This change in perspective should be relevant also for understanding the high-density DNA packing in viruses<xref ref-type="bibr" rid="b4">4</xref><xref ref-type="bibr" rid="b5">5</xref><xref ref-type="bibr" rid="b6">6</xref><xref ref-type="bibr" rid="b7">7</xref><xref ref-type="bibr" rid="b8">8</xref><xref ref-type="bibr" rid="b28">28</xref><xref ref-type="bibr" rid="b29">29</xref><xref ref-type="bibr" rid="b30">30</xref>, where DNA is under high osmotic pressure/high density conditions identical to those studied in our experiments, and on ejection driven by these osmotic stored forces undergoes a series of phase transitions<xref ref-type="bibr" rid="b7">7</xref><xref ref-type="bibr" rid="b8">8</xref> directly related to those addressed in this work. Indeed, the osmotic pressures (≈60 atm)<xref ref-type="bibr" rid="b28">28</xref><xref ref-type="bibr" rid="b29">29</xref> and the corresponding DNA interaxial spacings (≈26–27Å)<xref ref-type="bibr" rid="b31">31</xref> are right in the regime studied in this manuscript.</p><p>The details of the organization of DNA and genome regulation processes in eukaryotic cells are beyond the scope of this study. Briefly, in eukaryotic cells, DNA is packaged in repeating units, a length of DNA (≈50 nm) wound around nucleosomal core particles which consist of positively charged histone proteins<xref ref-type="bibr" rid="b32">32</xref>. In this <italic>diffuse</italic> packaging, the DNA molecule and its genome is organized in a way that specific genes are still available for transcription. However, in the compaction of DNA in eukaryotic sperm cells, the histones are replaced with much simpler arginine-rich protamines that pack DNA into a highly condensed hexagonal lattice<xref ref-type="bibr" rid="b34">34</xref>, identical to DNA-protamine condensation <italic>in vitro</italic><xref ref-type="bibr" rid="b14">14</xref><xref ref-type="bibr" rid="b33">33</xref>. The repeating unit of the hexagonal lattice consists of a length of DNA chain and an associated protamine polypeptide chain, as observed in x-ray diffraction of sperm chromatin<xref ref-type="bibr" rid="b34">34</xref>, with the polypeptide chain locked in the major groove of the DNA double helix so that the DNA charge is almost completely neutralized<xref ref-type="bibr" rid="b11">11</xref><xref ref-type="bibr" rid="b34">34</xref><xref ref-type="bibr" rid="b35">35</xref><xref ref-type="bibr" rid="b36">36</xref>. The volume occupied by DNA in the sperm cell is small compared with the volume of chromatin in somatic cells. The highly condensed, inactive state of the DNA in sperm nuclei confers additional protection against damage from the effects of mutagens and genotoxic factors<xref ref-type="bibr" rid="b37">37</xref><xref ref-type="bibr" rid="b38">38</xref><xref ref-type="bibr" rid="b39">39</xref>.</p><p>The study of highly concentrated DNA solutions is thus not only relevant from the fundamental biophysics point of view, but it also sheds light on the molecular mechanisms of DNA packing in bacteriophages and eukaryotic sperm cells. It also constrains possible mechanisms of gene delivery<xref ref-type="bibr" rid="b40">40</xref> and illuminates fundamental physical principles that extend to other areas of condensed matter physics<xref ref-type="bibr" rid="b17">17</xref><xref ref-type="bibr" rid="b18">18</xref>.</p><sec disp-level="1" sec-type="results"><title>Results</title><p>Our first significant observation is the abrupt change in the DNA density and order at the cholesteric → LH transition in NaCl solutions. The abrupt transition, from less-ordered cholesteric phase into the more-ordered LH phase, is evident in the x-ray diffraction behavior of unoriented<xref ref-type="bibr" rid="b26">26</xref> and oriented<xref ref-type="bibr" rid="b41">41</xref> long-fragment DNA samples. Higher-order peaks in the diffraction intensity profiles confirm hexagonal packing in the LH phase; hexagonal packing is assumed in the cholesteric phase. With our new experimental methodology of finely tuning the osmotic pressure (Π) of PEG solutions via temperature (T) variations<xref ref-type="bibr" rid="b25">25</xref>, the transitions are measured with high accuracy (see <xref ref-type="fig" rid="f1">Fig. 1</xref>). From DNA samples in NaCl solutions at different [PEG] and T (but approximately the same Π), the same interaxial distance (<italic>d<sub>int</sub></italic>) and full width at half-maximum (FWHM) are obtained. The impact of T (i.e., for 15°C ≤ T ≤ 45°C) on <italic>d<sub>int</sub></italic> and FWHM is thus only through its effect on Π; changing T in the cases considered does not translate into a direct effect on the interactions between the DNA chains as is the case for, e.g., Mn<sup>2+</sup> condensation<xref ref-type="bibr" rid="b42">42</xref>. This is evident in the data shown for [NaCl] = 0.1 M (see <xref ref-type="fig" rid="f1">Fig. 1</xref>) and a large number of similar measurements under 0.05 M ≤ [NaCl] ≤ 0.4 M. We undertake further measurements of the cholesteric → LH transitions based on this fact (see <xref ref-type="fig" rid="f2">Fig. 2</xref>).</p><p>In the cholesteric phase, there is a broad first-order x-ray diffraction peak (see <xref ref-type="fig" rid="f1">Fig. 1</xref>). Upon increasing Π, at the cholesteric → LH transition, the diffraction intensity profile changes abruptly, and a sharp peak (with FWHM approximately 5 times bigger than the instrumental resolution) is superimposed on the broad cholesteric peak. In addition, oriented DNA samples, with the helical axis parallel to the x-ray beam, give sixfold symmetric first-order diffraction peaks in the LH phase, indicating <italic>macroscopic</italic> bond orientational order<xref ref-type="bibr" rid="b1">1</xref> perpendicular to the local axis of the molecules. Disorder in the packing increases with increasing DNA density in the LH phase (see <xref ref-type="fig" rid="f1">Fig. 1</xref>), which points to the possibility of frustrated ordering at high densities<xref ref-type="bibr" rid="b27">27</xref>. After progressive disordering at high densities in LH phase, DNA eventually crystallizes through a LH → orthorhombic transition (discussed below) into an orthorhombic crystal. Conversely, in the cholesteric phase, we observed further broadening of the diffraction peak with decreasing DNA density. It is also worth emphasizing that in the cholesteric phase the diffraction peak width is sensitive to [NaCl], and increases with increasing [NaCl] at fixed DNA density (see <xref ref-type="fig" rid="f2">Fig. 2</xref>).</p><p>We measured the osmotic pressure of DNA arrays via the osmotic stress method<xref ref-type="bibr" rid="b19">19</xref><xref ref-type="bibr" rid="b43">43</xref><xref ref-type="bibr" rid="b44">44</xref>. The osmotic pressure data Π vs. <italic>d<sub>int</sub></italic>, for different [NaCl] are shown in <xref ref-type="fig" rid="f3">Fig. 3</xref>. In order to parameterize in a simple way the experimentally determined dependence of the osmotic pressure on <italic>d<sub>int</sub></italic> and ionic strength, we invoke a cylindrical cell model formulation of the linearized Poisson-Boltzmann theory<xref ref-type="bibr" rid="b45">45</xref>. This does not imply that this approximate theory can describe all the details of the DNA-DNA interaction appropriately. Nevertheless it serves the purpose of parsimoniously parameterizing a vast amount of data by intuitive effective parameters. In this simplified model, a molecule of radius <italic>a</italic> (≈10Å for B-DNA) is considered to be surrounded by a cylindrical cell of radius <italic>R</italic> = <italic>d<sub>int</sub></italic>/2, yielding the electrostatic part of the osmotic pressure as <disp-formula id="m1"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e423" xlink:href="srep06877-m1.jpg"/></disp-formula>to the leading order. Here <italic>K</italic><sub>0</sub>(<italic>x</italic>) and <italic>K</italic><sub>1</sub>(<italic>x</italic>) are the cylindrical Bessel functions of the second kind. <italic>λ<sub>D</sub></italic> is the Debye length and <inline-formula id="m12"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e448" xlink:href="srep06877-m12.jpg"/></inline-formula>, where <italic>σ</italic> is the effective surface charge density of DNA, and <inline-formula id="m13"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e455" xlink:href="srep06877-m13.jpg"/></inline-formula> is the dielectric permittivity of the medium (<inline-formula id="m14"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e458" xlink:href="srep06877-m14.jpg"/></inline-formula> is vacuum permittivity and <inline-formula id="m15"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e461" xlink:href="srep06877-m15.jpg"/></inline-formula> for water). The effect of T on <inline-formula id="m16"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e464" xlink:href="srep06877-m16.jpg"/></inline-formula>, over the range of temperatures used in the measurements, is small and can be ignored. For fully charged B-DNA, <italic>σ</italic> = <italic>e</italic><sub>0</sub>/(2<italic>πab</italic>), where <italic>e</italic><sub>0</sub> is the elementary charge and <italic>b</italic> ≈ 1.7Å is the linear density of phosphates on the DNA. In LH phase, where conformational fluctuation effects are negligible, the net repulsion is equal to the bare interaction osmotic pressure, i.e., Π<sub>0</sub>(<italic>R</italic>) = Π<italic><sub>h</sub></italic>(<italic>R</italic>) + Π<italic><sub>e</sub></italic>(<italic>R</italic>). The hydration repulsion<xref ref-type="bibr" rid="b46">46</xref>, Π<italic><sub>h</sub></italic>(<italic>R</italic>), being the universal short-range component of the interactions, can be described phenomenologically by the same formalism as the electrostatic repulsion, with <disp-formula id="m2"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e518" xlink:href="srep06877-m2.jpg"/></disp-formula>LH phase data sets for each [NaCl] are fitted simultaneously to Π<sub>0</sub>. The common hydration repulsion parameters (<italic>A<sub>h</sub></italic> and <italic>λ<sub>h</sub></italic>) are enforced to be the same for all [NaCl]. The dependencies of <italic>A<sub>e</sub></italic> and <italic>A<sub>h</sub></italic> on <italic>d<sub>int</sub></italic> were neglected, an approximation we discuss later. We simultaneously fitted different combinations of two out of four data sets. <italic>A<sub>h</sub></italic> and <italic>λ<sub>h</sub></italic> were linked for all [NaCl], while <italic>A<sub>e</sub></italic> was allowed to be different for different [NaCl]. In addition, for each [NaCl], the Debye decay length was allowed to vary within ±3% from its calculated value using <inline-formula id="m17"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e566" xlink:href="srep06877-m17.jpg"/></inline-formula>, where <italic>I</italic>(<italic>M</italic>) is the molar ionic concentration. We found <italic>λ<sub>h</sub></italic> ≈ 2.2Å with an error ≈ 10% and then performed a global fitting with four data sets. In this step, <italic>λ<sub>h</sub></italic> was fixed at 2.2Å, while <italic>A<sub>h</sub></italic> and <italic>A<sub>e</sub></italic> were free parameters. <italic>A<sub>h</sub></italic> was linked for all [NaCl] and <italic>A<sub>e</sub></italic> was allowed to be different for different [NaCl]. In this way <italic>A<sub>h</sub></italic> = 1019 atm and <italic>A<sub>e</sub></italic> ≈ 155 atm, about the same for all [NaCl], with an uncertainty ≈ 10%. The results of the simultaneous fits of the LH phase data to Π<sub>0</sub> are shown in <xref ref-type="fig" rid="f3">Fig. 3</xref> (see also <xref ref-type="supplementary-material" rid="s1">SI</xref> for details). The treatment of the cholesteric and the LH phase data separately was missing in the available fits in the literature. In addition, the forms for the repulsions and the functions used in those fits are not identical to the forms used in this study. Nevertheless, it was already noted in the literature<xref ref-type="bibr" rid="b47">47</xref> that the osmotic pressure data (i.e., Π vs. <italic>d<sub>int</sub></italic>) at small interaxial separations vary exponentially with the decay length reported as 2 to 3Å. We stress again that the above forms of the electrostatic and the hydration part of the total interaction osmotic pressure should be seen as parsimonious empirical fits rather than attempts at a comprehensive theoretical description of the complicated DNA-DNA interactions that have been reviewed extensively in the literature<xref ref-type="bibr" rid="b21">21</xref><xref ref-type="bibr" rid="b22">22</xref>.</p><p>The cholesteric phase of long DNA fragments, being less ordered, gives a broad x-ray diffraction peak with DNA chains fluctuating in their cylindrical cells, leading to positional disorder of the Bragg planes. The fluctuational free energy of DNA molecules is modulated by bare interactions with their neighbors and depends on their bending stiffness. It can be calculated from Frank's elastic free energy for polymer nematics<xref ref-type="bibr" rid="b48">48</xref> by integrating out the Gaussian fluctuations around a straight configuration<xref ref-type="bibr" rid="b2">2</xref>. The fluctuational free energy per DNA chain (<italic>F<sub>fl</sub></italic>), at densities where fluctuations are prominent, is then obtained as <disp-formula id="m3"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e647" xlink:href="srep06877-m3.jpg"/></disp-formula>where the wavelength cutoff (i.e., <italic>q<sub>max</sub></italic>) takes into account the molecular size. <italic>B<sub>b</sub></italic> is the bare bulk compressibility modulus of the DNA cholesteric phase (relative change in the bare interaction pressure with changing cell area), i.e., <disp-formula id="m4"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e661" xlink:href="srep06877-m4.jpg"/></disp-formula><italic>K</italic> is the bending elastic constant defined as <italic>K</italic> = <italic>ρK<sub>c</sub></italic>, where <italic>ρ</italic> is the 2D number density of DNA chains perpendicular to their helical axes and <italic>K<sub>c</sub></italic> = (k<sub>B</sub>T)<italic>L<sub>p</sub></italic> is the bending rigidity of a single DNA chain. We can ignore the effect of [NaCl] on the persistence length, <italic>L<sub>p</sub></italic> (≈500Å for B-DNA), as it is less than 5% for 0.1 M ≤ [NaCl] ≤ 0.4 M<xref ref-type="bibr" rid="b49">49</xref><xref ref-type="bibr" rid="b50">50</xref>. <italic>V<sub>DNA</sub></italic> is the volume per DNA chain in the cylindrical cell model, i.e., <italic>V<sub>DNA</sub></italic> = <italic>L</italic>(<italic>πR</italic><sup>2</sup>), where <italic>L</italic> is the length of DNA chains. We replace <italic>q<sub>max</sub></italic> with a scaling prefactor (<italic>c</italic>) times the Brillouin zone (per DNA chain) radius, i.e., <italic>q<sub>max</sub></italic> → <italic>c</italic> × (<italic>π</italic>/<italic>d<sub>int</sub></italic>), which is equivalent to replacing <italic>d<sub>int</sub></italic> with an effective separation, <italic>d<sub>eff</sub></italic> = <italic>d<sub>int</sub></italic>/<italic>c</italic>. Using the thermodynamic relation Π = −∂<italic>F</italic>/∂<italic>V</italic>, the osmotic pressure due to fluctuations is <disp-formula id="m5"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e770" xlink:href="srep06877-m5.jpg"/></disp-formula>with <disp-formula id="m6"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e774" xlink:href="srep06877-m6.jpg"/></disp-formula>when <italic>R</italic> and <italic>B<sub>b</sub></italic> are in units of Å and atm, respectively. The total repulsion in the cholesteric phase is described as Π<italic><sub>cho</sub></italic>(<italic>R</italic>) = Π<sub>0</sub>(<italic>R</italic>) + Π<italic><sub>fl</sub></italic>(<italic>R</italic>). The fits of the cholesteric phase osmotic pressure data to Π<italic><sub>cho</sub></italic>(<italic>R</italic>) (shown in <xref ref-type="fig" rid="f4">Fig. 4</xref>) yield <italic>c</italic> ≈ 3. Calculations, fits, and variations of <italic>B<sub>b</sub></italic>, <italic>F<sub>fl</sub></italic>, <inline-formula id="m18"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e830" xlink:href="srep06877-m18.jpg"/></inline-formula>, and Π<italic><sub>fl</sub></italic> with <italic>R</italic> for each [NaCl] are explained in detail in <xref ref-type="supplementary-material" rid="s1">SI</xref>.</p><p>A discontinuous change in <italic>d<sub>int</sub></italic>, i.e., from <inline-formula id="m19"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e852" xlink:href="srep06877-m19.jpg"/></inline-formula> to <inline-formula id="m20"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e855" xlink:href="srep06877-m20.jpg"/></inline-formula>, at the transition osmotic pressure Π<italic><sub>tr</sub></italic> for cholesteric → LH transition then results from the balance between the osmotic pressure in the cholesteric phase, Π<italic><sub>cho</sub></italic>, containing a strong fluctuation contribution, and the osmotic pressure in the LH phase, composed of the bare repulsive interaction pressure Π<sub>0</sub> plus an effective attractive component (Π<italic><sub>ea</sub></italic>) viz. diminished repulsion, analogous to the interaction decomposition in the case of the van der Waals gas transition. The contribution of the conformational fluctuations to the osmotic pressure in the much stiffer LH phase is assumed to be nil. Π<italic><sub>ea</sub></italic> increases with increasing [NaCl]. In addition, there is a common decay length at small interaxial distances, as can be discerned clearly in <xref ref-type="fig" rid="f5">Fig. 5</xref>. We propose that the effective attraction follows the same form as the bare interaction repulsion, i.e., the sum of two terms accounting for the two interactions of different origin, with different characteristic decay lengths, <disp-formula id="m7"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e881" xlink:href="srep06877-m7.jpg"/></disp-formula>where <inline-formula id="m21"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e884" xlink:href="srep06877-m21.jpg"/></inline-formula> and <inline-formula id="m22"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e888" xlink:href="srep06877-m22.jpg"/></inline-formula> are proportional to <italic>λ<sub>h</sub></italic> and <italic>λ<sub>D</sub></italic> in Π<sub>0</sub>, respectively, i.e., <inline-formula id="m23"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e904" xlink:href="srep06877-m23.jpg"/></inline-formula> and <inline-formula id="m24"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e907" xlink:href="srep06877-m24.jpg"/></inline-formula>. The parameters <inline-formula id="m25"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e911" xlink:href="srep06877-m25.jpg"/></inline-formula>, <inline-formula id="m26"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e914" xlink:href="srep06877-m26.jpg"/></inline-formula> as well as <italic>f<sub>h</sub></italic>, <italic>f<sub>e</sub></italic> were determined from the fits of Π<italic><sub>ea</sub></italic> vs. <italic>d<sub>int</sub></italic> data to <inline-formula id="m27"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e937" xlink:href="srep06877-m27.jpg"/></inline-formula> by further assuming <italic>f</italic> = <italic>f<sub>h</sub></italic> = <italic>f<sub>e</sub></italic> (see <xref ref-type="fig" rid="f5">Fig. 5</xref> and <xref ref-type="supplementary-material" rid="s1">SI</xref> for details). The effective attractive component in the total osmotic pressure, <inline-formula id="m28"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e961" xlink:href="srep06877-m28.jpg"/></inline-formula>, then yields a discontinuous first-order transition by the application of the standard Maxwell equal-area construction. We obtain a common <inline-formula id="m29"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e964" xlink:href="srep06877-m29.jpg"/></inline-formula> for all [NaCl], with an uncertainty ≈ 10%. The parameter attributed to the electrostatic interactions <inline-formula id="m30"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e967" xlink:href="srep06877-m30.jpg"/></inline-formula> for [NaCl] = 0.1, 0.2, 0.3, 0.4 M, respectively. The common short-range component suggests the structural adaptation of the DNA chains to hydration interactions at high densities. Furthermore, the variations of the magnitude and decay length of the effective attraction, as well as the variation of Π<italic><sub>tr</sub></italic>, with salt (as evidenced on <xref ref-type="fig" rid="f5">Fig. 5</xref>) imply the contribution due to electrostatic effects.</p><p>We proceed to thermodynamic analysis of the data. The change in the free energy per DNA chain (per unit length) upon changing the DNA density from cell radius <italic>R</italic><sub>1</sub> to <italic>R</italic><sub>2</sub> is <disp-formula id="m8"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e989" xlink:href="srep06877-m8.jpg"/></disp-formula>At the cholesteric → LH transitions, where the radius changes abruptly from <inline-formula id="m31"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e992" xlink:href="srep06877-m31.jpg"/></inline-formula> to <inline-formula id="m32"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e996" xlink:href="srep06877-m32.jpg"/></inline-formula>, the free energy change is <disp-formula id="m9"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e999" xlink:href="srep06877-m9.jpg"/></disp-formula>where <inline-formula id="m33"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1002" xlink:href="srep06877-m33.jpg"/></inline-formula> is the change in the cell area across the transition. The change in the number of Na<sup>+</sup> ions (per length) with the change in DNA cell radius, (Δ<italic>N</italic>/<italic>L</italic>), can be calculated using Maxwell cross relation, −(∂Π/∂<italic>μ</italic>) = (∂<italic>N</italic>/∂<italic>V</italic>), which with <italic>V</italic> = <italic>L</italic>(<italic>πR</italic><sup>2</sup>) reduces to <disp-formula id="m10"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1036" xlink:href="srep06877-m10.jpg"/></disp-formula>where <italic>μ</italic> is the chemical potential of the salt, i.e., NaCl. The above relation implies that as the concentration of DNA varies due to the imposed osmotic pressure, the salt is taken up from the bulk reservoir and is redistributed within the DNA subphase, all the while keeping the full electroneutrality of the system. While the detailed nature of this redistribution cannot be derived without a microscopic model of the ionic atmosphere around the DNA molecule, the change in the number of salt ions in the DNA subphase would indicate that it is indeed taking place. In addition, we will show that most of the changes in the distribution of ions around DNA takes place within the cholesteric phase.</p><p>In what follows we assume that the chemical potential of the salt has the ideal form, i.e., <italic>μ</italic> = k<sub>B</sub>T log[NaCl]. While in the concentration range 0.1 M to 0.4 M the activity coefficient of NaCl is clearly not equal to 1 and its behavior is not ideal, the chemical potential of the salt appears only in derivative and thus only its changes are important. The change in the activity coefficient of NaCl over the range of concentrations used in our experiments is about 10%<xref ref-type="bibr" rid="b51">51</xref> and the final effect of the assumption of ideality is less than other sources of error in the determination and analysis of the data.</p><p>We consider three regions separately in the analysis: (1) cholesteric phase (from <italic>R</italic> → ∞ to <inline-formula id="m34"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1057" xlink:href="srep06877-m34.jpg"/></inline-formula>); (2) cholesteric → LH transition region (from <inline-formula id="m35"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1060" xlink:href="srep06877-m35.jpg"/></inline-formula> to <inline-formula id="m36"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1063" xlink:href="srep06877-m36.jpg"/></inline-formula>); (3) LH phase (from <inline-formula id="m37"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1066" xlink:href="srep06877-m37.jpg"/></inline-formula> to <italic>R</italic> = <italic>R</italic><sub>0</sub>). We calculate the change in the number of Na<sup>+</sup> ions per DNA base pair, (Δ<italic>N</italic>/<italic>bp</italic>)<italic><sub>C</sub></italic>, (Δ<italic>N</italic>/<italic>bp</italic>)<italic><sub>tr</sub></italic>, and (Δ<italic>N</italic>/<italic>bp</italic>)<italic><sub>H</sub></italic>, in regions (1), (2), and (3), respectively. Here <italic>R</italic><sub>0</sub> is the smallest cell radius in LH phase, with clearly discernible hexagonal symmetry in the x-ray diffraction pattern. For larger DNA densities the hexagonal symmetry is lost and the LH → orthorhombic transition ensues. Using the quadratic and line fits in <xref ref-type="fig" rid="f6">Fig. 6</xref> (see also <xref ref-type="supplementary-material" rid="s1">SI</xref> for the details of the calculations), we find (Δ<italic>N</italic>/<italic>bp</italic>)<italic><sub>H</sub></italic> = 0.59, (Δ<italic>N</italic>/<italic>bp</italic>)<italic><sub>tr</sub></italic> = 0.01 and (Δ<italic>N</italic>/<italic>bp</italic>)<italic><sub>C</sub></italic> = 1.76 − 0.65 log[NaCl] with [NaCl] in mM.</p><p>These results suggest that the change in the number of Na<sup>+</sup> ions in the DNA subphase is independent of [NaCl] at the transition and in the LH phase (discussed later). However, (Δ<italic>N</italic>/<italic>bp</italic>)<italic><sub>C</sub></italic> varies with [NaCl] and upon changing the DNA density from infinite dilution up to the cholesteric → LH transition, about 23%, 13%, 7%, and 3% of the bare DNA charge is neutralized at [NaCl] = 0.1, 0.2, 0.3, 0.4 M, respectively, if we assume that of the salt ions taken up by the DNA subphase, Na<sup>+</sup> ends up being located in close proximity to the DNA charges. There is thus a detectable difference in the number of Na<sup>+</sup> ions near DNA phosphates under different [NaCl] at the infinite dilution limit and at a finite concentration in the cholesteric phase.</p><p>For short-fragment (≈50 nm long) DNA, in NaCl solutions, the hexagonal → orthorhombic transition occurs near DNA density corresponding to <italic>d<sub>int</sub></italic> = 23.7Å<xref ref-type="bibr" rid="b15">15</xref>. Lindsay <italic>et al.</italic><xref ref-type="bibr" rid="b52">52</xref> showed that long Na-DNA fibers also go through a similar transition at around 90% relative humidity that, in addition, coincides with a B to A conformational transition of DNA with a helical pitch length change from ≈34Å for B-form to ≈29Å for A-form. In the orthorhombic phase the DNA density does not change anymore upon further drying<xref ref-type="bibr" rid="b52">52</xref>. Our osmotic stress experiments, in which the water content of DNA is accurately controlled and the corresponding osmotic pressures are known, indicate that on increase of the osmotic pressure in LH phase the first-order x-ray diffraction peak first broadens, followed by the LH → orthorhombic transition at Π ≈ 170 atm, characterized by the complete disappearance of the hexagonal symmetry of the scattering intensity (see <xref ref-type="fig" rid="f7">Fig. 7</xref>).</p><p>The order of the cholesteric → LH transition in NaCl solutions, together with its equation of state and the pertinent Maxwell equal-area construction, are relevant also for DNA condensation transition induced by osmotic pressure at subcritical CoHex concentrations (see <xref ref-type="fig" rid="f7">Fig. 7</xref>). Obviously the subcritical condensation transition (i.e., abrupt change in the volume per base pair (<italic>v<sub>bp</sub></italic>) and the ensuing collapse of DNA into a highly ordered structure, induced by osmotic pressure, in the presence of low concentrations of polyvalent salts) bears a striking similarity to the cholesteric → LH transition in NaCl solutions even in terms of the identical diffraction fingerprint. The cholesteric → LH transition thus exists also at subcritical [CoHex] in osmotic pressure-induced condensation of long DNA, i.e., for any [CoHex] ≤ [CoHex]*, where [CoHex]* is the minimum critical [CoHex] necessary for condensation at zero osmotic pressure. We find that [CoHex]* = 28 mM in [NaCl] = 0.3 M solutions. The osmotic pressure-induced transitions for [CoHex] = 0, [CoHex] = 3 mM, and [CoHex] = 12 mM at [NaCl] = 0.3 M are shown in <xref ref-type="fig" rid="f7">Fig. 7</xref>. With increasing [CoHex], Δ<italic>v<sub>bp</sub></italic> increases and Π<italic><sub>tr</sub></italic> decreases. The effective attraction leading to the first-order condensation transition, that can be deduced in the same way as in the monovalent salt case, increases with the addition of CoHex in the solution.</p><p>These results point to a continuity of thermodynamic states between the cholesteric → LH transition in monovalent salts and DNA polyvalent salt-induced condensation, frequently viewed as completely distinct phenomena. In fact, addition of CoHex (at subcritical concentrations) simply increases Δ<italic>v<sub>bp</sub></italic> at the cholesteric → LH transition already present in NaCl solutions. As the polyvalent salt concentration increases, the cholesteric branch density first moves to higher values, following the LH branch, but then eventually starts moving toward lower densities. At the critical concentration of the polyvalent salt, it moves out to infinity, i.e., the DNA condenses at zero external osmotic pressure. This last part of the polyvalent salt dependence is experimentally difficult to quantify<xref ref-type="bibr" rid="b53">53</xref>, but the existence of the condensation at zero imposed osmotic pressure indicates that indeed the cholesteric branch must recede to infinite dilution at the critical concentration.</p></sec><sec disp-level="1" sec-type="discussion"><title>Discussion</title><p>With increasing osmotic pressure, delicately controlled with small temperature variations<xref ref-type="bibr" rid="b25">25</xref>, DNA undergoes a transition from a less ordered fluctuating (cholesteric) state to a more ordered LH state with negligible conformational fluctuations. Changes in DNA density and packing at the cholesteric → LH transition are discontinuous, as observed in the x-ray scattering of long DNA fragments. We measured this transition with high accuracy and investigated its sensitivity to solution conditions. The sixfold symmetric azimuthal intensity profile of the first-order diffraction peak in the x-ray images of oriented DNA suggests that long-fragment DNA packs in a straight parallel untwisted arrangement in the LH phase, with long-range bond orientational order perpendicular to the axis of the molecules<xref ref-type="bibr" rid="b1">1</xref><xref ref-type="bibr" rid="b24">24</xref>. At the transition from the more ordered LH phase to the cholesteric phase, the sixfold azymuthal symmetry in the diffraction peak disappears; the vanishing of long-range bond orientational order and the abrupt change in DNA density occur simultaneously.</p><p>The osmotic pressure and inverse ionic strength dependence of the cholesteric → LH transition is similar to the pressure and temperature dependence of the gas-liquid transition, i.e., cholesteric → LH transition is shifted to higher osmotic pressures upon decreasing ionic strength of the solution. Furthermore, Δ<italic>v<sub>bp</sub></italic> at the cholesteric → LH transition decreases, and DNA osmotic pressure curves become progressively more flat around the transition region with decreasing ionic strength. When [NaCl] = 50 mM (the smallest [NaCl] for which we measured the cholesteric → LH transition), and for <inline-formula id="m38"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1245" xlink:href="srep06877-m38.jpg"/></inline-formula> the first-order x-ray diffraction peak is lost because of strong electrostatic repulsion. For this reason, [NaCl] = 50 mM data (available only for <inline-formula id="m39"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1248" xlink:href="srep06877-m39.jpg"/></inline-formula> and shown in <xref ref-type="supplementary-material" rid="s1">SI</xref>) were not used in the simultaneous Π<italic><sub>cho</sub></italic>(<italic>d<sub>int</sub></italic>) fits. For [NaCl] = 50 mM, the transition in fact occurs at Π<italic><sub>tr</sub></italic> = 9.7 atm, and the abrupt change in the interaxial distance (Δ<italic>d<sub>int</sub></italic>) is 1.2Å (<inline-formula id="m40"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1274" xlink:href="srep06877-m40.jpg"/></inline-formula> and <inline-formula id="m41"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1277" xlink:href="srep06877-m41.jpg"/></inline-formula>). With increasing [NaCl], Π<italic><sub>tr</sub></italic> decreases and Δ<italic>d<sub>int</sub></italic> increases. For [NaCl] = 0.4 M, Π<italic><sub>tr</sub></italic> = 5.8 atm and Δ<italic>d<sub>int</sub></italic> = 2Å, they do not vary any more upon further increase in [NaCl].</p><p>The estimated value for the effective charge <inline-formula id="m42"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1301" xlink:href="srep06877-m42.jpg"/></inline-formula> (see <xref ref-type="disp-formula" rid="m1">Eq. 1</xref>) from the simultaneous fits of LH phase data is ≈155 atm (approximately the same for all [NaCl], with an uncertainty ≈ 10%). For fully charged DNA chains, <italic>A<sub>e</sub></italic> would be approximately twice as large as what we extracted from the fits. If the parameter <italic>A<sub>e</sub></italic> is a measure of the net charge, then about half the bare DNA charge is neutralized in the LH phase. Conversely, if the net charge decreases with increasing (<italic>N</italic>/<italic>bp</italic>)<italic><sub>H</sub></italic>, then one would also expect <italic>A<sub>e</sub></italic> to decrease with increasing DNA density. The change in the number of Na<sup>+</sup> ions from cholesteric → LH transition up to the LH → orthorhombic transition, is (Δ<italic>N</italic>/<italic>bp</italic>)<italic><sub>H</sub></italic> = 0.59 and is independent of [NaCl].</p><p>The fact that (Δ<italic>N</italic>/<italic>bp</italic>)<italic><sub>H</sub></italic> is independent of [NaCl] is instructive. Combined with the observation that the osmotic pressure curves converge at high pressures, it suggests that the number of salt ions per base pair is already the same for all [NaCl] at the cholesteric → LH transition and that the transition occurs when the net charge drops to a certain value. <inline-formula id="m43"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e1359" xlink:href="srep06877-m43.jpg"/></inline-formula> is zero in the cholesteric phase, i.e., when the net DNA charge is above a certain limit. Lee <italic>et.al.</italic><xref ref-type="bibr" rid="b54">54</xref> predict similar transitions of DNA in NaCl solutions and underline the role of structural adaptation of DNA helices to the interactions which is a function of ionic conditions and <italic>d<sub>int</sub></italic>. Their theory predicts a ratio of ≈2 between the decay lengths for attractive and repulsive interactions<xref ref-type="bibr" rid="b21">21</xref>. In our experiments, the measured effective attraction Π<italic><sub>ea</sub></italic> is approximately an order of magnitude smaller then the net repulsion in LH phase Π<sub>0</sub>. The magnitude of Π<italic><sub>ea</sub></italic> is also sensitive to the ionic strength and decreases with decreasing [NaCl] (see <xref ref-type="fig" rid="f5">Fig. 5</xref>). The ratio of the decay length of Π<italic><sub>ea</sub></italic> to the decay length of Π<sub>0</sub> is extracted by fitting the osmotic pressure data. The obtained factor <italic>f</italic> = <italic>f<sub>h</sub></italic> = <italic>fe</italic> ≈ 2.4 is reasonable, although our analysis was based on several simplifying assumptions.</p><p>The emerging connection between the cholesteric → LH transition in univalent NaCl solution and the DNA condensation transition in the presence of polyvalent salts<xref ref-type="bibr" rid="b26">26</xref>, such as CoHex, at subcritical concentrations indicates a major change in DNA behavior in various solution conditions. We observe that osmotic pressure-induced DNA condensation at subcritical [CoHex] occurs in the same way as the cholesteric → LH transition in NaCl solutions. Our experiments now reveal that the osmotic pressure-induced condensation of long DNA indeed occurs at all polyvalent salt concentrations, for example at any [CoHex] ≤ [CoHex]*, where [CoHex]* is the minimum [CoHex] necessary for condensation at zero osmotic pressure. It depends also on the concentration of monovalent salt so that when [NaCl] = 0.3 M, [CoHex]* = 28 mM. With increasing [CoHex], Δ<italic>v<sub>bp</sub></italic> increases slightly, and the transition pressure Π<italic><sub>tr</sub></italic> decreases. With the addition of CoHex to the bathing solution, the effective attraction therefore increases.</p><p>The general similarity between these two transitions therefore points to a continuity of thermodynamic states between the cholesteric → LH transition and the osmotic pressure-induced DNA condensation transition at subcritical [CoHex], thus bridging the gap between superficially distinctly different behaviors of DNA in monovalent and polyvalent salt bathing solutions.</p></sec><sec disp-level="1" sec-type="methods"><title>Methods</title><sec disp-level="2"><title>Sample preparation and data collection</title><p>Oriented<xref ref-type="bibr" rid="b41">41</xref> and unoriented<xref ref-type="bibr" rid="b25">25</xref><xref ref-type="bibr" rid="b26">26</xref> DNA samples are prepared using calf thymus or salmon sperm DNA (molecular weight ≈ 10<sup>7</sup> Daltons). Oriented fibers are prepared by wet-spinning using the apparatus (designed by A. Rupprecht) in ILL (Grenoble, France). X-ray diffraction measurements are made using our in-house setup<xref ref-type="bibr" rid="b25">25</xref> at UMass Amherst. Diffraction peak fits are done using IGOR Pro multi-peak fitting package. Brief explanations of x-ray diffraction data collection and analysis are given in the caption to <xref ref-type="fig" rid="f1">Fig. 1</xref>. See also ref. <xref ref-type="bibr" rid="b25">25</xref> and <xref ref-type="supplementary-material" rid="s1">SI</xref> for more details on sample preparations and data collection.</p></sec><sec disp-level="2"><title>Osmotic pressure data</title><p>Temperature-dependent osmotic pressure data of PEG (molecular weight of 8000 Daltons) solutions are from ref. <xref ref-type="bibr" rid="b19">19</xref>. Osmotic pressure decreases almost linearly with increasing T at constant [PEG] for 20°C ≤ T ≤ 40°C. The osmotic pressure of PEG, as well as the temperature dependence of the osmotic pressure of PEG, are not new. They have been described, analyzed, and used for decades, being also extensively documented in various publications and reproduced by a variety of experimental methods (e.g., vapor pressure osmometer, membrane osmometer). In addition, the effect of salt on PEG activity (measured at room temperature by Wescor vapor pressure osmometer 5600) is insignificant at salt concentrations used in the experiments.</p></sec><sec disp-level="2"><title>Cholesteric → LH transition measurements</title><p>The novel methodology used in this study takes advantage of the dependence of PEG (molecular weight of 8000 Daltons) osmotic pressure on temperature in order to achieve a heretofore unattained accuracy in fixing the value of this pressure. This enables also a much-improved accuracy in the determination of the equation of state of DNA (i.e., Π vs. <italic>d<sub>int</sub></italic>), which reveals additional fine features of this equation of state that have been previously missed, enabling a deeper insight into the behavior of DNA at high concentrations. Note that using temperature to vary the osmotic pressure of the PEG solution in equilibrium with a DNA subphase is possible because under certain conditions, DNA-DNA interactions are almost independent of temperature over the range 15°C to 45°C. We therefore take advantage of the fact that temperature has no <italic>detectable</italic> effect on the DNA-DNA interactions to use it to accurately set the osmotic pressure of the DNA solution. The use of temperature variation methodology to measure the cholesteric → LH transitions is described briefly in the captions to <xref ref-type="fig" rid="f1">Fig. 1</xref> and <xref ref-type="fig" rid="f2">Fig. 2</xref>. See also ref. <xref ref-type="bibr" rid="b25">25</xref> and <xref ref-type="supplementary-material" rid="s1">SI</xref> for more details.</p></sec></sec><sec disp-level="1"><title>Author Contributions</title><p>S.Y., R.P. and V.A.P. designed research; S.Y., R.P. and J.V.O. performed research; J.V.O. and M.J. contributed new reagents and tools; S.Y., R.P. and V.A.P. analyzed data; S.Y. prepared figures; and S.Y. and R.P. wrote the paper. All authors reviewed the manuscript.</p></sec><sec sec-type="supplementary-material" id="s1"><title>Supplementary Material</title><supplementary-material id="d33e89" content-type="local-data"><caption><title>Supplementary Information</title><p>Supplementary Information for ‘Continuity of states between the cholesteric → line hexatic transition and the condensation transition in DNA solutions’</p></caption><media xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="srep06877-s1.pdf"/></supplementary-material></sec> |
Retracted: Why Fish Oil Fails: A Comprehensive 21st Century Lipids-Based Physiologic Analysis | Could not extract abstract | <contrib contrib-type="author"><name><surname>Journal of Lipids</surname><given-names/></name><xref ref-type="corresp" rid="cor1">*</xref></contrib> | Journal of Lipids | <p>The article titled “<italic>Why Fish Oil Fails: A Comprehensive 21st Century Lipids-Based Physiologic Analysis</italic>” [<xref rid="B1" ref-type="bibr">1</xref>], published in Journal of Lipids has been retracted as a result of an undeclared competing interest on the part of the manuscript's author.</p> |
Effects of cobalt-chromium everolimus eluting stents or bare metal stent on fatal and non-fatal cardiovascular events: patient level meta-analysis | <p><bold>Objectives</bold> To examine the safety and effectiveness of cobalt-chromium everolimus eluting stents compared with bare metal stents.</p><p><bold>Design</bold> Individual patient data meta-analysis of randomised controlled trials. Cox proportional regression models stratified by trial, containing random effects, were used to assess the impact of stent type on outcomes. Hazard ratios with 95% confidence interval for outcomes were reported.</p><p><bold>Data sources and study selection</bold> Medline, Embase, the Cochrane Central Register of Controlled Trials. Randomised controlled trials that compared cobalt-chromium everolimus eluting stents with bare metal stents were selected. The principal investigators whose trials met the inclusion criteria provided data for individual patients.</p><p><bold>Primary outcomes</bold> The primary outcome was cardiac mortality. Secondary endpoints were myocardial infarction, definite stent thrombosis, definite or probable stent thrombosis, target vessel revascularisation, and all cause death.</p><p><bold>Results</bold> The search yielded five randomised controlled trials, comprising 4896 participants. Compared with patients receiving bare metal stents, participants receiving cobalt-chromium everolimus eluting stents had a significant reduction of cardiac mortality (hazard ratio 0.67, 95% confidence interval 0.49 to 0.91; P=0.01), myocardial infarction (0.71, 0.55 to 0.92; P=0.01), definite stent thrombosis (0.41, 0.22 to 0.76; P=0.005), definite or probable stent thrombosis (0.48, 0.31 to 0.73; P<0.001), and target vessel revascularisation (0.29, 0.20 to 0.41; P<0.001) at a median follow-up of 720 days. There was no significant difference in all cause death between groups (0.83, 0.65 to 1.06; P=0.14). Findings remained unchanged at multivariable regression after adjustment for the acuity of clinical syndrome (for instance, acute coronary syndrome <italic>v</italic> stable coronary artery disease), diabetes mellitus, female sex, use of glycoprotein IIb/IIIa inhibitors, and up to one year <italic>v</italic> longer duration treatment with dual antiplatelets.</p><p><bold>Conclusions</bold> This meta-analysis offers evidence that compared with bare metal stents the use of cobalt-chromium everolimus eluting stents improves global cardiovascular outcomes including cardiac survival, myocardial infarction, and overall stent thrombosis.</p> | <contrib contrib-type="author" corresp="yes"><name><surname>Valgimigli</surname><given-names>Marco</given-names></name><role>associate professor of medicine</role><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Sabaté</surname><given-names>Manel</given-names></name><role>associate professor of medicine</role><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Kaiser</surname><given-names>Christoph</given-names></name><role>professor of medicine</role><xref ref-type="aff" rid="aff3">3</xref></contrib><contrib contrib-type="author"><name><surname>Brugaletta</surname><given-names>Salvatore</given-names></name><role>assistant professor of medicine</role><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>de la Torre Hernandez</surname><given-names>Jose Maria</given-names></name><role>associate professor of medicine</role><xref ref-type="aff" rid="aff4">4</xref></contrib><contrib contrib-type="author"><name><surname>Galatius</surname><given-names>Soeren</given-names></name><role>interventional cardiologist</role><xref ref-type="aff" rid="aff5">5</xref></contrib><contrib contrib-type="author"><name><surname>Cequier</surname><given-names>Angel</given-names></name><role>associate professor of medicine</role><xref ref-type="aff" rid="aff6">6</xref></contrib><contrib contrib-type="author"><name><surname>Eberli</surname><given-names>Franz</given-names></name><role>professor of medicine</role><xref ref-type="aff" rid="aff7">7</xref></contrib><contrib contrib-type="author"><name><surname>de Belder</surname><given-names>Adam</given-names></name><role>interventional cardiologist</role><xref ref-type="aff" rid="aff8">8</xref></contrib><contrib contrib-type="author"><name><surname>Serruys</surname><given-names>Patrick W</given-names></name><role>professor of medicine</role><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Ferrante</surname><given-names>Giuseppe</given-names></name><role>interventional cardiologist</role><xref ref-type="aff" rid="aff9">9</xref></contrib><aff id="aff1"><label>1</label>Thoraxcenter, Erasmus Medical Center, 3015 CE Rotterdam, Netherlands</aff><aff id="aff2"><label>2</label>University Hospital Clinic, IDIBAPS, Cardiology Department, 08036 Barcelona, Spain</aff><aff id="aff3"><label>3</label>University Hospital 4003 Basel, Switzerland</aff><aff id="aff4"><label>4</label>Hospital Marqués de Valdecilla, 39008 Santander, Spain</aff><aff id="aff5"><label>5</label>Gentofte Hospital, 2900 Hellerup, Denmark</aff><aff id="aff6"><label>6</label>University Hospital of Bellvitge, 08907 Barcelona, Spain</aff><aff id="aff7"><label>7</label>Stadtspital Triemli, 8063 Zürich, Switzerland</aff><aff id="aff8"><label>8</label>Brighton and Sussex University Hospitals NHS Trust, Brighton BN1 6AG, UK</aff><aff id="aff9"><label>9</label>Interventional Cardiology, Humanitas Clinical and Research Institute IRCCS, 20089 Rozzano, Milan, Italy</aff> | The BMJ | <sec sec-type="intro"><title>Introduction</title><p>Randomised controlled trials,<xref rid="ref1" ref-type="bibr">1</xref>
<xref rid="ref2" ref-type="bibr">2</xref> a meta-analysis,<xref rid="ref3" ref-type="bibr">3</xref> and an observational study<xref rid="ref4" ref-type="bibr">4</xref> have consistently shown reduced rates of target vessel revascularisation because of angiographic restenosis and ischaemia after treatment with drug eluting stents compared with bare metal stents. As a result, most percutaneous coronary interventions are done with drug eluting stents rather than bare metal stents. The higher rates of late stent thrombosis and the concern about higher risks of late stent thrombosis after early discontinuation of dual antiplatelet therapy with first generation drug eluting stents, however, have raised safety concerns.<xref rid="ref5" ref-type="bibr">5</xref>
<xref rid="ref6" ref-type="bibr">6</xref> New drug eluting stents have been developed with novel materials, designs, and delivery systems, with biocompatible polymers and new antiproliferative agents. The box gives details of the types of stents. Most of these second generation stents, however, were approved after non-inferiority trials in which they were compared with first generation drug eluting stents.<xref rid="ref7" ref-type="bibr">7</xref>
<xref rid="ref8" ref-type="bibr">8</xref>
<xref rid="ref9" ref-type="bibr">9</xref>
<xref rid="ref10" ref-type="bibr">10</xref> Few studies have directly compared second generation drug eluting stents with bare metal stents, and their comparative safety profile remains unclear.</p><boxed-text position="float" content-type="style2"><sec><title>Bare metal stents</title><p>A bare metal stent is vascular stent without a coating (as used in drug eluting stents). It is a mesh-like tube of thin wire. The first stents licensed for use in cardiac arteries were bare metal—often 316L stainless steel. More recent (“second generation”) stents use cobalt chromium alloy. The major limitation of this coronary device is the so called risk for in stent restenosis, caused by proliferation of cells in the media layer of the vessel wall leading to the growth of new tissue within the stent struts, called intimal hyperplasia, finally narrowing the vessel lumen in a sizable proportion of patients. The growth of intimal hyperplasia peaks on average a few months after stent implantation and remains thereafter stable. This stent requires the use of dual antiplatelet therapy for 30 days. </p></sec><sec><title>First generation drug eluting stents</title><p>They were primarily conceived to reduce in stent neointimal formation and therefore minimise the occurrence of restenosis, the major drawback of percutaneous coronary interventions with bare metal stents. They consist of a metallic vascular stent coated with a permanent polymer, which slowly releases a drug, such as sirolimus or paclitaxel, to block cell proliferation and thus limit intimal hyperplasia. The permanent presence of these polymers has been associated with inflammatory responses and local toxicity in preclinical analysis. The optimal duration of dual antiplatelet therapy after first generation drug eluting stents has never been conclusively determined, but it is likely to be more than a year as this device has been associated with a fourfold to fivefold risk for very late (that is, after one year) stent thrombosis compared with bare metal stents. On the other hand, rates of acute, subacute, and late stent thrombosis have been shown to be equal or even lower than with bare metal stents. </p></sec><sec><title>Second generation drug eluting stents</title><p>They have been developed with thinner, more biocompatible, permanent, or bioabsorbable polymers, and new antiproliferative agents. Whether technology based on erodible polymers—such as poly-l-lactic acid and poly-D,L-lactide, which control drug release after implantation and vanish thereafter—is truly superior to technology engineered with biocompatible, thin, and durable material such as the fluorinated polymer present in the cobalt-chromium everolimus eluting stent is still matter of debate. Second generation drug eluting stents have largely been compared with first generation devices and some of them have been shown to be associated with a much lower risk of stent thrombosis, both early and late. As with first generation drug eluting stents, the optimal duration of dual antiplatelet therapy after second generation drug eluting stents has never been conclusively determined. No randomised study has so far reported safety issues in patients who discontinued dual antiplatelet therapy after six or even three months after their implantation. </p><p>While first generation drug eluting stents have now been withdrawn from the market, clinical decision making regarding second generation drug eluting stents versus bare metal stents currently depends on a complex interplay of several factors, including perceived risk for in stent restenosis—if the perceived risk is low, a bare metal stent might be preferable, contraindications to long term dual antiplatelet therapy, and costs. </p></sec></boxed-text><p>Recent studies have reported a possible lower risk of stent thrombosis after implantation with cobalt-chromium everolimus eluting stents compared with bare metal stents.<xref rid="ref11" ref-type="bibr">11</xref>
<xref rid="ref12" ref-type="bibr">12</xref>
<xref rid="ref13" ref-type="bibr">13</xref> The implications of this finding on clinical outcomes are currently unknown.</p><p>We undertook a collaborative analysis of data from randomised trials of patients who underwent random allocation to either cobalt-chromium everolimus eluting stents or bare metal stents to assess the comparative safety and efficacy of these two devices on fatal and non-fatal cardiovascular outcomes.</p></sec><sec sec-type="methods"><title>Methods</title><sec><title>Search strategy and study selection</title><p>Two investigators (MV and GF) independently searched Medline, Embase, and the Cochrane Central Register of Controlled Trials (from database inception to December 2013) using the terms everolimus eluting stent and bare metal stent. A sensitive filter for randomised controlled trials was used. There were no restrictions by language, publication date, or publication status. We hand searched conference proceedings (from 2006 to 2013) of the American Heart Association, the American College of Cardiology, the Transcatheter Cardiovascular Therapeutics, and the European Society of Cardiology. All suitable non-published completed registered studies were considered for inclusion. We checked reference lists of identified articles, recent editorials, and related reviews and contacted experts for further eligible trials.</p></sec><sec><title>Trial eligibility and quality assessment</title><p>Two investigators (MV and GF) independently assessed trial eligibility. Randomised trials comparing two or more different drug eluting stents or drug eluting stents with bare metal stents were eligible for inclusion. Inclusion criteria comprised randomised treatment allocation, comparison of cobalt-chromium everolimus eluting stent versus bare metal stent, and length of follow-up of at least a year. Exclusion criteria were equivocal or non-random treatment allocation, lack of outcome data up to one year, and duplicate reports. The two reviewers resolved disagreements by consensus. All included trials were reviewed and approved by ethics committees, and all participants had given written informed consent. Two investigators (MV and GF) systematically assessed the presence of sources of bias by following the approach recommended by the Cochrane Collaboration that identifies selection, performance, attrition, detection, reporting bias, and other sources of bias for each study, and classifies each of these as low, unclear, or high by analysing the following domains: random sequence generation, allocation concealment, blinding of participants and of outcome assessment, incomplete outcome data, selective outcome reporting, and “other issues.”<xref rid="ref14" ref-type="bibr">14</xref> Disagreements were resolved through consensus. A risk of bias summary for each item for each included study was reported.</p><p>The meta-analysis was performed in agreement with the PRISMA statement.<xref rid="ref15" ref-type="bibr">15</xref></p></sec><sec><title>Data extraction</title><p>We invited the principal investigators whose trials met the inclusion criteria to participate in this collaborative analysis, and they provided the following data for individual patients: a set of core clinical variables consisting of demographics (age, sex, body mass index (BMI)), cardiac risk factors (diabetes, smoking, hypertension, and hypercholesterolaemia), clinical manifestations (stable coronary artery disease, cardiac markers negative unstable coronary artery disease, cardiac markers positive unstable coronary artery disease without persistent ST segment elevation myocardial infarction and persistent ST segment myocardial infarction), angiographic factors (number and type of diseased vessels, number of treated lesions, number of implanted stents, overlapping stents implantation, total stent length and average stent diameter), use of antithrombotic drugs (actual duration of clopidogrel after treatment and use of glycoprotein IIb/IIIa inhibitors during the procedure), randomised treatment assignment, and outcomes in follow-up (death, cardiac death, myocardial infarction, stent thrombosis, repeat revascularisation, and last follow-up contact).</p><p>Two investigators (MV, GF) extracted data independently on trial design, baseline characteristics, outcomes, and safety events. After resolution of data queries, we recoded data from each trial in a single dataset and checked data summaries from individual trials against the associated publications for accuracy.</p></sec><sec><title>Endpoints</title><p>The primary outcome was cardiac mortality at the longest available follow-up. We selected cardiac mortality and not all cause mortality as primary outcome measure to minimise the confounding effect of non-cardiac events on stent performance in the context of a relatively old and unselected population of patients followed-up through two years. Importantly, cardiac mortality was homogenously classified across the five included studies according to the ARC (Academic Research Consortium) criteria.<xref rid="ref16" ref-type="bibr">16</xref></p><p>Secondary outcomes were all cause death, myocardial infarction (fatal and non-fatal), stent thrombosis (definite and definite or probable), target vessel revascularisation, a composite of cardiac death and myocardial infarction, and a composite of all cause death and myocardial infarction. The definition of stent thrombosis was in alignment with the ARC criteria in four out of five studies,<xref rid="ref11" ref-type="bibr">11</xref>
<xref rid="ref12" ref-type="bibr">12</xref>
<xref rid="ref13" ref-type="bibr">13</xref>
<xref rid="ref17" ref-type="bibr">17</xref>
<xref rid="ref18" ref-type="bibr">18</xref>
<xref rid="ref19" ref-type="bibr">19</xref> whereas in the SPIRIT I trial<xref rid="ref20" ref-type="bibr">20</xref> stent thrombosis was defined as a total coronary artery occlusion by angiography at the stent site with abrupt onset of symptoms, raised biochemical markers, and changes on electrocardiography consistent with myocardial infarction. The definition of myocardial infarction, which is summarised in table A in appendix 1, differed among included trials largely because of different criteria used to adjudicate peri-procedural events.<xref rid="ref11" ref-type="bibr">11</xref>
<xref rid="ref12" ref-type="bibr">12</xref>
<xref rid="ref13" ref-type="bibr">13</xref>
<xref rid="ref17" ref-type="bibr">17</xref>
<xref rid="ref18" ref-type="bibr">18</xref>
<xref rid="ref19" ref-type="bibr">19</xref>
<xref rid="ref20" ref-type="bibr">20</xref> For all five studies, fatal and non-fatal cardiovascular endpoints were confirmed on the basis of documentation collected at each hospital and were centrally adjudicated by clinical events committees, whose members were unaware of patients’ assignments to treatment group.</p></sec><sec><title>Statistical analysis</title><p>We carried out an individual patient level data meta-analysis using a “one stage” approach. Briefly, all data from individual participants were combined in a single meta-analysis based on a simple Cox proportional regression model stratified by trial, containing random effects (frailty models) to assess the impact of stent type—for instance, cobalt-chromium everolimus eluting stent versus bare metal stent on outcome measures.<xref rid="ref21" ref-type="bibr">21</xref> We calculated hazard ratios and 95% confidence interval for the endpoints.</p><p>We also assessed the impact of stent type on outcomes by multivariable Cox regression stratified by trial, with random effects, entering into the model the following prespecified clinically relevant covariates: clinical syndrome (that is, acute coronary syndrome <italic>v</italic> stable syndrome), history of diabetes mellitus, female sex, use of glycoprotein IIb/IIIa inhibitors, and up to one year versus longer duration of dual antiplatelet therapy.</p><p>For each endpoint, we performed time to first event analysis. When assessing each individual endpoint, we censored patients who died from any cause beyond the time of death—that is, they were not at risk anymore for the endpoint assessed after they died.</p><p>We verified the proportional hazard assumptions by applying a test for non-proportional hazards with the Schoenfeld residuals and by visual estimation after plotting graphs of the scaled Schoenfeld residuals against transformed time at follow-up after index procedure and by adding time-by-covariate interactions for each variable of the model, by introducing products between the variables and a linear function of time.</p><p>As secondary analyses, we performed competing risk regression models for endpoints other than death, considering all cause mortality as the competing event. We calculated subhazard ratios with 95% confidence interval (according to the method of Fine and Gray,<xref rid="ref22" ref-type="bibr">22</xref> accounting for the clustered nature of the data with trial as cluster) and have reported them in a supplemental table. Subhazard ratios refer to the hazard that generates failure events of interest while keeping participants who experience competing events “at risk” so that they can be adequately counted as not having any chance of failing.</p><p>In case of violation of the proportional hazard assumptions, we calculated and report time dependent hazard ratios.</p><p>For target vessel revascularisation, and the composite of target vessel revascularisation or myocardial infarction, for which stent type effect presented a violation in proportional hazard assumptions in both main and competing risk analyses, we report a plot depicting Schoenfeld-like residuals against time with a lowess smooth fitted through it from simple competing risk regression model.</p><p>For outcomes not treated as competing events, we report cumulative hazard curves of events, derived directly from the simple Cox regression model built for hazard ratio calculation. For endpoints assessed in competing risk analyses, we report cumulative incidence curves of events, derived from the corresponding simple regression model.</p><p>The numbers of patients needed to treat for an additional beneficial or harmful outcome were calculated according to the formula: NTT=1/{[Sc1(t)]−Sc0(t)}, where the Sc0(t) is the survival probability at two years in the bare metal group and [Sc1(t)] is the survival probability at two years in the cobalt-chromium everolimus eluting stent group. Survival probabilities were derived from simple Cox regression model for mortality endpoints after estimation of baseline survival functions and from simple competing risk regression model for non-mortality endpoints.</p><p>We evaluated the presence of heterogeneity among studies with the variance of random effects estimated by multivariable stratified Cox regression models with random effects. Additionally, we performed classic meta-analytic estimates of heterogeneity with the Cochran Q χ<sup>2</sup> test, where a P value ≤0.10 was considered as significant; with estimation of the variance between studies with τ<sup>2</sup> and by using I<sup>2</sup> test to evaluate the inconsistency. The I<sup>2</sup> statistic is derived from the Q statistic ([Q−df/Q]*100) and describes the percentage of total variation across studies attributable to heterogeneity: values of 25%, 50%, and 75% correspond to low, moderate, and high heterogeneity.<xref rid="ref23" ref-type="bibr">23</xref></p><p>We could not statistically assess publication bias because we had fewer than 10 studies. All analyses were conducted according to the intention to treat principle. Categorical variables are reported as counts and percentages and were compared by χ<sup>2</sup> or Fisher’s exact tests as appropriate. Continuous variables are reported as means and SD or median and interquartile ranges whenever appropriate and were compared by <italic>t</italic> tests or Mann Whitney U test as appropriate.</p></sec><sec><title>Subgroup analyses</title><p>We tested for interactions between assigned treatment and baseline characteristics for the primary outcome measure of cardiac death using stratified Cox models with random effects that included treatment assignment, the baseline characteristic of interest, and their interaction. We report coefficient interactions with standard errors and P values for interaction. The following covariates were tested for interaction: age ≥65, sex, diabetes, use of glycoprotein IIb/IIIa inhibitors, duration of dual antiplatelet therapy up to one year versus longer, overlapping stent, clinical syndrome (acute coronary syndrome/stable angina), treated culprit vessel, left descending artery versus no left descending artery, and multi-vessel disease. For descriptive purposes only, we have provided a plot of treatment effects within each subgroup.</p></sec><sec><title>Sensitivity analyses</title><p>In a sensitivity analysis, we assessed the impact of treatment assignment on main outcome measure after censoring all patients with cardiac fatalities temporarily associated (that is, occurring within one week) with the occurrence of definite or probable stent thrombosis.</p><p>In another sensitivity analysis we excluded one study (SPIRIT I) with a sample size of fewer than 100 patients and assessed the main outcome measure.</p><p>The level of significance for the summary treatment effect estimate was two tailed P<0.05. We used R version 2.13.2 (the R foundation for statistical computing), Stata 11.2 statistical software (Statacorp LP, College Station, TX), and Review Manager (RevMan) version 5.1 (2011, Nordic Cochrane Centre, Cochrane Collaboration, Copenhagen) for statistical analyses.</p></sec></sec><sec sec-type="results"><title>Results</title><p>Of the 346 citations screened, we selected five randomised controlled trials (fig 1<xref ref-type="fig" rid="fig1"/>).<xref rid="ref11" ref-type="bibr">11</xref>
<xref rid="ref12" ref-type="bibr">12</xref>
<xref rid="ref13" ref-type="bibr">13</xref>
<xref rid="ref17" ref-type="bibr">17</xref>
<xref rid="ref18" ref-type="bibr">18</xref>
<xref rid="ref19" ref-type="bibr">19</xref>
<xref rid="ref20" ref-type="bibr">20</xref></p><fig id="fig1" position="float"><caption><p><bold>Fig 1</bold> Flow diagram of literature search for studies included in meta-analysis</p></caption><graphic xlink:href="valm019503.f1_default"/></fig><p>The five participating trials provided data on 4896 patients (tables 1-3<xref ref-type="table" rid="tbl1"/>
<xref ref-type="table" rid="tbl2"/>
<xref ref-type="table" rid="tbl3"/>). The average age of the study population was 67, with about 57% and 33% patients aged 65 or 75 or older, respectively. The XIMA population had the highest mean age (83), followed by PRODIGY (69), BASKET PROVE (64), SPIRIT I (63), and EXAMINATION (61). Table 1 shows the baseline characteristics of the patients included in the trials.<xref ref-type="table" rid="tbl1"/> Overall, the proportion of women was slightly over 24% and ranged from almost 40% in XIMA to 17% in EXAMINATION. Diabetes mellitus was present in roughly 19% of overall included patients. As a result of including two all comer percutaneous coronary intervention studies<xref rid="ref11" ref-type="bibr">11</xref>
<xref rid="ref13" ref-type="bibr">13</xref> and one all comer ST segment myocardial infarction trial,<xref rid="ref12" ref-type="bibr">12</xref> 44% of patients (n=2129) received stenting in the setting of primary percutaneous coronary intervention (table 2<xref ref-type="table" rid="tbl2"/>) and more than 87% (n=4279) of patients underwent coronary treatment for an unstable presentation, including 22% (n=1083) of patients with non-ST segment myocardial infarction. The recommended duration of treatment with clopidogrel after stenting differed across studies, from one year for BASKET PROVE, at least one year for EXAMINATION, at least three months in SPIRIT I, and up to 6 or 24 months, according to the randomisation scheme, in PRODIGY (table 3<xref ref-type="table" rid="tbl3"/>. In the XIMA trial, the recommended duration of clopidogrel was three months after bare metal stents and 12 months after cobalt-chromium everolimus eluting stents.</p><table-wrap id="tbl1" position="float"><label>Table 1</label><caption><p> Clinical characteristics in 4896 patients in five trials in meta-analysis of effects of bare metal stent (BMS) or cobalt-chromium everolimus eluting stents (DES) on fatal and non-fatal cardiovascular events. Figures are numbers (percentages) unless stated otherwise</p></caption><table frame="hsides" rules="groups"><thead><tr><th colspan="1" rowspan="2" align="left" valign="bottom"/><th colspan="1" rowspan="2" align="center" valign="bottom">Overall (n=4896)</th><th colspan="2" rowspan="1" align="center" valign="bottom">PRODIGY</th><th colspan="1" rowspan="2" align="center" valign="top"/><th colspan="2" rowspan="1" align="center" valign="bottom">EXAMINATION</th><th colspan="1" rowspan="2" align="center" valign="top"/><th colspan="2" rowspan="1" align="center" valign="bottom">BASKET PROVE</th><th colspan="1" rowspan="2" align="center" valign="top"/><th colspan="2" rowspan="1" align="center" valign="bottom">SPIRIT I</th><th colspan="1" rowspan="2" align="center" valign="top"/><th colspan="2" rowspan="1" align="center" valign="bottom">XIMA </th></tr><tr><th colspan="1" rowspan="1" align="center" valign="bottom">BMS (n=502)</th><th colspan="1" rowspan="1" align="center" valign="bottom">DES (n=501)</th><th colspan="1" rowspan="1" align="center" valign="bottom">BMS (n=747)</th><th colspan="1" rowspan="1" align="center" valign="bottom">DES (n=751)</th><th colspan="1" rowspan="1" align="center" valign="bottom">BMS (n=765)</th><th colspan="1" rowspan="1" align="center" valign="bottom">DES (n=774)</th><th colspan="1" rowspan="1" align="center" valign="bottom"> BMS (n=29)</th><th colspan="1" rowspan="1" align="center" valign="bottom">DES (n=27)</th><th colspan="1" rowspan="1" align="center" valign="bottom">BMS (n=401)</th><th colspan="1" rowspan="1" align="center" valign="bottom">DES (n=399)</th></tr></thead><tbody><tr><td colspan="1" rowspan="1" align="left" valign="top">Mean (SD) age (years)</td><td colspan="1" rowspan="1" align="center" valign="top">67 (13)</td><td colspan="1" rowspan="1" align="center" valign="top">68.9 (11.2)</td><td colspan="1" rowspan="1" align="center" valign="top">68.1 (11.3)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">61.6 (12.6)</td><td colspan="1" rowspan="1" align="center" valign="top">60.8 (12.2)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">64.1 (10.8)</td><td colspan="1" rowspan="1" align="center" valign="top">63.6 (10.7)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">61.4 (9.3)</td><td colspan="1" rowspan="1" align="center" valign="top">64.2 (9.6)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">83.4 (3.1)</td><td colspan="1" rowspan="1" align="center" valign="top">83.6 (3.2)</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">Female </td><td colspan="1" rowspan="1" align="center" valign="top">1200 (24.5)</td><td colspan="1" rowspan="1" align="center" valign="top">139 (27.7)</td><td colspan="1" rowspan="1" align="center" valign="top">107 (21.4)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">137 (18.3)</td><td colspan="1" rowspan="1" align="center" valign="top">117 (15.6)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">179 (23.4)</td><td colspan="1" rowspan="1" align="center" valign="top">187 (24.2)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">7 (24.1)</td><td colspan="1" rowspan="1" align="center" valign="top">8 (29.6)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">164 (40.9)</td><td colspan="1" rowspan="1" align="center" valign="top">155 (38.8)</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">Diabetes </td><td colspan="1" rowspan="1" align="center" valign="top">914 (18.7)</td><td colspan="1" rowspan="1" align="center" valign="top">111 (22.1)</td><td colspan="1" rowspan="1" align="center" valign="top">113 (22.5)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">121 (16.2)</td><td colspan="1" rowspan="1" align="center" valign="top">137 (18.3)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">108 (14.1)</td><td colspan="1" rowspan="1" align="center" valign="top">119 (15.4)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">3 (10.3)</td><td colspan="1" rowspan="1" align="center" valign="top">3 (11.1)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">97 (24.2)</td><td colspan="1" rowspan="1" align="center" valign="top">102 (25.6)</td></tr><tr><td colspan="16" rowspan="1" align="left" valign="top">Smoking habit:</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"> Current smoker</td><td colspan="1" rowspan="1" align="center" valign="top">1581 (32.3)</td><td colspan="1" rowspan="1" align="center" valign="top">126 (25.2)</td><td colspan="1" rowspan="1" align="center" valign="top">117 (23.4)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">386 (51.7)</td><td colspan="1" rowspan="1" align="center" valign="top">372 (49.6)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">261 (34.1)</td><td colspan="1" rowspan="1" align="center" valign="top">267 (34.5)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">9 (31.0)</td><td colspan="1" rowspan="1" align="center" valign="top">7 (25.9)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">16 (3.9)</td><td colspan="1" rowspan="1" align="center" valign="top">20 (5.0)</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"> Former smoker</td><td colspan="1" rowspan="1" align="center" valign="top">1323 (27.0)</td><td colspan="1" rowspan="1" align="center" valign="top">101 (20.2)</td><td colspan="1" rowspan="1" align="center" valign="top">124 (24.8)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">152 (20.3)</td><td colspan="1" rowspan="1" align="center" valign="top">172 (22.9)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">203 (26.5)</td><td colspan="1" rowspan="1" align="center" valign="top">229 (29.6)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">7 (24.1)</td><td colspan="1" rowspan="1" align="center" valign="top">2 (7.4)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">168 (41.9)</td><td colspan="1" rowspan="1" align="center" valign="top">165 (41.3)</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"> Never</td><td colspan="1" rowspan="1" align="center" valign="top">1987 (40.6)</td><td colspan="1" rowspan="1" align="center" valign="top">272 (54.5)</td><td colspan="1" rowspan="1" align="center" valign="top">259 (51.8)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">209 (27.9)</td><td colspan="1" rowspan="1" align="center" valign="top">206 (27.5)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">301 (39.3)</td><td colspan="1" rowspan="1" align="center" valign="top">278 (35.9)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">13 (44.8)</td><td colspan="1" rowspan="1" align="center" valign="top">18 (66.7)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">217 (54.1)</td><td colspan="1" rowspan="1" align="center" valign="top">214 (53.6)</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">Hypertension </td><td colspan="1" rowspan="1" align="center" valign="top">3055 (62.4)</td><td colspan="1" rowspan="1" align="center" valign="top">376 (74.9)</td><td colspan="1" rowspan="1" align="center" valign="top">356 (71.1)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">378 (50.6)</td><td colspan="1" rowspan="1" align="center" valign="top">347 (46.3)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">485 (63.4)</td><td colspan="1" rowspan="1" align="center" valign="top">469 (60.6)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">15 (51.7)</td><td colspan="1" rowspan="1" align="center" valign="top">19 (70.4)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">311 (77.6)</td><td colspan="1" rowspan="1" align="center" valign="top">299 (75.1)</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">Hypercholesterolaemia </td><td colspan="1" rowspan="1" align="center" valign="top">2680 (54.7)</td><td colspan="1" rowspan="1" align="center" valign="top">254 (50.6)</td><td colspan="1" rowspan="1" align="center" valign="top">294 (58.7)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">301 (40.3)</td><td colspan="1" rowspan="1" align="center" valign="top">354 (47.2)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">495 (64.7)</td><td colspan="1" rowspan="1" align="center" valign="top">498 (64.3)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">22 (75.9)</td><td colspan="1" rowspan="1" align="center" valign="top">20 (70.1)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">212 (52.9)</td><td colspan="1" rowspan="1" align="center" valign="top">230 (57.6)</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">Previous MI </td><td colspan="1" rowspan="1" align="center" valign="top">724 (14.8)</td><td colspan="1" rowspan="1" align="center" valign="top">114 (22.8)</td><td colspan="1" rowspan="1" align="center" valign="top">143 (28.7)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">47 (6.3)</td><td colspan="1" rowspan="1" align="center" valign="top">33 (4.4)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">100 (13.1)</td><td colspan="1" rowspan="1" align="center" valign="top">82 (10.6)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">0</td><td colspan="1" rowspan="1" align="center" valign="top">0</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">86 (21.5)</td><td colspan="1" rowspan="1" align="center" valign="top">119 (29.8)</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">Previous CABG </td><td colspan="1" rowspan="1" align="center" valign="top">201 (4.1)</td><td colspan="1" rowspan="1" align="center" valign="top">45 (8.9)</td><td colspan="1" rowspan="1" align="center" valign="top">61 (12.2)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">7 (0.9)</td><td colspan="1" rowspan="1" align="center" valign="top">3 (0.4)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">20 (2.6)</td><td colspan="1" rowspan="1" align="center" valign="top">20 (2.6)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">0</td><td colspan="1" rowspan="1" align="center" valign="top">0</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">17 (4.2)</td><td colspan="1" rowspan="1" align="center" valign="top">28 (7.0)</td></tr></tbody></table><table-wrap-foot><p>MI=myocardial infarction; CABG=coronary artery bypass grafting.</p></table-wrap-foot></table-wrap><table-wrap id="tbl2" position="float"><label>Table 2</label><caption><p> Clinical presentation and treatment in 4896 patients in five trials in meta-analysis of effects of bare metal stent (BMS) or cobalt-chromium everolimus eluting (DES) on fatal and non-fatal cardiovascular events. Figures are numbers (percentages) unless stated otherwise</p></caption><table frame="hsides" rules="groups"><thead><tr><th colspan="1" rowspan="2" align="left" valign="bottom"/><th colspan="1" rowspan="2" align="center" valign="bottom">Overall (n=4896)</th><th colspan="2" rowspan="1" align="center" valign="bottom">PRODIGY</th><th colspan="1" rowspan="2" align="center" valign="top"/><th colspan="2" rowspan="1" align="center" valign="bottom">EXAMINATION</th><th colspan="1" rowspan="2" align="center" valign="top"/><th colspan="2" rowspan="1" align="center" valign="bottom">BASKET PROVE</th><th colspan="1" rowspan="2" align="center" valign="top"/><th colspan="2" rowspan="1" align="center" valign="bottom">SPIRIT I</th><th colspan="1" rowspan="2" align="center" valign="top"/><th colspan="2" rowspan="1" align="center" valign="bottom">XIMA </th></tr><tr><th colspan="1" rowspan="1" align="center" valign="bottom"> BMS (n=502)</th><th colspan="1" rowspan="1" align="center" valign="bottom"> DES (n=501)</th><th colspan="1" rowspan="1" align="center" valign="bottom"> BMS (n=747)</th><th colspan="1" rowspan="1" align="center" valign="bottom"> DES (n=751)</th><th colspan="1" rowspan="1" align="center" valign="bottom"> BMS (n=765)</th><th colspan="1" rowspan="1" align="center" valign="bottom"> DES (n=774)</th><th colspan="1" rowspan="1" align="center" valign="bottom"> BMS (n=29)</th><th colspan="1" rowspan="1" align="center" valign="bottom">DES (n=27)</th><th colspan="1" rowspan="1" align="center" valign="bottom">BMS (n=401)</th><th colspan="1" rowspan="1" align="center" valign="bottom">DES (n=399)</th></tr></thead><tbody><tr><td colspan="1" rowspan="1" align="left" valign="top">Stable angina pectoris</td><td colspan="1" rowspan="1" align="center" valign="top">615 (15.6)</td><td colspan="1" rowspan="1" align="center" valign="top">111 (22.1)</td><td colspan="1" rowspan="1" align="center" valign="top">118 (23.6)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">0</td><td colspan="1" rowspan="1" align="center" valign="top">0</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">34 (4.4)</td><td colspan="1" rowspan="1" align="center" valign="top">41 (5.3)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">29 (100)</td><td colspan="1" rowspan="1" align="center" valign="top">27 (100)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">127 (31.7)</td><td colspan="1" rowspan="1" align="center" valign="top">128 (32.2)</td></tr><tr><td colspan="16" rowspan="1" align="left" valign="top">Acute coronary syndrome (ACS):</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"> Non-ST elevation ACS</td><td colspan="1" rowspan="1" align="center" valign="top">2150 (43.9)</td><td colspan="1" rowspan="1" align="center" valign="top">211 (42.0)</td><td colspan="1" rowspan="1" align="center" valign="top">220 (43.9)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">0</td><td colspan="1" rowspan="1" align="center" valign="top">0</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">615 (80.4)</td><td colspan="1" rowspan="1" align="center" valign="top">619 (79.9)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">0</td><td colspan="1" rowspan="1" align="center" valign="top">0</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">237 (59.2)</td><td colspan="1" rowspan="1" align="center" valign="top">248 (62.3)</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"> ST segment elevation MI</td><td colspan="1" rowspan="1" align="center" valign="top">2129 (43.5)</td><td colspan="1" rowspan="1" align="center" valign="top">180 (35.9)</td><td colspan="1" rowspan="1" align="center" valign="top">163 (32.5)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">747 (100)</td><td colspan="1" rowspan="1" align="center" valign="top">751 (100)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">116 (15.1)</td><td colspan="1" rowspan="1" align="center" valign="top">114 (14.7)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">0</td><td colspan="1" rowspan="1" align="center" valign="top">0</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">36 (9.0)</td><td colspan="1" rowspan="1" align="center" valign="top">22 (5.5)</td></tr><tr><td colspan="16" rowspan="1" align="left" valign="top">Angiographic features:</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"> Single vessel disease </td><td colspan="1" rowspan="1" align="center" valign="top">2795 (57.1)</td><td colspan="1" rowspan="1" align="center" valign="top">170 (33.9)</td><td colspan="1" rowspan="1" align="center" valign="top">145 (28.9)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">653 (87.4)</td><td colspan="1" rowspan="1" align="center" valign="top">634 (84.4)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">438 (57.2)</td><td colspan="1" rowspan="1" align="center" valign="top">455 (58.8)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">23 (79.3)</td><td colspan="1" rowspan="1" align="center" valign="top">20 (74.1)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">127 (31.7)</td><td colspan="1" rowspan="1" align="center" valign="top">130 (32.6)</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"> Multi-vessel disease </td><td colspan="1" rowspan="1" align="center" valign="top">2096 (42.8)</td><td colspan="1" rowspan="1" align="center" valign="top">332 (66.1)</td><td colspan="1" rowspan="1" align="center" valign="top">356 (71.1)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">94 (12.6)</td><td colspan="1" rowspan="1" align="center" valign="top">117 (15.6)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">327 (42.7)</td><td colspan="1" rowspan="1" align="center" valign="top">319 (41.2)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">6 (20.7)</td><td colspan="1" rowspan="1" align="center" valign="top">7 (25.9)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">270 (67.3)</td><td colspan="1" rowspan="1" align="center" valign="top">268 (67.2)</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">LAD/LMCA treated </td><td colspan="1" rowspan="1" align="center" valign="top">2523 (51.5)</td><td colspan="1" rowspan="1" align="center" valign="top">287 (57.2)</td><td colspan="1" rowspan="1" align="center" valign="top">260 (51.9)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">309 (41.4)</td><td colspan="1" rowspan="1" align="center" valign="top">326 (43.4)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">404 (52.8)</td><td colspan="1" rowspan="1" align="center" valign="top">417 (53.9)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">13 (44.8)</td><td colspan="1" rowspan="1" align="center" valign="top">13 (48.1)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">248 (61.8)</td><td colspan="1" rowspan="1" align="center" valign="top">246 (246)</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">CFX treated </td><td colspan="1" rowspan="1" align="center" valign="top">1155 (23.6)</td><td colspan="1" rowspan="1" align="center" valign="top">147 (29.3)</td><td colspan="1" rowspan="1" align="center" valign="top">186 (37.1)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">107 (14.3)</td><td colspan="1" rowspan="1" align="center" valign="top">102 (13.6)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">203 (26.5)</td><td colspan="1" rowspan="1" align="center" valign="top">202 (26.1)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">6 (20.7)</td><td colspan="1" rowspan="1" align="center" valign="top">6 (22.2)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">97 (24.2)</td><td colspan="1" rowspan="1" align="center" valign="top">99 (24.8)</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">RCA treated </td><td colspan="1" rowspan="1" align="center" valign="top">1934 (39.5)</td><td colspan="1" rowspan="1" align="center" valign="top">190 (37.8)</td><td colspan="1" rowspan="1" align="center" valign="top">177 (35.3)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">329 (44.0)</td><td colspan="1" rowspan="1" align="center" valign="top">322 (42.9)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">325 (42.5)</td><td colspan="1" rowspan="1" align="center" valign="top">310 (40.0)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">10 (34.5)</td><td colspan="1" rowspan="1" align="center" valign="top">8 (29.6)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">131 (32.7)</td><td colspan="1" rowspan="1" align="center" valign="top">132 (33.1)</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">No of treated lesions </td><td colspan="1" rowspan="1" align="center" valign="top">1.3 (0.69)</td><td colspan="1" rowspan="1" align="center" valign="top">1.5 (0.8)</td><td colspan="1" rowspan="1" align="center" valign="top">1.5 (0.9)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">1.1 (0.3)</td><td colspan="1" rowspan="1" align="center" valign="top">1.2 (0.4)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">1.5 (0.8)</td><td colspan="1" rowspan="1" align="center" valign="top">1.4 (0.8)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">1</td><td colspan="1" rowspan="1" align="center" valign="top">1</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">1.4 (0.6)</td><td colspan="1" rowspan="1" align="center" valign="top">1.4 (0.6)</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">No of stents implanted</td><td colspan="1" rowspan="1" align="center" valign="top">1.6 (1.0)</td><td colspan="1" rowspan="1" align="center" valign="top">1.8 (1.1)</td><td colspan="1" rowspan="1" align="center" valign="top">1.9 (1.2)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">1.5 (0.7)</td><td colspan="1" rowspan="1" align="center" valign="top">1.5 (0.8)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">1.7 (1.1)</td><td colspan="1" rowspan="1" align="center" valign="top">1.7 (1.1)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">1</td><td colspan="1" rowspan="1" align="center" valign="top">1</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">1.6 (1.1)</td><td colspan="1" rowspan="1" align="center" valign="top">1.6 (1.1)</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">Total stent length (mm)</td><td colspan="1" rowspan="1" align="center" valign="top">30.9 (22.2)</td><td colspan="1" rowspan="1" align="center" valign="top">36.9 (30.1)</td><td colspan="1" rowspan="1" align="center" valign="top">39.6 (28.3)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">27.0 (13.2)</td><td colspan="1" rowspan="1" align="center" valign="top">27.9 (14.8)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">31.1 (22.5)</td><td colspan="1" rowspan="1" align="center" valign="top">31.1 (23.3)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">18</td><td colspan="1" rowspan="1" align="center" valign="top">18</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">29.1 (20.6)</td><td colspan="1" rowspan="1" align="center" valign="top">28.5 (22.9)</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">Stent size (mm)</td><td colspan="1" rowspan="1" align="center" valign="top">3.2 (0.4)</td><td colspan="1" rowspan="1" align="center" valign="top">2.9 (0.5)</td><td colspan="1" rowspan="1" align="center" valign="top">2.9 (0.5)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">3.2 (0.4)</td><td colspan="1" rowspan="1" align="center" valign="top">3.2 (0.4)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">3.5 (0.3)</td><td colspan="1" rowspan="1" align="center" valign="top">3.5 (0.4)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">3</td><td colspan="1" rowspan="1" align="center" valign="top">3</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">2.9 (0.3)</td><td colspan="1" rowspan="1" align="center" valign="top">2.9 (0.3)</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">Overlapping stents </td><td colspan="1" rowspan="1" align="center" valign="top">1149 (23.5)</td><td colspan="1" rowspan="1" align="center" valign="top">139 (27.7)</td><td colspan="1" rowspan="1" align="center" valign="top">142 (28.3)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">206 (27.6)</td><td colspan="1" rowspan="1" align="center" valign="top">198 (26.4)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">150 (19.6)</td><td colspan="1" rowspan="1" align="center" valign="top">155 (20.0)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">0</td><td colspan="1" rowspan="1" align="center" valign="top">0</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">84 (20.9)</td><td colspan="1" rowspan="1" align="center" valign="top">75 (18.8)</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">Use of IIb/IIIa inhibitors</td><td colspan="1" rowspan="1" align="center" valign="top">1652 (33.8)</td><td colspan="1" rowspan="1" align="center" valign="top">232 (46.2)</td><td colspan="1" rowspan="1" align="center" valign="top">219 (43.7)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">385 (51.5)</td><td colspan="1" rowspan="1" align="center" valign="top">400 (53.3)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">168 (21.9)</td><td colspan="1" rowspan="1" align="center" valign="top">176 (22.7)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">1 (3.4)</td><td colspan="1" rowspan="1" align="center" valign="top">2 (7.4)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">37 (9.4)</td><td colspan="1" rowspan="1" align="center" valign="top">32 (8.1)</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">No of patients who continued DAPT beyond one year</td><td colspan="1" rowspan="1" align="center" valign="top">707 (14.4)</td><td colspan="1" rowspan="1" align="center" valign="top">230 (46.1)</td><td colspan="1" rowspan="1" align="center" valign="top">258 (51.6)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">98 (13.1)</td><td colspan="1" rowspan="1" align="center" valign="top">115 (15.3)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">0</td><td colspan="1" rowspan="1" align="center" valign="top">0</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">4 (13.8)</td><td colspan="1" rowspan="1" align="center" valign="top">2 (7.4)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">0</td><td colspan="1" rowspan="1" align="center" valign="top">0</td></tr></tbody></table><table-wrap-foot><p>DAPT=dual antiplatelet therapy; MI=myocardial infarction; RCA=right coronary artery; CFX=circumflex artery; LAD=left anterior descending artery; LMCA=left main coronary artery.</p></table-wrap-foot></table-wrap><table-wrap id="tbl3" position="float"><label>Table 3</label><caption><p> Recommended duration (months) of dual antiplatelet therapy and follow-up treatment in three of five trials in meta-analysis of effects of bare metal stent (BMS) or cobalt-chromium everolimus eluting (DES) on fatal and non-fatal cardiovascular events</p></caption><table frame="hsides" rules="groups"><thead><tr><th colspan="1" rowspan="1" align="left" valign="bottom"/><th colspan="1" rowspan="1" align="center" valign="bottom">PRODIGY (n=1003)</th><th colspan="1" rowspan="1" align="center" valign="bottom">EXAMINATION (n=1498)</th><th colspan="1" rowspan="1" align="center" valign="bottom">BASKET PROVE (n=1539)</th><th colspan="1" rowspan="1" align="center" valign="bottom">SPIRIT I (n=56)</th><th colspan="1" rowspan="1" align="center" valign="bottom">XIMA (n=800)</th></tr></thead><tbody><tr><td colspan="1" rowspan="1" align="left" valign="middle">Experimental arm</td><td colspan="1" rowspan="1" align="center" valign="middle">6 or 24*</td><td colspan="1" rowspan="1" align="center" valign="middle">12</td><td colspan="1" rowspan="1" align="center" valign="middle">12</td><td colspan="1" rowspan="1" align="center" valign="middle">≥3</td><td colspan="1" rowspan="1" align="center" valign="middle">12</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="middle">Control arm</td><td colspan="1" rowspan="1" align="center" valign="middle">Up to 6 or 24*</td><td colspan="1" rowspan="1" align="center" valign="middle">12</td><td colspan="1" rowspan="1" align="center" valign="middle">12</td><td colspan="1" rowspan="1" align="center" valign="middle">≥3</td><td colspan="1" rowspan="1" align="center" valign="middle">3</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="middle">Follow-up (years)</td><td colspan="1" rowspan="1" align="center" valign="middle">2</td><td colspan="1" rowspan="1" align="center" valign="middle">2</td><td colspan="1" rowspan="1" align="center" valign="middle">2</td><td colspan="1" rowspan="1" align="center" valign="middle">5</td><td colspan="1" rowspan="1" align="center" valign="middle">1</td></tr></tbody></table><table-wrap-foot><p>*1:1 randomisation.</p></table-wrap-foot></table-wrap><p>Notably, the comparator bare metal stent arm was homogenised with the use of cobalt-chromium Vision stent in BASKET PROVE, EXAMINATION, SPIRIT I, and XIMA<xref rid="ref17" ref-type="bibr">17</xref> whereas the choice of bare metal stent type was left at the discretion of the treating physician in PRODIGY.</p><p>Median duration of follow-up was 720 days (interquartile range 719-720) in both groups. Complete two year follow-up data were available for four studies,<xref rid="ref11" ref-type="bibr">11</xref>
<xref rid="ref18" ref-type="bibr">18</xref>
<xref rid="ref19" ref-type="bibr">19</xref>
<xref rid="ref20" ref-type="bibr">20</xref> including 3933 (80.3%) patients. Thirty nine (0.8%) patients (19 in the bare metal and 17 in the cobalt-chromium everolimus eluting stent group) were lost to follow-up.</p><sec><title>Bias assessment</title><p>Figure 2 shows the systematic bias analysis<xref ref-type="fig" rid="fig2"/>. There was a low risk of bias for most items for each study, except for the presence of performance bias because of the lack of the double blind design in each study. Further the potential for selection bias in the SPIRIT I study was acknowledged.</p><fig id="fig2" position="float"><caption><p><bold>Fig 2</bold> Risk of bias summary reporting each risk of bias item for each included study</p></caption><graphic xlink:href="valm019503.f2_default"/></fig></sec><sec><title>Mortality endpoints</title><p>At two years, the risk of cardiac death was significantly reduced in the cobalt-chromium everolimus eluting stent group at 2.7% compared with 4.0% in the bare metal stent arm (hazard ratio 0.67, 95% confidence interval 0.49 to 0.91; P=0.01; fig 3<xref ref-type="fig" rid="fig3"/>, table 4<xref ref-type="table" rid="tbl4"/>. With multivariable regression, cardiac mortality remained significantly lower in the cobalt-chromium everolimus eluting stent group (0.69, 0.50 to 0.94; P=0.02; table 4).</p><fig id="fig3" position="float"><caption><p><bold>Fig 3</bold> Cumulative hazard for cardiac death and forest plot with point estimates and 95% confidence intervals for each included study and pooled estimate from simple Cox regression model stratified by trial with random effects. Co-Cr EES=cobalt-chromium everolimus eluting; BMS=bare metal stent</p></caption><graphic xlink:href="valm019503.f3_default"/></fig><table-wrap id="tbl4" position="float"><label>Table 4</label><caption><p> Overall clinical outcomes in 4896 patients in five trials in meta-analysis by treatment assignment to bare metal stent (BMS) or cobalt chromium-everolimus eluting stent (DES). Figures are numbers (percentage) of patients and hazard ratios (HR) from multivariable Cox regression stratified by trial</p></caption><table frame="hsides" rules="groups"><thead><tr><th colspan="1" rowspan="2" align="left" valign="bottom">Outcomes</th><th colspan="2" rowspan="1" align="center" valign="bottom">Event rate</th><th colspan="1" rowspan="2" align="center" valign="bottom"/><th colspan="2" rowspan="1" align="center" valign="bottom">Unadjusted</th><th colspan="1" rowspan="2" align="center" valign="bottom"/><th colspan="2" rowspan="1" align="center" valign="bottom">Adjusted*</th></tr><tr><th colspan="1" rowspan="1" align="center" valign="bottom">BMS (n=2444)</th><th colspan="1" rowspan="1" align="center" valign="bottom">DES (n=2452)</th><th colspan="1" rowspan="1" align="center" valign="bottom">HR (95% CI)</th><th colspan="1" rowspan="1" align="center" valign="bottom">P value</th><th colspan="1" rowspan="1" align="center" valign="bottom">HR (95% CI)</th><th colspan="1" rowspan="1" align="center" valign="bottom">P value</th></tr></thead><tbody><tr><td colspan="1" rowspan="1" align="left" valign="top">Cardiac death </td><td colspan="1" rowspan="1" align="center" valign="top">99 (4.1)</td><td colspan="1" rowspan="1" align="center" valign="top">67 (2.7)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">0.67 (0.49 to 0.91)</td><td colspan="1" rowspan="1" align="center" valign="top">0.01</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">0.69 (0.50 to 0.94)</td><td colspan="1" rowspan="1" align="center" valign="top">0.02</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">Death from any cause </td><td colspan="1" rowspan="1" align="center" valign="top">144 (5.9)</td><td colspan="1" rowspan="1" align="center" valign="top">121 (4.9)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">0.83 (0.65 to 1.06)</td><td colspan="1" rowspan="1" align="center" valign="top">0.14</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">0.84 (0.66 to 1.07)</td><td colspan="1" rowspan="1" align="center" valign="top">0.16</td></tr><tr><td colspan="9" rowspan="1" align="left" valign="top">Myocardial infarction:</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"> All</td><td colspan="1" rowspan="1" align="center" valign="top">136 (5.6)</td><td colspan="1" rowspan="1" align="center" valign="top">98 (4.0)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">0.71 (0.55 to 0.92)</td><td colspan="1" rowspan="1" align="center" valign="top">0.01</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">0.71 (0.55 to 0.93)</td><td colspan="1" rowspan="1" align="center" valign="top">0.01</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"> Non-fatal</td><td colspan="1" rowspan="1" align="center" valign="top">118 (4.8)</td><td colspan="1" rowspan="1" align="center" valign="top">96 (3.9)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">0.81 (0.61 to 1.05)</td><td colspan="1" rowspan="1" align="center" valign="top">0.12</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">0.80 (0.61 to 1.05)</td><td colspan="1" rowspan="1" align="center" valign="top">0.12</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top"> Fatal</td><td colspan="1" rowspan="1" align="center" valign="top">18 (0.8)</td><td colspan="1" rowspan="1" align="center" valign="top">2 (0.1)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">0.11 (0.03 to 0.48)</td><td colspan="1" rowspan="1" align="center" valign="top">0.003</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">0.11 (0.03 to 0.49)</td><td colspan="1" rowspan="1" align="center" valign="top">0.004</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">Definite stent thrombosis </td><td colspan="1" rowspan="1" align="center" valign="top">33 (1.4)</td><td colspan="1" rowspan="1" align="center" valign="top">14 (0.6)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">0.42 (0.22 to 0.78)</td><td colspan="1" rowspan="1" align="center" valign="top">0.006</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">0.41 (0.22 to 0.76)</td><td colspan="1" rowspan="1" align="center" valign="top">0.005</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">Definite or probable stent thrombosis </td><td colspan="1" rowspan="1" align="center" valign="top">63 (2.6)</td><td colspan="1" rowspan="1" align="center" valign="top">31 (1.3)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">0.48 (0.31 to 0.74)</td><td colspan="1" rowspan="1" align="center" valign="top">0.001</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">0.48 (0.31 to 0.73)</td><td colspan="1" rowspan="1" align="center" valign="top">0.001</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">Target vessel revascularisation </td><td colspan="1" rowspan="1" align="center" valign="top">250 (10.2)</td><td colspan="1" rowspan="1" align="center" valign="top">105 (4.3)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">0.29 (0.20 to 0.42)</td><td colspan="1" rowspan="1" align="center" valign="top"><0.001</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">0.29 (0.20 to 0.41)</td><td colspan="1" rowspan="1" align="center" valign="top"><0.001</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">Target vessel myocardial infarction</td><td colspan="1" rowspan="1" align="center" valign="top">44 (1.8)</td><td colspan="1" rowspan="1" align="center" valign="top">22 (0.9)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">0.27 (0.12 to 0.58)</td><td colspan="1" rowspan="1" align="center" valign="top">0.001</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">0.27 (0.12 to 0.59)</td><td colspan="1" rowspan="1" align="center" valign="top">0.001</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">Cardiac death or myocardial infarction</td><td colspan="1" rowspan="1" align="center" valign="top">207 (8.5)</td><td colspan="1" rowspan="1" align="center" valign="top">156 (6.4)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">0.75 (0.61 to 0.92)</td><td colspan="1" rowspan="1" align="center" valign="top">0.006</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">0.76 (0.61 to 0.93)</td><td colspan="1" rowspan="1" align="center" valign="top">0.008</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">Death or myocardial infarction</td><td colspan="1" rowspan="1" align="center" valign="top">250 (10.2)</td><td colspan="1" rowspan="1" align="center" valign="top">206 (8.4)</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">0.81 (0.68 to 0.98)</td><td colspan="1" rowspan="1" align="center" valign="top">0.028</td><td colspan="1" rowspan="1" align="center" valign="top"/><td colspan="1" rowspan="1" align="center" valign="top">0.81 (0.68 to 0.98)</td><td colspan="1" rowspan="1" align="center" valign="top">0.03</td></tr></tbody></table><table-wrap-foot><p>*Adjusted for clinical syndrome (that is, acute coronary syndrome <italic>v</italic> stable syndrome), history of diabetes mellitus, female sex, use of glycoprotein IIb/IIIa inhibitors, and up to 1 year <italic>v</italic> longer duration of dual antiplatelet therapy. </p></table-wrap-foot></table-wrap><p>Overall mortality was not significantly reduced in the cobalt-chromium everolimus eluting stent group (4.9% <italic>v</italic> 5.9%; unadjusted hazard ratio 0.83, 95% confidence interval 0.65 to 1.06; P=0.14; adjusted hazard ratio 0.84, 0.66 to 1.07; P=0.16) (table 4<xref ref-type="table" rid="tbl4"/>, fig A in appendix 2). There was evidence for small non-significant heterogeneity for cardiac mortality (table A in appendix 1).</p></sec><sec><title>Other fatal and/or non-fatal cardiovascular endpoints</title><p>At two years, the risk of myocardial infarction was significantly lower in the cobalt-chromium everolimus eluting stent group (4.0% <italic>v</italic> 5.6% in the bare metal stent group; unadjusted hazard ratio 0.71, 95% confidence interval 0.55 to 0.92; P=0.01; adjusted hazard ratio 0.71, 0.55 to 0.93; P=0.01 (table 4<xref ref-type="table" rid="tbl4"/>, fig 4<xref ref-type="fig" rid="fig4"/>).</p><fig id="fig4" position="float"><caption><p><bold>Fig 4</bold> Cumulative incidence curve for myocardial infarction and forest plot with point estimates and 95% confidence intervals for each included study and pooled estimate from simple Cox regression model stratified by trial with random effects. Co-Cr EES=cobalt-chromium everolimus eluting; BMS=bare metal stent</p></caption><graphic xlink:href="valm019503.f4_default"/></fig><p>The risk of fatal myocardial infarction was significantly lower in the cobalt-chromium everolimus eluting stent group (unadjusted hazard ratio 0.11, 95% confidence interval 0.03 to 0.48, P=0.003; adjusted hazard ratio 0.11, 0.03 to 0.49, P=0.004; table 4, fig 5<xref ref-type="fig" rid="fig5"/>). No significant difference in the risk of non-fatal myocardial infarction between groups was found (table 4, fig B in appendix 2).<xref ref-type="table" rid="tbl4"/></p><fig id="fig5" position="float"><caption><p><bold>Fig 5</bold> Cumulative incidence curve for fatal myocardial infarction and forest plot with point estimates and 95% confidence interval for each included study and pooled estimate from simple Cox regression model stratified by trial with random effects. Co-Cr EES=cobalt-chromium everolimus eluting; BMS=bare metal stent</p></caption><graphic xlink:href="valm019503.f5_default"/></fig><p>Similarly, at two years the risk of definite stent thrombosis (0.6% <italic>v</italic> 1.4%; adjusted hazard ratio 0.41, 95% confidence interval 0.22 to 0.76; P=0.005; table 4<xref ref-type="table" rid="tbl4"/>, fig 6<xref ref-type="fig" rid="fig6"/>), definite or probable stent thrombosis (1.3% <italic>v</italic> 2.6%; 0.48, 0.31 to 0.73; P=0.001; table 4, fig 7<xref ref-type="fig" rid="fig7"/>), and the need for re-intervention in the previously treated vessel (4.3% <italic>v</italic> 10.2%; adjusted hazard ratio 0.29, 0.20 to 0.41; P<0.001; table 4, fig 8<xref ref-type="fig" rid="fig8"/>) were all consistently at least halved in the cobalt-chromium everolimus eluting stent group.</p><fig id="fig6" position="float"><caption><p><bold>Fig 6</bold> Cumulative incidence curve for definite stent thrombosis and forest plot with point estimates and 95% confidence interval for each included study and pooled estimate from simple Cox regression model stratified by trial with random effects. Co-Cr EES=cobalt-chromium everolimus eluting; BMS=bare metal stent</p></caption><graphic xlink:href="valm019503.f6_default"/></fig><fig id="fig7" position="float"><caption><p><bold>Fig 7</bold> Cumulative incidence curve for definite or probable stent thrombosis and forest plot with point estimates and 95% confidence interval for each included study and pooled estimate from simple Cox regression model stratified by trial with random effects. Co-Cr EES=cobalt-chromium everolimus eluting; BMS=bare metal stent</p></caption><graphic xlink:href="valm019503.f7_default"/></fig><fig id="fig8" position="float"><caption><p><bold>Fig 8</bold> Cumulative incidence curve for target vessel revascularisation and forest plot with point estimates and 95% confidence interval for each included study and pooled estimate from simple Cox regression model stratified by trial with random effects. Co-Cr EES=cobalt-chromium everolimus eluting; BMS=bare metal stent</p></caption><graphic xlink:href="valm019503.f8_default"/></fig><p>Rates of the composite of target vessel revascularisation or myocardial infarction, cardiac death or myocardial infarction, death, or myocardial infarction were significantly lower in the cobalt-chromium everolimus eluting stent group (table 4<xref ref-type="table" rid="tbl4"/>, figs C-E in appendix 2).</p><p>Subhazard ratios calculated in competing risk analyses were consistent with results of the main analysis for each endpoint (table B in appendix 1).</p><p>For target vessel revascularization, and the composite of target vessel revascularisation or myocardial infarction, we report a plot depicting Schoenfeld-like residuals against time because of the presence of time dependent hazard ratios (fig F in appendix 2). Table C in appendix 1 shows the numbers needed to treat for each endpoint.</p><p>Heterogeneity was null or minimal for the endpoints assessed. We found a small non-significant heterogeneity for target vessel revascularisation only (table A in appendix 1).</p></sec><sec><title>Subgroups and sensitivity analyses</title><p>As shown in figure 9<xref ref-type="fig" rid="fig9"/>, the benefit of treatment with cobalt-chromium everolimus eluting stents was consistent across all analysed subgroups, including sex, age ≥65, presence of diabetes, extension of coronary artery disease, need for overlapping stents during the procedure, use of glycoprotein IIb/IIIa inhibitors, treatment of left anterior descending artery, and duration of dual antiplatelet therapy up to one year or beyond.</p><fig id="fig9" position="float"><caption><p><bold>Fig 9</bold> Subgroup analyses for treatment effect, with P value for interaction. IIb/IIIa=glycoprotein IIb/IIIa inhibitors</p></caption><graphic xlink:href="valm019503.f9_default"/></fig><p>We found an interaction for the type of coronary artery disease at the time of intervention, indicating that patients presenting with a stable disease phenotype might have a greater benefit from treatment with cobalt-chromium everolimus eluting stents with respect to cardiac death (fig 9<xref ref-type="fig" rid="fig9"/>, table D in appendix 1).</p><p>At sensitivity analysis, the occurrence of definite or probable stent thrombosis explained 47 (28.3%) fatal cardiac events. After we censored these stent related events, the cardiac mortality rate was lower at 2.8% in the bare metal stent group and 2.1% in the cobalt-chromium everolimus eluting stent group, which is not a significant difference (hazard ratio 0.76, 95% confidence interval 0.53 to 1.10; P=0.15 by multivariable Cox regression). After exclusion of the SPIRIT I study, the main outcome measure remained unchanged.</p></sec></sec><sec sec-type="discussion"><title>Discussion</title><p>Our combined analysis of data from individual patients, most of whom had acute coronary syndrome, from five randomised open label or single blinded trials in which events were independently and blinded adjudicated, shows that, compared with bare metal stents, cobalt-chromium everolimus eluting stent implantation is associated with a reduction in cardiac mortality. This benefit came with a significant reduction in myocardial infarction, fatal myocardial infarction, stent thrombosis, and target vessel revascularisation.</p><p>Randomised clinical trials provide the reference standard for comparing the effectiveness of treatments for a given clinical condition. Yet the effectiveness of treatments on relatively rare events, such as overall or cardiac mortality, might not be tested adequately in a single study because of limited statistical power. Combining individual patient data from multiple randomised trials helps to overcome this limitation by increasing the number of patients available for analysis, thus enhancing statistical power.</p><p>In particular, individual patient data meta-analyses are considered to be an ideal standard of systematic review as they have been shown to produce more reliable results.<xref rid="ref24" ref-type="bibr">24</xref> Compared with conventional meta-analyses that use aggregate data from publications, the availability of individual patient data allowed us to provide a more informative analysis of time dependent data derived from the whole period of follow-up with calculation of hazard ratios. Moreover, an individual patient data meta-analysis made possible an accurate definition of prespecified subgroup of patients, allowing for consistency assessment of study findings across predefined patient subgroups. Finally, it allowed us to investigate the effect of stent type on cardiac mortality after censoring specific fatal events related to stent or procedure, as well as assessing the robustness of our main study results by implementing both a multivariable and competing risk assessment analyses.</p><sec><title>Possible mechanisms</title><p>To get some insights into the possible mechanism leading to a cardiac mortality benefit after cobalt-chromium everolimus eluting stent implantation, at sensitivity analysis we censored all cardiac fatalities temporarily associated (that is, occurring within one week) with the occurrence of definite or probable stent thrombosis. We chose a seven day time window to maximise specificity even if it is known to underestimate the actual burden of stent thrombosis in terms of cardiac fatalities.<xref rid="ref25" ref-type="bibr">25</xref> These events explained roughly a quarter of all cardiac fatalities observed up to two years. After we censored these stent related events, cardiac mortality was no longer different between the two stent groups, suggesting a mechanistic interpretation of our clinical observations. The lack of a significant difference in all cause mortality, despite the difference in cardiac mortality, is of interest. Crude non-cardiac mortality was 0.19%, 1.79%, and 4.02% in first, second, and last third of the age distribution, respectively (P<0.001).</p><p>A potential diluting effect of benefit observed for cobalt-chromium everolimus eluting stent on all cause mortality could be ascribed to the higher non-cardiac mortality rate among older patients.</p><p>Treatment effect was not altered by other characteristics of patients, including sex, age, presence of diabetes, extension of coronary artery disease, the need for overlapping stents, use of glycoprotein IIb/IIIa inhibitors, treatment of left anterior descending artery, or duration of clopidogrel therapy after coronary stenting.</p><p>We found evidence of an interaction between treatment effect and stent type for the acuity of coronary artery disease, indicating that patients with stable coronary artery disease might have greater benefit from treatment with cobalt-chromium everolimus eluting stents in terms of cardiac mortality. This could be chance a finding, however, because of the small sample size of stable patients (n=610) and the multiple explored subgroups (thereby inflating type I error). Further studies are warranted to verify this hypothesis.</p></sec><sec><title>Comparison with other studies</title><p>After the widespread use of first generation drug eluting stents in clinical practice, concern was raised that the reduction in restenosis associated with these stents was achieved at the cost of a potential increase in adverse clinical events, including death and myocardial infarction.<xref rid="ref26" ref-type="bibr">26</xref>
<xref rid="ref27" ref-type="bibr">27</xref>
<xref rid="ref28" ref-type="bibr">28</xref> Long term treatment with dual antiplatelets has been therefore advocated after implantation of drug eluting stents,<xref rid="ref29" ref-type="bibr">29</xref> despite the absence of supporting evidence. Considerable efforts have gone into the development of new generation drug eluting stents that would retain the powerful anti-restenosis properties of sirolimus and paclitaxel eluting stents, yet provide improved deliverability and regain safety profiles comparable with bare metal stents.<xref rid="ref30" ref-type="bibr">30</xref></p><p>Most randomised trials, however, have compared second generation drug eluting stents with their first generation counterparts, and rarely with bare metal stents, thus restricting a fully comparative understanding of their outcomes. No single study comparing drug eluting with bare metal stents or different types of drug eluting stents has ever been powered for mortality. Hence, the lack of evidence coming from a single, albeit relatively large, randomised controlled trial regarding the proposed association between stent thrombosis and mortality should be interpreted with the knowledge of the likelihood of type II error.</p><p>Recently, a network meta-analysis of 49 randomised trials, including 50 844 patients, suggested that cobalt-chromium everolimus eluting stent was associated with a significant reduction of definite stent thrombosis during two years of follow-up compared with bare metal stent.<xref rid="ref31" ref-type="bibr">31</xref> That analysis, however, included only two direct randomised trials comparing cobalt-chromium everolimus eluting and bare metal stents,<xref rid="ref11" ref-type="bibr">11</xref>
<xref rid="ref12" ref-type="bibr">12</xref> and follow-up data for over a year were available for only one<xref rid="ref11" ref-type="bibr">11</xref> of these two studies. Therefore, this network meta-analysis fell short in dealing with the concern that very late stent thrombosis (that is, beyond one year) might counterweigh early reduction of thrombotic events. Notably this paradigm has also been observed in pooled analyses of first generation drug eluting versus bare metal stent.<xref rid="ref6" ref-type="bibr">6</xref> Different durations of dual antiplatelet therapy in patients treated with the two types of stent or differential use of antithrombotics at the time of intervention are also possible confounding factors in this study, which were not corrected for.<xref rid="ref31" ref-type="bibr">31</xref> Therefore, whether cobalt-chromium everolimus eluting stents would provide sustained improved safety over bare metal stents and most importantly to what extent this could translate into an improved survival rate remains so far largely elusive.</p></sec><sec><title>Strengths and limitations</title><p>Our pooled analysis of 4896 patients, most of whom had acute coronary syndrome, is the first to show a cardiac mortality benefit associated with the use of a specific second generation drug eluting stent—namely, cobalt-chromium everolimus eluting—compared with a bare metal stent. This treatment effect persisted after multivariable adjustment of confounders, including duration of dual antiplatelet therapy after stenting and use of glycoprotein IIb/IIIa inhibitors during the procedure.</p><p>Our observation that a durable polymer based drug eluting stent, such as the cobalt-chromium everolimus eluting stent, offers improved safety and efficacy profiles compared with non-active polymer-free stents supports a major paradigm shift in our understanding about coronary devices.</p><p>Firstly, stent safety and efficacy can no longer be disconnected at least for some newer generation devices. Two studies have so far randomised patients to first or second generation drug eluting stents, including cobalt-chromium everolimus eluting stent, to evaluate the optimal duration of treatment with clopidogrel.<xref rid="ref32" ref-type="bibr">32</xref>
<xref rid="ref33" ref-type="bibr">33</xref> These trials were largely underpowered to conclusively assess the optimal duration of treatment after each of the single stent to which patients have been randomised. Yet, a distinct signal towards a possible increase of ischaemic events after discontinuation of clopidogrel at six months has been specifically observed after sirolimus or paclitaxel eluting stents but not after cobalt-chromium everolimus eluting stents.</p><p>Additionally, because the durable polymer might not necessarily harbour potential for thrombogenesis, it does not need to be avoided in future. Recently, the rates of stent thrombosis were reduced with fluorinated polymer coated stents compared with equivalent bare metal stents in an experimental setting.<xref rid="ref34" ref-type="bibr">34</xref> Four out of the five studies included in our pooled analysis compared cobalt-chromium everolimus eluting stent with the uncoated otherwise identical metallic stent, and they showed a reduction in fatal and non-fatal adverse events consistent with the results of our pooled analysis. It is therefore conceivable to attribute the benefit for ischaemic events and cardiac mortality noted here and elsewhere to the presence of the fluorinated copolymer/drug coatings. Bare metal stent thrombogenicity has long been recognised.<xref rid="ref35" ref-type="bibr">35</xref> Copolymer coatings in cobalt-chromium everolimus eluting stent are durable because of the hydrophobic nature of fluorinated polymers, and they can act as a mechanical barrier excluding metallic surface potential for thrombogenesis. Moreover, fluorinated copolymer coatings are highly electronegative and as such repel protein adsorption because of their high electronegativity and elicit a cellular response conducive to healing with minimal chronic inflammation.<xref rid="ref36" ref-type="bibr">36</xref></p><p>No single study has so far suggested a safety issue through over three<xref rid="ref37" ref-type="bibr">37</xref>
<xref rid="ref38" ref-type="bibr">38</xref> or even five years<xref rid="ref39" ref-type="bibr">39</xref> after cobalt-chromium everolimus eluting stent implantation compared with first generation drug eluting stents. Longer duration follow-up is required to confirm the durability of the benefit observed here at a medium term time point when cobalt-chromium everolimus eluting stents are compared with bare metal stents.</p><p>Actual dual antiplatelet therapy status at follow-up was not<xref rid="ref11" ref-type="bibr">11</xref> or was suboptimally<xref rid="ref17" ref-type="bibr">17</xref> recorded in two of the five included studies. Hence, the role of concomitant antiplatelet therapy should be further explored, bearing in mind that no single study has so far reported a cardiac mortality benefit of clopidogrel therapy beyond one month. No detailed information is available from any of the included studies regarding patients who underwent surgery throughout follow-up.</p><p>While an explanation for the observed reduction in cardiac mortality in favour of cobalt-chromium everolimus eluting stent can be only speculative at the present time, an interplay between reduced device thrombegenicity as suggested by the lower stent thrombosis and myocardial infarction rates coupled with improved efficacy (that is, reduced need for target vessel reintervention because of neointimal proliferation) might account for the observed medium term cardiac survival benefit.</p></sec><sec><title>Conclusions and policy implications</title><p>This collaborative individual patient data meta-analysis of five randomised controlled trials, including 4896 patients most with acute coronary syndrome, shows a consistent reduction of several fatal and non-fatal ischaemic endpoints in favour of cobalt-chromium everolimus eluting stent compared with bare metal stents, including cardiac mortality, stent thrombosis (both definite and definite or probable), myocardial infarction, and target vessel revascularisation throughout two year follow-up. Our analysis challenges the current belief that bare metal stents are safer than drug eluting stents. Stent safety and efficacy can no longer be disconnected, at least for some newer generation devices.</p><boxed-text position="float" content-type="style3"><sec><title>What is already known on this topic</title><list list-type="simple"><list-item><p>Coronary stents are widely used to treat patients with coronary artery disease, with drug eluting stents being more efficacious than bare metal stents in preventing in stent restenosis and subsequent need for reintervention</p></list-item><list-item><p>As first generation drug eluting stents were associated with higher rates of stent thrombosis than bare metal stents, newer generation drug eluting stents were developed to improve the safety and main the efficacy </p></list-item><list-item><p>Most randomised trials compared newer generation drug eluting stents with first generation stents, and rarely with bare metal stents, restricting a fully comparative understanding of their outcomes</p></list-item></list></sec><sec><title>What this study adds</title><list list-type="simple"><list-item><p>This individual patient data meta-analysis shows a consistent reduction of several fatal and non-fatal ischaemic endpoints in favour of the newer generation everolimus eluting stent compared with bare metal stents, including significant reductions of cardiac mortality, stent thrombosis, myocardial infarction, and target vessel revascularisation throughout two year follow-up</p></list-item></list></sec></boxed-text></sec></sec> |
Maximizing Social Model Principles in Residential Recovery Settings | <title>Abstract</title><p>Peer support is integral to a variety of approaches to alcohol and drug problems. However, there is limited information about the best ways to facilitate it. The “social model” approach developed in California offers useful suggestions for facilitating peer support in residential recovery settings. Key principles include using 12-step or other mutual-help group strategies to create and facilitate a recovery environment, involving program participants in decision making and facility governance, using personal recovery experience as a way to help others, and emphasizing recovery as an interaction between the individual and their environment. Although limited in number, studies have shown favorable outcomes for social model programs. Knowledge about social model recovery and how to use it to facilitate peer support in residential recovery homes varies among providers. This article presents specific, practical suggestions for enhancing social model principles in ways that facilitate peer support in a range of recovery residences.
</p> | <contrib contrib-type="author"><name><surname>Polcin</surname><given-names>Douglas</given-names></name><degrees>Ed.D.</degrees><xref ref-type="aff" rid="AFF0001">
<sup>a</sup>
</xref><xref ref-type="corresp" rid="AN0001">
<sup>*</sup>
</xref></contrib><contrib contrib-type="author"><name><surname>Mericle</surname><given-names>Amy</given-names></name><degrees>Ph.D.</degrees><xref ref-type="aff" rid="AFF0002">
<sup>b</sup>
</xref></contrib><contrib contrib-type="author"><name><surname>Howell</surname><given-names>Jason</given-names></name><degrees>M.B.A., P.R.S.</degrees><xref ref-type="aff" rid="AFF0003">
<sup>c</sup>
</xref></contrib><contrib contrib-type="author"><name><surname>Sheridan</surname><given-names>Dave</given-names></name><xref ref-type="aff" rid="AFF0004">
<sup>d</sup>
</xref></contrib><contrib contrib-type="author"><name><surname>Christensen</surname><given-names>Jeff</given-names></name><degrees>B.S.</degrees><xref ref-type="aff" rid="AFF0004">
<sup>d</sup>
</xref></contrib><aff id="AFF0001"><label><sup>a</sup></label><institution><named-content content-type="institution-name">Senior Scientist, Alcohol Research Group</named-content></institution>, <named-content content-type="city">Emeryville</named-content>, <named-content content-type="state">CA</named-content></aff><aff id="AFF0002"><label><sup>b</sup></label><institution><named-content content-type="institution-name">Alcohol Research Group</named-content></institution>, <named-content content-type="city">Emeryville</named-content>, <named-content content-type="state">CA</named-content></aff><aff id="AFF0003"><label><sup>c</sup></label><institution><named-content content-type="institution-name">National Alliance for Recovery Residences</named-content></institution>, <named-content content-type="city">Austin</named-content>, <named-content content-type="state">TX</named-content></aff><aff id="AFF0004"><label><sup>d</sup></label><institution><named-content content-type="institution-name">Sober Living Network</named-content></institution>, <named-content content-type="city">Los Angeles</named-content>, <named-content content-type="state">CA</named-content></aff> | Journal of Psychoactive Drugs | <p>Most programs for alcohol and drug problems emphasize the importance of peer support, which is sometimes known as “mutual aid” (Borkman <xref rid="CIT0005" ref-type="bibr">1999</xref>) or “self-help” (Kurtz <xref rid="CIT0016" ref-type="bibr">1997</xref>; Riessman & Carroll <xref rid="CIT0024" ref-type="bibr">1995</xref>). Peer support involves interpersonal sharing of information and personal experiences, offering practical help, and interacting in ways that enhance emotional and social well-being. However, the strategies for facilitating peer support within alcohol and drug programs vary. Some programs build peer support primarily by offering group counseling or on-site 12-step meetings, such as Alcoholics Anonymous (AA). Other programs require that participants attend outside 12-step or other types of mutual aid meetings in the community. Less common are well conceptualized ways of enhancing peer influences within programs. The “social model” approach to recovery (Wittman & Polcin <xref rid="CIT0026" ref-type="bibr">2014</xref>; Shaw & Borkman, <xref rid="CIT0025" ref-type="bibr">1990</xref>; Borkman <xref rid="CIT0003" ref-type="bibr">1983</xref>) provides a starting point for understanding peer influences and facilitating peer support in residential recovery settings.</p><p>Social model recovery emerged in California primarily as a grassroots movement that was built upon the principles of AA (Wittman & Polcin <xref rid="CIT0026" ref-type="bibr">2014</xref>; Borkman et al. <xref rid="CIT0006" ref-type="bibr">1998</xref>). Although there is limited professional literature on social model recovery, a number of studies have shown favorable outcomes. Programs that self-identified as social model were shown to have similar or better outcomes than clinically oriented treatment programs that were typically more expensive (Kaskutas et al. <xref rid="CIT0015" ref-type="bibr">2008</xref>; Kaskutas, Ammon & Weisner <xref rid="CIT0013" ref-type="bibr">2003</xref>-2004; Borkman et al. <xref rid="CIT0006" ref-type="bibr">1998</xref>). Studies of sober living houses (SLHs) that used a social model approach showed significant resident improvements on a variety of outcomes that were maintained at 18-month follow-up (Polcin et al. <xref rid="CIT0023" ref-type="bibr">2010</xref>). Moreover, these studies found factors central to social model recovery (i.e., involvement in 12-step groups and social network characteristics) were related to outcome.</p><p>The purpose of this paper is threefold. First, we provide a brief overview of the history and principles of the social model approach to recovery. Second, we describe four different levels of recovery residences based on standards developed by the National Alliance for Recovery Residences (NARR <xref rid="CIT0019" ref-type="bibr">2012</xref>). Finally, we provide guidance on how aspects of social model can be used to address challenges encountered across all four levels of NARR residences. Examples include house meetings, decision making, establishment and enforcement of house rules, and admission and termination of residents. We also emphasize using social model as parallel social processes among providers and between providers and the surrounding community. Conceptualization of peer support and suggestions for enhancing it draw upon 11 years of research on recovery homes and decades of experience among the co-authors operating recovery homes and recovery home organizations.</p><sec id="S0002"><title>HISTORY OF SOCIAL MODEL RECOVERY AND BASIC CONCEPTS</title><p>Although the term “social model” did not emerge until the 1970s, the basic elements of this approach were being practiced as early as the 1940s (Wittman & Polcin <xref rid="CIT0026" ref-type="bibr">2014</xref>). Social model recovery emerged as a grassroots movement in California, largely as an offshoot of Alcoholics Anonymous (AA). Many individuals attempting to abstain from substances through attendance at AA lacked an affordable alcohol-free living environment. Their efforts were often undermined by destructive living environments that promoted substance use. In response to this need, recovering persons involved in AA created group living environments, which they called “12-step” houses. Drinking and drug use were prohibited and residents were expected to work a 12-step recovery program. Because residents typically shared bedrooms, owners were able to keep rents affordable. By the 1970s, 12-step houses became known as “sober living houses” (SLHs).</p><p>Beginning in the 1970s, conferences and publications in the addiction field began to use the term “social model” to describe SLHs and similar programs (O’Briant & Lennard <xref rid="CIT0020" ref-type="bibr">1973</xref>). The primary rationale for this term was that it emphasized social and interpersonal aspects of recovery rather than approaches that were more individually oriented. It also emphasized peer-to-peer rather than practitioner-client relationships and replaced the concept of treatment plan with “recovery plans” (Borkman <xref rid="CIT0004" ref-type="bibr">1998</xref>). The latter term emphasized actions the person will take to achieve and maintain recovery instead of the types of professional services they will receive. The primary characteristics of social model recovery programs were summarized by Wright (<xref rid="CIT0027" ref-type="bibr">1990</xref>) as follows:
<list list-type="bullet"><list-item><p>There is an emphasis on experiential knowledge gained through one’s recovery experience. Residents draw on that experience as a way to help others.</p></list-item><list-item><p>Recovery operates via connections between residents, not between an individual resident and a professional caregiver.</p></list-item><list-item><p>All residents are consumers and providers, both giving and receiving help.</p></list-item><list-item><p>As with the early 12-step recovery houses, involvement in AA creates the basic framework for recovery.</p></list-item><list-item><p>A positive sober environment that encourages support for abstinence is crucial.</p></list-item><list-item><p>Alcoholism is viewed as being centered in the reciprocal relationship between the individual and his or her surrounding social unit.</p></list-item></list>
</p><sec id="S0002-S2001"><title>Social Model Recovery Scale</title><p>A variety of residential programs, including those offering formal treatment, adopted different aspects of social model into their approaches. Kaskutas et al. (<xref rid="CIT0014" ref-type="bibr">1998</xref>) developed the Social Model Philosophy Scale (SMPS) as a way to assess the extent to which programs used a social model approach to recovery as well as what aspects of social model were used. The 33-item SMPS has been shown to have high internal reliability (α = 92). The SMPS assesses six program domains:
<list list-type="order"><list-item><p>Physical environment: the extent to which the program facility offers a homelike environment.</p></list-item><list-item><p>Staff role: the extent to which staff are seen as recovering peers.</p></list-item><list-item><p>Authority base: the extent to which experiential knowledge about recovery is valued.</p></list-item><list-item><p>View of substance abuse problems: the extent to which residents view substance abuse as a disease and are involved in 12-step groups.</p></list-item><list-item><p>Governance: the extent to which the program empowers residents in decision making.</p></list-item><list-item><p>Community orientation: the extent to which the program interacts with the surrounding community in a mutually beneficial manner.</p></list-item></list>
</p><p>Although the SMPS was based on data obtained from California social model programs, the principles are relevant to a variety of recovery home models throughout the U.S. Some of these models are reviewed below along with initial research examining services offered and outcomes.</p></sec><sec id="S0002-S2002"><title>Social Model Recovery in Other Residential Recovery Settings</title><p>There are a variety of residential approaches to recovery that emphasize characteristics similar to social model principles. One example is the Oxford House model, which began in 1975 (O’Neill <xref rid="CIT0021" ref-type="bibr">1990</xref>). Like SLHs, Oxford Houses offer long-term recovery in a residential, homelike environment that is free of alcohol and drugs. They are financially self-sustained by residents and do not offer on-site formal treatment services. Although they do not have individual house managers or operators, Oxford Houses are supported externally through a system of regional managers responsible for the welfare of groups of homes. All residents are required to have some type of a recovery plan and most attend AA or other 12-step groups.</p><p>A study of individuals who had been residing at Oxford Houses for varying lengths of time (several days to over 10 years) showed good longitudinal outcomes at four-month follow-up intervals (Jason et al. <xref rid="CIT0009" ref-type="bibr">2007</xref>). Oxford Houses have also been found to be effective as an aftercare service for clients who completed long-term residential treatment (Jason et al. <xref rid="CIT0010" ref-type="bibr">2006</xref>). Today there are more than 1,500 Oxford Houses nationwide; SLHs in California that are affiliated with associations such as the Sober Living Network and California Association of Addiction Recovery Resources number close to 800.</p><p>A heterogeneous mix of other types of recovery residences has emerged throughout the U.S. that use social model principles to varying degrees. Mericle et al. (<xref rid="CIT0018" ref-type="bibr">2014</xref>) used the SMPS to assess the extent to which recovery residences in Philadelphia used practices that were consistent with social model principles. While only 11% met criteria as social model recovery residences using a cutoff score on the SMPS, some characteristics of social model were strong across most houses (e.g., view of substance abuse problems and authority base) and others were relatively weak (e.g., governance).</p></sec></sec><sec id="S0003"><title>NATIONAL ALLIANCE OF RECOVERY RESIDENCE LEVELS</title><p>The Mericle et al. investigation was unique in that it assessed social model recovery principles across different levels of recovery residences as defined by the National Alliance of Recovery Residences (NARR). Briefly, NARR (National Association of Recovery Residences <xref rid="CIT0019" ref-type="bibr">2012</xref>) describes four levels of recovery residences:
<list list-type="bullet"><list-item><p>Level I residences are peer-managed houses located in residential neighborhoods. They are democratically run by the residents themselves and there are no paid staff members or on-site services. Although most residents are involved in 12-step recovery groups, attendance is not mandatory. Oxford Houses (Jason, Olson & Foli <xref rid="CIT0011" ref-type="bibr">2008</xref>) are a good example of Level I residences.</p></list-item><list-item><p>Level II residences are also typically located in residential neighborhoods. Unlike Level I houses, they are managed by a house manager or senior resident who is either paid or receives a reduction of rent. There are typically no services offered on-site and residents are usually mandated or strongly encouraged to attend 12-step recovery groups. California Sober Living Houses (Polcin et al. <xref rid="CIT0023" ref-type="bibr">2010</xref>) are good examples of Level II residences.</p></list-item><list-item><p>Level III residences employ paid staff who provide on-site services, such as linkage to resources in the community, recovery wellness planning, recovery support groups, and life skills training. In California, these residences are required to be licensed as treatment programs. Mericle et al. (<xref rid="CIT0018" ref-type="bibr">2014</xref>) pointed out that these can be considered hybrid programs that combine social model recovery and additional services delivered by trained staff. A recovery approach that has become known as the “Florida model” combines intensive outpatient or day treatment services with residence in a sober living house. Some Level III residences exist as private households in residential neighborhoods while others operate in multifamily, commercial or other environments.</p></list-item><list-item><p>Level IV residences are best understood as residential treatment programs that are more structured than Level III and that provide a variety of on-site clinical services. Although some staff may be in recovery, Level IV’s employ licensed or credentialed professionals. A number of social model characteristics are emphasized: (1) peer support; (2) resident involvement in upkeep of the facility; and (3) resident input into establishing and enforcing rules and policies. Therapeutic communities (De Leon <xref rid="CIT0007" ref-type="bibr">2000</xref>) are a good example of Level IV facilities. These facilities are typically not zoned as ordinary housing in residential neighborhoods.</p></list-item></list>
</p><p>Each of the subheadings below addresses ways that social model principles can be implemented within and across the four levels described by NARR. Particular emphasis is placed on using social model as a way of understanding issues in recovery residences and mobilizing peer support to address them.</p></sec><sec id="S0004"><title>CONCEPTUALIZING ISSUES ACROSS NARR LEVELS</title><p>Central to a social model perspective is maintaining a focus that emphasizes the quality of the household as a recovery environment rather than a focus primarily on individual residents. Although there are some differences related to understanding and addressing issues between NARR levels, much of what promotes social model is relevant to all four levels.</p><sec id="S0004-S2001"><title>Fostering a Culture of Recovery</title><p>Viewing issues from a broader, environmental perspective requires deliberate focus. In the U.S., there is a cultural norm to view alcohol and drug problems as a personal failing. “Individual responsibility” is an often repeated term among government officials at all levels as well as by the general public as a way of conceptualizing and addressing multiple problems, including those related to alcohol and drug use. That approach to alcohol and drug problems results in lost opportunities to mobilize community and peer influences that can have a strong salutary impact.</p><p>The social model approach to alcohol and drug problems shifts the focus to the household and community environment as a way to foster a culture of recovery. Residents are invited to draw on the strengths of the household and utilize peer support to shed their addictive lifestyle and reconstruct their self-identity as a person in recovery. Because recovery is a reality that is exemplified by recovering peers and their staff, recovery grows out of hope and results in a process of self-redefinition and the rebuilding of a life in the community. The success of this approach is dependent upon the household’s ability to address issues within a framework that enhances peer support within programs. It also requires successful collaboration with neighbors, outside service providers, and the local community. Each section in the following advocates for a vision of issues in recovery residences that includes broad ownership of problems and solutions to the benefit of residents and the surrounding community.</p></sec><sec id="S0004-S2002"><title>Facilitating Social Model Perspectives among Residents and Staff</title><p>Whether the leadership in a recovery residence is a house manager, treatment professional, or residents who function in rotating leadership positions, social model can be facilitated by the leadership articulating problems and issues from a household or program perspective rather than one focused primarily on individuals. All four levels of NARR residences have expectations and rules that apply to individuals (e.g., abstinence, attendance at house meetings, and participation in house chores and upkeep). However, from a social model perspective, it is important that residents understand rules and expectations in terms of how they impact the overall community as a group of recovering persons. When expectations and responsibilities are ignored, the residence does not function as a successful household or as a forum that facilitates recovery. The ultimate goal is to create an environment where residents articulate that perspective themselves rather than relying on the leadership to do it. In this scenario, developing a recovery lifestyle is conceptualized among residents as more than avoiding addictive substances and improving personal health, it is characterized by citizenship—the importance of living one’s life with regard and respect for those around you (Betty Ford Institute <xref rid="CIT0002" ref-type="bibr">2007</xref>). Doing one’s fair share in terms of contributing to the household as a recovery environment and recognizing how one’s behavior affects that environment is a key tenet across all social model programs.</p><p>Developing and maintaining a social model environment cannot be a function of the leadership alone. Residents must play a central role in helping each other understand household operations and dynamics from a social model perspective and translate that understanding into action. Most residents will have had experience with 12-step or other mutual-help programs and can draw on recovery principles used in those programs as a guide. Like 12-step programs, there is an informal “oral tradition” process that occurs where residents who are more experienced with social model programs (sometimes called “senior peers”) pass their knowledge on to new residents. The leadership in the program needs to consistently emphasize and reinforce these processes. As new residents observe how issues in the household are understood and addressed, they sharpen their social model skills. As they learn more, they are empowered to contribute more to the welfare of the household and the individuals who live there.</p></sec><sec id="S0004-S2003"><title>Understanding House Meetings from a Social Model Perspective</title><p>Mandatory house meetings are a staple of all types of recovery residences. They offer opportunities for residents and staff to understand and discuss issues from a social model perspective and reinforce a recovery-oriented culture. Typically, a variety of individual, interpersonal, and house issues are presented. Regardless of the NARR level, it is important that persons in leadership positions (e.g., house manager, treatment professional, or peer leader) avoid being overly directive or offering solutions to problems prematurely. Instead, residents should be engaged in a collaborative process where various perspectives can be explored. Peer empowerment and support are strengthened when residents are involved in defining problems, identifying options, and implementing plans to resolve them. Peers with more experience take leadership roles in helping to guide conversations and decisions. Peer involvement in decision making creates a sense of resident ownership and connection to house operations that counteracts an “us versus them” mentality dividing residents and staff.</p><p>When interactions in house meetings are limited to a sole focus on individual issues and behaviors or the meeting gets bogged down in interpersonal struggles, it is important for the facilitator to shift the discussion toward a broader, social model perspective. As issues are discussed, the facilitator should consider questions such as how does this issue impact the overall house? How can house members be mobilized to address the issue? Should we discuss changes in house rules or operations to address the issue? How would such changes affect the recovery culture of the household? Addressing these types of questions facilitates shared ownership of problems and reliance on the resident community as a way to address them.</p></sec><sec id="S0004-S2004"><title>House Rules and Policies from a Social Model Viewpoint</title><p>Maintaining a recovery-supportive community requires house rules, recovery-oriented social norms, and peer accountability. These are frequent house meeting topics in all types of recovery residences. Often they are brought up in terms of complaints about individuals being noncompliant. The result can be administration of consequences or warnings about noncompliant behaviors. However, there is an opportunity during these discussions to articulate the purposes of rules and policies from social model perspective that links them to household functioning and principles of recovery. Examples include linking policies and rules to issues such as safety, maintaining an alcohol- and drug-free environment, and the role of accountability in recovery.</p><p>Rules and policies can also be linked to AA principles such as “giving back” to the community from which one receives help, accepting powerlessness over some situations, taking an inventory of one’s weaknesses or flaws, and asking for help from others. Not everyone in recovery residences works an AA or other type of 12-step program, but the majority in NARR Levels I and II residences are involved in some type of 12-step program (NARR <xref rid="CIT0019" ref-type="bibr">2012</xref>). In addition, some programs not explicitly identified as 12-step-oriented (mostly NARR Levels III and IV) have similar recovery concepts that can be related to program rules and policies. For example, therapeutic communities (TCs), like 12-step-oriented programs, emphasize the importance of “giving back” to newcomers, demonstrating commitment to the community through one’s behavior, and taking responsibility for the ways that one contributes to problems and conflicts. Ideally, the residents themselves would take the lead in these discussions. Programs with relatively newer residents and those in early recovery will need role modeling of this process from senior residents or staff.</p><p>To the extent that residents have input into formation, modification, and enforcement of house rules and policies they are more likely to feel ownership and a commitment to their implementation. Creating a social model environment in which peers hold each other accountable to house rules and social norms is the ideal. NARR Level I and II programs are likely to allow for significant input into rules and policies through discussions in house meetings or other forums. Although Level III and IV houses are likely to have paid staff who are ultimately held accountable for implementation of rules and policies, most have forums where residents can have input into modification and enforcement of rules and policies. For example, therapeutic communities often have some version of a resident government that helps enforce rules and make recommendations to staff for modifications.</p></sec></sec><sec id="S0005"><title>USING SOCIAL MODEL TO ADDRESS SPECIFIC ISSUES</title><p>Recovery residences provide individual level interventions for a variety of issues (e.g., noncompliance with house rules, referral to outside services, and development of individual recovery plans). However, the leadership within houses can work to create a context where individual issues are also addressed by drawing upon the strengths of the resident community, or what Laudet and White (<xref rid="CIT0017" ref-type="bibr">2008</xref>) have referred to as “recovery capital.” In this section, we describe ways to mobilize peer support to address issues commonly encountered in residential recovery settings, including relapse, resident conflicts, and personal crises. This section also addresses the advantages of mobilizing residents to influence two of the most important decisions for any recovery residence: when to admit a person as a new resident and when a resident should be asked to leave due to noncompliance.</p><sec id="S0005-S2001"><title>Applicant Interviews and Resident Evictions</title><p>Few decisions are more important to a recovery residence than who is allowed to enter and who is asked to leave. NARR Level I houses typically make these decisions by democratic vote of residents. Level II houses will involve the house manager or owner, but there may also be a mechanism for resident input as well. Similarly, decisions about admission and termination among Level III and IV houses will involve paid staff, but there also may be mechanisms for residents to have input.</p><p>From a social model perspective, there are advantages to including residents in these processes. First, it empowers residents to take part in a critically important household decision. Second, admissions that include current residents in the process can help create a sense of commitment to the new person. Finally, it facilitates the new person feeling a sense of accountability to the entire household, not just individuals in leadership positions. A practical consideration is that involvement of current residents draws upon the perceptions of the entire community, not just one individual who may not recognize potential problems or assets.</p><p>There are similar advantages to involving current residents in decisions about involuntary eviction. It helps create a sense that each resident is accountable to the community, not just to the staff, house manager, or others in leadership positions. It also invites discussion about the importance of maintaining an abstinent living environment.</p></sec><sec id="S0005-S2002"><title>Relapse</title><p>One of the most difficult issues faced across all types of recovery residences is alcohol and drug relapse. At the individual level, there are a variety of responses that might be implemented. Depending on the circumstances and the facility, the individual who relapses may be asked to leave the residence. A temporary eviction is the policy of most houses. However, there may be an invitation to reapply for admission after some minimum period of time has passed. In some circumstances, the individual might be referred to a different type of setting, particularly one with more structure and oversight that might prevent additional relapses.</p><p>The recovery field has moved away from stigmatizing relapse toward viewing it as part of the addiction process. At least some individuals in the household will have experienced relapse at some point in their recovery. Social model recovery suggests that it can be helpful for these individuals to share their experience of relapse and how they were able to resume recovery. Importantly, it can help decrease the sense of self-loathing experienced by some persons who relapse and refocus their energies toward reestablishing abstinence.</p><p>Relapse is also a household issue because it affects other residents. It therefore needs to be discussed in house meetings or other forums. There may be expressions of fear, anxiety, loss, anger, guilt, or increased vulnerability about residents own potential for relapse, all of which need to be met with empathy and understanding. There might also be discussions about ways residents can enhance the recovery environment and increase support for sobriety. The social model concept of mutuality (i.e., everyone is a consumer and provider of help) is important here. Each resident is a giver and receiver of help and to the maximum extent possible there should be cultivation of norms in the house that reinforce asking for and receiving help. Putting an emphasis on the importance of residents recognizing and responding to vulnerability in themselves and others is imperative. In this way, an individual relapse can be mobilized to influence the household in ways that enhance recovery.</p></sec><sec id="S0005-S2003"><title>Resident Conflicts</title><p>Residential recovery settings invite a certain amount of interpersonal conflict and thus offer opportunities to practice recovery skills as they emerge during day-to-day activities. Conflicts occur as a result of sharing a room, failing to complete assigned chores, personal jealosies, and a host of other reasons. These are opportunities for residents to apply 12-step or other recovery principles to real-life situations. Newer residents and those in early recovery can benefit from senior residents with longer recovery sharing examples of how they worked the steps and applied other recovery principles to similar situations. This might involve consideration of recovery concepts such as taking an inventory and owning one’s part in the conflict, making amends, and accepting powerlessness over other people and situations. In Level I and II residences, these activities are most likely to be implemented among peers. In Level III and Level IV houses, they may be implemented by staff or peers.</p><p>It is important to facilitate a house-wide perspective that the emergence of conflict is expected and an ordinary part of life. The task in developing a recovery lifestyle is to manage conflict in healthy ways that enhance or at least do not undermine recovery. To the extent that residents are able to resolve conflicts and apply 12-step or other recovery principles to them, the household will function more efficiently and the quality of the house in terms of a source for recovery will be stronger. In addition, learning conflict management skills helps residents learn valuable life skills that will help them outside the house as they manage their recovery across their lifespan.</p></sec><sec id="S0005-S2004"><title>Resident Crises</title><p>Resident crises are not uncommon in recovery residences and can include relapse, onset of psychiatric symptoms problems such as suicidality, family crises, problems with intimate partners, or loss of a job, just to name a few. The community of residents can be mobilized to help residents prevent or cope with crises. Just having awareness about the issues residents are going through is important. Simple things like being available to talk and showing concern can be helpful. Whether one is part of the household as a peer or staff member, sharing of one’s own experiences in dealing with similar problems is important, especially in terms of the application of recovery principles to manage the crises. As residents help others prevent and cope with crises they also prepare themselves for how to deal with their own future crises.</p><p>An additional way that peers and staff can assist residents who are in crises is through suggestions for accessing outside services. Assistance can help in terms of sharing practical information, such as providers with whom they are familiar who may be helpful, suggestions for transportation to services and options for paying for services. Most importantly peers who have used the needed service can share their experiences and help the individual understand what to expect.</p></sec></sec><sec id="S0006"><title>TRAINING AND INTERACTIVE LEARNING</title><p>Recovery organizations in California (e.g., the Sober Living Network and the California Association of Addiction Recovery Resources) have for many years recognized the need for managers of SLHs to receive training in how to facilitate social model dynamic within houses. As such, they offer regular workshops that cover essential aspects for understanding and implementing a social model. However, there is also an appreciation for the value of experiential knowledge gained as a result of having lived in a SLH and the knowledge gained from managing houses. In this way, there is a type of parallel social model process that occurs at the manager as well as resident level.</p><p>While yearly conferences are one such mechanism for sharing experiences, more systematic and regular peer trainings might be even more beneficial. Facilitating manager visits to other sober living houses on a regular basis beyond formal inspections each year might be one way to increase cross-fertilization of ideas and experiences. Without interactive learning on a regular basis, there is a significant danger that houses can become disconnected, unfocussed, and out of date in their approaches as well as noncompliant with network standards. Thus, social model needs to be conceptualized beyond the resident level to include the larger recovery community.</p></sec><sec id="S0007"><title>INTERACTING WITH THE NEIGHBORHOOD AND LOCAL COMMUNITY</title><p>Social model posits that drug and alcohol problems operate in a reciprocal fashion between individuals and their surrounding environment (Wittman & Polcin <xref rid="CIT0026" ref-type="bibr">2014</xref>). It suggests that low-income, high-crime communities with high densities of alcohol outlets and readily available access to drugs contributes to substance use among individuals. In turn, substance use contributes to destructive characteristics of the environment (e.g., crime, availability of drugs, unemployment). We posit that recovery operates in a similar manner. Individuals in residential recovery settings need to use positive characteristics of their community environment that can benefit recovery. In addition, supporters of residential recovery services need to show positive impacts on the local community, which, in turn, can contribute to more support for recovery residences.</p><sec id="S0007-S2001"><title>Accessing Community Services</title><p>Although social model programs emerged in part as an alternative to formal clinical and medical treatments, they need to view themselves within a larger continuum of community-based services. Historically, social model programs avoided offering on-site services, in part to elevate the peer-support aspects of recovery. Instead, there was an emphasis on helping residents access needed services in the community. This continues to be the approach taken by NARR Level I and II residences.</p><p>NARR Level III and IV residences provide on-site services beyond peer support, although few programs meet all of the needs that residents present. Because persons with alcohol and drug problems frequently need help in a variety of areas, all of which cannot be met in one setting, it is important for residences to have good relationships with service providers in the surrounding community. Conversely, many of these providers serve persons in the community with alcohol and drug problems who could benefit from residence in a recovery setting and can therefore be sources for referrals.</p></sec><sec id="S0007-S2002"><title>Disseminating Beneficial Impact of Recovery Residences</title><p>Despite the existence of research showing favorable outcomes across all four types of recovery residences (NARR <xref rid="CIT0019" ref-type="bibr">2012</xref>), NIMBY (not in my back yard) resistances continue to plague many residences. Community resistance occurs despite documentation that recovery residences do not decrease property values or increase crime (American Planning Association <xref rid="CIT0001" ref-type="bibr">2003</xref>). In addition, research on Level I and Level II residences show they enjoy supportive relationships with neighbors (Heslin et al. <xref rid="CIT0008" ref-type="bibr">2012</xref>; Polcin et al. <xref rid="CIT0022" ref-type="bibr">2012</xref>; Jason, Roberts & Olson <xref rid="CIT0012" ref-type="bibr">2005</xref>). These studies show when neighbors are familiar with the recovery homes in their neighborhoods and the residents who live there they tend to be more supportive. Professional treatment providers have similar responses. A study of mental health professionals and certified addiction counselors found those who were most familiar with recovery houses were most supportive (Polcin et al. <xref rid="CIT0022" ref-type="bibr">2012</xref>).</p><p>Resistance to recovery homes is often based on stigma from persons who have little or no experience with the houses or residents who live there. Stigma feeds upon negative news reports in the media about problem houses, even if these are rare exceptions. There is therefore an urgent need for recovery residences at all levels to be associated with peer-based associations that monitor health, safety, and operational standards, such as NARR. Advocacy organizations have procedures in place to address problem residences quickly, especially complaints from neighbors. They also have resources to advise houses about their legal rights and advocate for houses that are targeted by NIMBY groups.</p><p>Because familiarity is associated with improved perceptions, there is a need for advocacy groups to organize formal interaction between operators of residences and key stakeholders: (1) neighbors of residences; (2) the general public; (3) local and state officials; and (4) mental health and other service provider groups. Examples of such interaction include dissemination of information about the goals and operation of recovery residences, advice to persons in the community who have family or friends suffering from addictive disorders, education about addiction and recovery more broadly, and encouraging house residents to volunteer for community service activities (neighborhood clean-up, holiday events, etc.) (Heslin et al. <xref rid="CIT0008" ref-type="bibr">2012</xref>; Polcin et al. <xref rid="CIT0022" ref-type="bibr">2012</xref>). Interaction of recovery homes and recovery home organizations with surrounding communities is another example of how social model dynamics need to occur as parallel processes across different levels of social interaction, including residents, staff and managers, and the larger community.</p></sec></sec><sec id="S0008"><title>CONCLUSION</title><p>Recovery residences for alcohol and drug problems universally emphasize peer support. However, few articles have provided suggestions for how to maximize positive peer influences in recovery settings. The California Social Model approach to recovery provides a framework for understanding and addressing issues in residential settings from a peer-based perspective. This paper has drawn on social model principles to develop specific suggestions for how recovery residences can involveand empower residents to address critical issues, such as applicant interviews, involuntary eviction, management of house meetings, resident conflicts, and a variety of crises. Additional work is needed to better understand how facilitation of peer support varies among different level of recovery residences as defined by NARR (<xref rid="CIT0019" ref-type="bibr">2012</xref>). In addition to using social model concepts to improve peer support within recovery home settings, social model theory can be used to enhance interaction within recovery home organizations and with the surrounding community.</p></sec> |
Long-term, treatment-free survival in select patients with distant metastatic papillary thyroid cancer | <p>Well-differentiated thyroid carcinoma (WDTC) generally has a favorable prognosis. However, patients with distant metastatic disease experience progression of disease with a higher mortality. A subset of patients not previously described may challenge the conventional dogma regarding the progressive nature of all metastatic WDTC. Through analysis of our database, we identified patients with distant metastatic WDTC and persistent, minimally progressive disease. In all patients, persistent metastatic disease was confirmed via tissue biopsy, abnormal PET scan, and/or biochemical elevations in thyroglobulin or antibody levels. Progression of disease was monitored clinically and with repeat imaging. We describe five patients with WDTC and pulmonary metastases, aged 8–43 years at diagnosis. All patients underwent initial surgery and radioactive iodine (RAI) ablation, with some receiving multiple treatments. Persistent pulmonary metastatic disease was confirmed over decades (mean 22 years, range 8–42 years) with minimal progression despite no further treatment beyond thyroid hormone suppression. Persistent disease was biopsy-proven in all patients at a mean of 9.6 years from last RAI treatment. All patients had elevated thyroglobulin or anti-thyroglobulin antibody levels, while three demonstrated metabolically active disease with positive FDG uptake on PET scan, and one patient with persistent radioactive iodine avid pulmonary metastasis 36 years after her last RAI treatment. This case series demonstrates that some patients with distant metastases, even if metabolically active and radioactive iodine resistant, remain stable for decades without further treatment. Clinical awareness of such patients and continual reassessment of disease risk following initial therapy are crucial as aggressive treatment may not be necessary.</p> | <contrib contrib-type="author"><name><surname>Kwong</surname><given-names>Norra</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Marqusee</surname><given-names>Ellen</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Gordon</surname><given-names>Michael S</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Larsen</surname><given-names>P Reed</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Garber</surname><given-names>Jeffrey R</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="aff" rid="aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Kim</surname><given-names>Matthew I</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Alexander</surname><given-names>Erik K</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="corresp" rid="cor1"/></contrib> | Endocrine Connections | <sec sec-type="intro"><title>Introduction</title><p>Well-differentiated thyroid cancer is common, easily identified, and often curable. While the incidence of thyroid cancer has nearly tripled in the past three decades, mortality rates have remained stable <xref rid="bib1" ref-type="bibr">(1, 2, 3)</xref>. This underscores the widespread success in identifying, treating, and thereby limiting disease-related harm in most affected individuals <xref rid="bib4" ref-type="bibr">(4, 5)</xref>. Yet paradoxically, such features may have simultaneously hindered our ability to prospectively investigate the natural history of this illness and define the optimal extent of necessary treatment. This is true for both localized and metastatic disease. For nearly seven decades, the recommended treatment for biopsy-proven disease has been surgical thyroidectomy, and radioactive iodine (RAI) (<sup>131</sup>I) ablation, followed by thyroid hormone suppression therapy <xref rid="bib6" ref-type="bibr">(6, 7, 8, 9)</xref>. Though there may be benefits to this approach, especially for patients with advanced disease, increasing evidence confirms that such a standardized approach to care may not be equally effective for all patients <xref rid="bib10" ref-type="bibr">(10, 11, 12)</xref>.</p><p>For example, attention has recently been focused upon the necessity for (and the optimal dosing of) <sup>131</sup>I in the treatment of papillary thyroid carcinoma. Two separate prospective trials confirmed the equivalency of 30 and 100 mCi <sup>131</sup>I dosing for post-surgical remnant ablation <xref rid="bib13" ref-type="bibr">(13, 14)</xref>. At a follow-up of two years, both studies demonstrated effective remnant ablation in 85–90% of patients, depicting lower side-effect profiles, costs, and complication rates when 30 mCi <sup>131</sup>I was administered. These findings have led to frequent adoption of 30 mCi <sup>131</sup>I for the treatment of low-risk patients <xref rid="bib15" ref-type="bibr">(15, 16)</xref>. In addition, a recent study has also shown an equivalent efficacy by low-dose RAI on tumor outcome in patients with intermediate tumor risk <xref rid="bib17" ref-type="bibr">(17)</xref>. More broadly, such prospective and randomized data have also raised questions about the necessity, extent, and duration of treatment for all thyroid cancer patients, even those with advanced or metastatic disease. Importantly, there currently exist no randomized controlled trials comparing <sup>131</sup>I with placebo. As a consequence of these studies, there has been a paradigm shift over the past two decades to individualize risk assessment and treatment strategies in an effort to avoid potential harm, especially in patients with low-risk disease <xref rid="bib10" ref-type="bibr">(10, 11, 12, 18, 19, 20)</xref>.</p><p>However, these concepts are more difficult to apply in patients with advanced disease, given their guarded prognosis. The 10-year survival rate for patients with distant metastatic thyroid cancer ranges from 26 to 60% <xref rid="bib21" ref-type="bibr">(21, 22, 23)</xref>. As a result, more aggressive and repeated therapies are often employed. For instance, most patients with pulmonary metastases are considered for repeated RAI treatment so long as iodine avidity is still confirmed. This approach, while seemingly logical, nonetheless remains untested when compared with a more conservative regimen.</p><p>Observations have suggested that a subset of such patients may challenge the conventional dogma regarding the progressive nature of all thyroid malignancies with distant metastases. Some patients, who have widely metastatic and seemingly persistent thyroid cancer, demonstrate minimal progression of their disease over years despite no further treatment beyond TSH suppression. Clinician awareness of this unique group is crucial as conventional treatment protocols may not be fully applicable. In this paper, we present a case series of patients with distant metastatic thyroid cancer whose disease remained quiescent without observable progression of pulmonary metastases for decades. While standard initial therapy was administered, such therapy did not eliminate all malignant tissues as proven by biopsies of metastatic foci and evidence of metabolic activity on PET and radionuclide scans. These data suggest that persistent distant metastatic thyroid cancer in some patients can be effectively managed over time with only TSH-suppressive therapy.</p></sec><sec sec-type="methods"><title>Methods</title><p>We searched in the thyroid nodule database of the Brigham and Women's Hospital (BWH) to identify patients with long-standing yet stable thyroid cancer metastases. To expand our search, we also surveyed thyroidologists at BWH and at affiliated institutions. Criteria used for identification included the following. All patients were initially diagnosed with well-differentiated thyroid cancer (papillary or follicular carcinoma), confirmed histopathologically following thyroidectomy. Distant metastatic disease was first identified by imaging at the time of initial staging or during follow-up surveillance. All patients have pulmonary metastatic disease and some have additional regional nodal disease. Importantly, metastatic foci were confirmed by tissue biopsy in all cases. Of the 6665 patients consecutively enrolled in the BWH thyroid nodule database, 791 patients proved to have papillary thyroid carcinoma (PTC). Of these, ∼25–35 patients have been found to have confirmed or suspected distant metastatic disease. Several post-<sup>131</sup>I nuclear scans have shown possible, though not conclusive, iodine uptake at distant metastatic sites. As we cannot definitively confirm metastatic disease without tissue sampling (which many did not receive), we acknowledge a level of uncertainty to this calculation.</p><p>All patients received standard therapy for their illness, including thyroidectomy followed by <sup>131</sup>I administration. All patients were prepared for <sup>131</sup>I therapy using thyroid hormone withdrawal. Typically, doses of ∼75–200 mCi <sup>131</sup>I were administered, and post-therapy whole-body scanning was performed 3–9 days thereafter. Patient follow-up and repeat assessments were performed as per the practice of the treating endocrinologist. In general, patients were monitored by biochemical testing including serum thyroglobulin measurement, ultrasound, and cross-sectional imaging. Monitoring generally occurred biannually or annually.</p><p>Ideally, the goal of <sup>131</sup>I therapy was to destroy thyroid follicular cells, including those that were malignant. When this happens, metastatic foci or adenopathy can remain anatomically abnormal due to fibrosis or scarring. In such cases, while malignant cells are not present, imaging may detect persistent abnormality. Our goal was to exclude such cases, as they represent effectively treated disease. Thus, we included only subjects for whom we could identify persistent and/or metabolically active metastatic disease despite initial treatment. For this study, persistent disease was confirmed histologically with lung biopsy at a time distant from initial therapy. Lesions were also noted on cross-sectional imaging and increasing thyroglobulin concentration (or new anti-thyroglobulin antibodies) was commonly detected after decades. Metabolically active metastatic disease was defined as positive FDG uptake on PET scanning or lung radioactive iodine uptake upon radionuclide scanning consistent with metastatic disease. Permission was obtained from the BWH Institutional Review Board to perform this analysis.</p></sec><sec><title>Results</title><p>A case series of five patients is described below and in <xref rid="tbl1" ref-type="table">Table 1</xref>.</p><sec><title>Patient no. 1</title><p>A 45-year-old woman was referred for evaluation of persistent metastatic papillary thyroid carcinoma. The patient was first diagnosed with papillary carcinoma and diffuse pulmonary metastases in 1975 at age eight. Owing to the risks associated with therapy, observation was initially favored. Three years later (age 10 years), chest X-ray demonstrated progression of pulmonary metastatic disease. Near-total thyroidectomy and left neck dissection were performed. Histology reported it as a papillary carcinoma with extensive extrathyroidal involvement invading into the trachea and with extensive cervical lymph node metastases. Shortly thereafter, 80 mCi of <sup>131</sup>I was administered, followed by a second dose of 150 mCi 5 months later. Post-therapy scans confirmed diffuse pulmonary and left neck uptake consistent with distant metastatic carcinoma. Suppressive doses of levothyroxine were administered.</p><p>Thereafter, from age 11 to 45 (34 years), the patient received no further therapy and remained in good health, but demonstrated persistently elevated thyroglobulin levels (range 25–57 ng/ml) on intermittent follow-up. At the age of 45, the patient re-established medical care. Evaluation revealed a serum thyroglobulin level of 25 ng/ml. Chest computed tomography (CT) demonstrated innumerable bilateral pulmonary nodules ranging from 5 to 10 mm. Whole-body scanning performed with <sup>123</sup>I revealed faint uptake in the left neck as well as 1.5% uptake in each lung field (3% total lung uptake). PET scan confirmed some lung foci to be FDG avid. Ultrasound of the neck was negative for signs of local recurrence. CT-guided core needle biopsy of the pulmonary nodules confirmed persistent metastatic papillary thyroid carcinoma. The patient has been monitored for 2 years, with follow-up testing demonstrating variable thyroglobulin levels (25–40 ng/ml), though without upward trend. Her most recent thyroglobulin concentration was 33 ng/ml. Repeated chest CT revealed no change in nodularity. Aside from chronic, moderate shortness of breath attributed to vocal cord dysfunction, the patient remains asymptomatic despite her persistent, stable, iodine avid pulmonary metastases that have remained untreated for 36 years.</p></sec><sec><title>Patient no. 2</title><p>A 28-year-old female presented with a thyroid mass. Evaluation led to total thyroidectomy and neck dissection. Multifocal papillary thyroid carcinoma, up to 3 cm in size, with capsular invasion was diagnosed. Fifteen cervical lymph nodes were positive for local metastatic disease. The patient received 150 mCi of <sup>131</sup>I revealing extensive (5%) neck uptake highly suspicious for residual nodal disease. No uptake was noted in the lung fields or bony structures. During follow-up, thyroglobulin was persistently detectable with concentrations of ∼19 ng/ml. A CT scan revealed bilateral pulmonary nodules ranging from 2 to 5 mm in diameter. No further treatment was provided at that time. One year later (age 29), the patient underwent a second right neck dissection due to persistent disease. A second 150 mCi dose of <sup>131</sup>I was administered, with no post-treatment evidence of lung uptake. Two additional neck surgeries were performed at ages 33 and 35 for persistent malignant lymphadenopathy. Since age 29, no further <sup>131</sup>I or systemic treatment was administered beyond TSH suppression.</p><p>The patient remained stable for 11 years with unchanged pulmonary nodules on repeat cross-sectional imaging. Stimulated thyroglobulin ranged from 1.9 to 2.5 ng/ml over the first 6 years, though was once reported as undetectable. However, she later developed anti-thyroglobulin antibodies, which have since persisted. At the age of 40, PET scanning revealed multiple pulmonary nodules that remained unchanged in comparison to CT scans 13 years prior, though one 0.6 mm left lower nodule was FDG-avid. The patient was referred for a diagnostic video-assisted thoracoscopic surgery (VATS) wedge resection of the left pulmonary nodule because of concerns for a possible new malignancy. Histopathology confirmed persistent, metastatic papillary thyroid carcinoma. Currently, at the age of 41, the patient is asymptomatic and fully functional. Her biopsy-proven pulmonary metastases have remained stable for 12 years.</p></sec><sec><title>Patient no. 3</title><p>A 41-year-old female sought medical attention for a thyroid mass. Neck ultrasound revealed a 3.8 cm left thyroid nodule and FNA proved cytologically suspicious. A near-total thyroidectomy and left neck dissection were performed. Pathology revealed a 4 cm papillary thyroid carcinoma with extensive invasion into skeletal muscle and subcutaneous soft tissues. The surgical resection margin involved carcinoma and ten cervical lymph nodes were positive for metastases. She received 142 mCi of <sup>131</sup>I with post-treatment scans revealing only neck (remnant) uptake. Anti-thyroglobulin antibodies were detected after therapy with increasing concentrations 4 years later (age 45), leading to chest CT evaluation. CT imaging demonstrated innumerable bilateral pulmonary nodules, ranging from 1 to 4 mm in diameter. The patient was treated with a second 151 mCi dose of <sup>131</sup>I, though post-therapy scans demonstrated no pulmonary or neck uptake. The patient underwent diagnostic VATS exploration and wedge biopsy 1 month later (4 years after her initial therapy), because of concerns for a possible new malignancy. Lung histology was consistent with metastatic papillary thyroid carcinoma. No further treatment beyond TSH suppression was provided. The patient has since been followed for 8 years without further treatment. TSH was maintained suppressed. Despite increasing anti-thyroglobulin antibody concentrations up to a level of 1000 IU/ml, her repeat neck ultrasounds and CT scans of the lungs have shown stable pulmonary metastatic disease for 8 years.</p></sec><sec><title>Patient no. 4</title><p>A 29-year-old female was observed to have a thyroid mass in 1968. At that time, she underwent near-total thyroidectomy revealing papillary thyroid carcinoma. Three years after initial diagnosis, she underwent right neck radical dissection for metastatic lymphadenopathy. At the age of 33, a chest X-ray showed bilateral pulmonary nodules. A decision was made to treat her with 75 mCi of <sup>131</sup>I. Post-therapy radionuclide scanning demonstrated no pulmonary uptake. Given the concern for another malignancy, the patient was referred for diagnostic wedge resection of the two right lung nodules. Histopathology confirmed metastatic papillary thyroid carcinoma. After her radioactive iodine therapy at the age of 33, the patient has received no further treatment beyond thyroid hormone-suppressive therapy. Since age 53, the patient has undergone annual CT scans of the lungs revealing persistent, but stable, pulmonary metastases measuring 2–4 mm in diameter without new nodules or progressive enlargement of the existing nodules. Repeat neck sonography has revealed no evidence of local recurrent disease. Anti-thyroglobulin antibodies developed at the age of 59, and concentrations have remained positive, though variable. Despite biopsy-proven pulmonary metastasis, the patient has been asymptomatic and healthy. At the age of 75, patient had stable pulmonary metastases for 42 years.</p></sec><sec><title>Patient no. 5</title><p>In 1966, a 43-year-old female underwent hemi-thyroidectomy revealing papillary thyroid carcinoma. No other therapy was recommended at that time. After 12 years, she developed recurrent disease and underwent complete thyroidectomy. She did not receive further treatment except for thyroid hormone suppression and experienced an unremarkable course for the next 34 years until a mediastinal mass was detected at the age of 77 years. A chest CT confirmed a 2.5 cm mediastinal mass as well as numerous micro- and macro-pulmonary nodules measuring 3–12 mm in diameter. A PET scan showed intense FDG avidity in the sternal mass and mild avidity in a left lung nodule. She underwent resection of the mediastinal mass, confirming PTC with tall cell features. The patient was treated with radioactive iodine therapy (203 mCi dose) but demonstrated no pulmonary uptake. Given concerns for possible pulmonary sarcoidosis and a separate malignancy, diagnostic thoracoscopic wedge resection of the left lower lung was performed 2 months later. This confirmed metastatic papillary carcinoma. No further treatment was provided. For the next 8 years, the patient was monitored with CT scans of the chest demonstrating persistent, but stable, pulmonary metastases. Follow-up testing demonstrated variable thyroglobulin levels (2.2–4.8 ng/ml), though without upward trend. She did have recurrent neck metastases that were resected at ages 81 and 85 years respectively. Pathology confirmed metastatic papillary carcinoma. From age 85 to 87, CT scans showed mild enlargement of pulmonary metastasis but stabilization thereafter. At the age of 89, the patient was unfortunately diagnosed with breast carcinoma and biopsy-proven breast cancer metastasis to the liver and bone. She died within 12 months from rapidly progressing breast cancer. At the time of death, the patient had thyroid cancer for over 47 years, with persistent, stable pulmonary metastases disease for 13 years.</p></sec></sec><sec><title>Discussion</title><p>The incidence of thyroid cancer has been increasing <xref rid="bib1" ref-type="bibr">(1, 3, 24)</xref>. Most patients demonstrate low-risk, localized disease, which confers an outstanding long-term survival following treatment <xref rid="bib4" ref-type="bibr">(4, 5)</xref>. However, a minority of patients present with distant metastatic disease, often viewed as life threatening. While the majority of deaths from thyroid carcinoma <xref rid="bib2" ref-type="bibr">(2)</xref> are indeed those with distant metastatic disease <xref rid="bib23" ref-type="bibr">(23, 25, 26)</xref>, our data demonstrate a remarkable diversity in the natural progression of patients with advanced thyroid cancer. We identified five individuals initially diagnosed with well-differentiated thyroid carcinoma (WDTC), who underwent surgery and <sup>131</sup>I therapy. However, concerns for separate malignancies led to biopsy of metastatic foci at time-points far distant from the initial diagnosis (mean: 19 years). In each case, histopathological analysis confirmed persistent, generally stable pulmonary metastases long after the completion of initial therapy. Persistent disease was further supported by elevated serum thyroglobulin levels in two patients and the development of new (or rising) anti-thyroglobulin antibodies in the other three patients. Abnormal uptake on PET scanning (<italic>n</italic>=3) and/or persistent iodine uptake on radionuclide scanning (<italic>n</italic>=1) confirmed metabolic activity in these persistent, yet stable, foci of cancer. The lack of disease progression was assessed by a careful review of repeat imaging and the subsequent clinical follow-up, while no further therapy was administered beyond thyroid hormone suppression. Together, these data confirm the remarkable ability of select distant metastatic WDTC to persist yet remain stable for decades (mean 22 years, range 8–42 years) despite evidence of metabolic activity.</p><p>Our data do not provide metrics allowing us to determine the prevalence of such patients. We estimate that ∼4% of the patients diagnosed with PTC at BWH have distant metastatic disease. However, our study is notable for the inclusion criteria, which mandated lung biopsy and histological confirmation of persistent metastatic cancer at a later time point. While this protocol was necessary to prove that persistent lung nodules were not simply effectively treated (and destroyed) WDTC tissue, these criteria almost certainly underestimate the number of similar cases that may exist. Although studies have reported high mortality rates in patients with metastatic thyroid cancer, those who survived probably shared a fate similar to our population. In support of this, others have estimated that up to ∼30% of patients with metastatic thyroid cancer remain stable during 10 years of follow-up <xref rid="bib27" ref-type="bibr">(27)</xref>. In addition, many patients with thyroid cancer and lung nodules do not undergo invasive lung biopsy or wedge resection. Most of them are simply monitored by serial clinical examinations and chest imaging. We speculate that at our institution, such cases number at least one per year. Extrapolation to a national level allows one to postulate that hundreds of patients may prove to have persistent, yet non-progressive distant metastatic disease.</p><p>Though impossible to identify the predictive characteristics from such a small patient population, it is worth pointing out some striking findings. One notable finding was the paucity of iodine uptake on post-therapy scans in the metastases of four patients (patients nos 2–5). Typically, non-iodine-avid distant metastases are associated with worse prognoses. However, those in our series demonstrated stable disease <xref rid="bib28" ref-type="bibr">(28, 29)</xref>. Separately, FDG positivity on PET scan is also known to be associated with unfavorable prognoses <xref rid="bib30" ref-type="bibr">(30)</xref>. However, all three patients with FDG-avid pulmonary metastasis experienced prolonged stability. We also note that the patients described in this series are all women and were relatively young at the time of diagnosis (mean 29 years). It is well recognized that younger patients respond better to initial therapy and experience a longer life expectancy compared with older patients with a similar disease <xref rid="bib6" ref-type="bibr">(6, 31, 32, 33)</xref>. This case series also lends support to long-term TSH-suppressive therapy in patients with distant metastatic thyroid cancer, as all patients were treated solely by this modality for decades. However, we acknowledge that no prospective, randomized study has yet proven the long-term survival benefit of TSH suppression <xref rid="bib34" ref-type="bibr">(34, 35, 36)</xref>.</p><p>Very few published reports exist on this topic. Vassilopoulou-Sellin <italic>et al</italic>. <xref rid="bib37" ref-type="bibr">(37)</xref> reported distant metastatic papillary carcinoma in a 9-year-old female. She was treated with surgery, external radiation therapy, and <sup>131</sup>I ablation. Long-term follow-up was unremarkable, though a new provider detected a lung nodule at the age of 40. Biopsy demonstrated thyroid carcinoma ultimately proven to be metabolically active and iodine avid. This is remarkably similar to patient no. 1 described above. However, few other comparable reports <xref rid="bib18" ref-type="bibr">(18)</xref> exist despite epidemiological data confirming young patients (<45 years) with distant metastatic disease may experience a 63–85% 10-year survival rate after initial therapy <xref rid="bib33" ref-type="bibr">(33, 38, 39)</xref>. We are confident that there are many patients who may experience a similar disease course.</p><p>These data have important implications for the clinical care of patients with WDTC. Importantly, distant metastatic disease occurs in 2–7% of cases <xref rid="bib22" ref-type="bibr">(22, 40, 41, 42)</xref> and should be treated aggressively. It is notable that all patients in our series underwent initial therapy including thyroidectomy and neck dissection, followed by <sup>131</sup>I ablation. Some subjects (patients nos 2 and 5) have substantial or recurrent neck disease that was aggressively treated and required multiple surgeries. Complete resection of the local disease (including adenopathy) appears to be important <xref rid="bib15" ref-type="bibr">(15, 30, 31, 34, 43, 44)</xref>. When resection is not complete following initial surgery, repeated local dissections may prove beneficial <xref rid="bib15" ref-type="bibr">(15, 45)</xref>. It is likely that such therapy was important to their outcome and should be recommended for similar patients. However, the utility of repeated <sup>131</sup>I dosing, and possible use of systemic chemotherapy should be questioned, with perhaps more judicious decision-making favored based upon continued individual risk assessment and response to treatment, as advocated by the American Thyroid Association <xref rid="bib15" ref-type="bibr">(15)</xref>. In support of this, repeat RAI dosing in the absence of iodine avidity in metastatic disease has been associated with a low efficacy on tumor outcome <xref rid="bib46" ref-type="bibr">(46)</xref>. Furthermore, some patients in our study may have qualified for enrollment in clinical trials investigating the use of tyrosine kinase inhibitors, especially if disease appeared to have anatomically progressed (patient no. 5). However, a decision for TKI for treating advanced or even advancing DTC must be based on continuous assessment of treatment benefit vs possible toxicity. All patients who have completed initial surgery and ablation should be monitored by serial examinations and imaging. Those who remain asymptomatic and show no signs of anatomic progression may be candidates for expectant monitoring. We recognize that some patients will progress. Therefore, we do not advocate that a singular approach be utilized in all patients, but rather individualized therapy be provided to optimize treatment benefit and minimize morbidity.</p><p>In conclusion, although the diagnosis of advanced thyroid cancer usually confers a worse prognosis, this case series demonstrates the remarkable ability of some patients with metabolically active distant pulmonary metastases to persist and remain stable for decades without further treatment apart from TSH suppression. Though our data are not sufficiently robust to ascertain the prevalence or predictive characteristics of this cohort, this study may serve to promote physician awareness of such a unique subset of patients and highlight the treatment dilemmas that often arise while managing such patients. We hope that this case series may serve as a platform upon which further studies can build to better understand this complex disease.</p></sec> |
Palliation in metastatic non-small cell lung cancer: Early integration with standard oncological care is the key | Could not extract abstract | <contrib contrib-type="author"><name><surname>Singh</surname><given-names>Navneet</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Behera</surname><given-names>Digambar</given-names></name><xref ref-type="aff" rid="aff1"/></contrib> | Lung India : Official Organ of Indian Chest Society | <p>Lung cancer remains one of the most common malignancies globally (and within India also), amongst males as well as for both gender combined.[<xref rid="ref1" ref-type="bibr">1</xref><xref rid="ref2" ref-type="bibr">2</xref>] Approximately, 80% of cases are of non-small cell lung cancer (NSCLC) histology and present with advanced/unresectable disease (stages IIIB and IV) in which traditional treatment options like chemotherapy and radiation therapy are aimed at disease and symptom control rather than at achieving a cure.[<xref rid="ref3" ref-type="bibr">3</xref><xref rid="ref4" ref-type="bibr">4</xref>] For advanced and metastatic NSCLC, the last two decades have witnessed the emergence of molecularly targeted therapy as an important addition to the therapeutic armamentarium. In particular, adenocarcinoma patients should undergo testing for the presence of sensitizing mutations in the epidermal growth factor receptor (EGFR) gene and for rearrangements in the anaplastic lymphoma kinase (ALK) gene since these are two actionable (‘druggable’) targets wherein use of EGFR tyrosine kinase inhibitors (EGFR-TKIs like gefitinib, erlotinib and afatinib) and of ALK inhibitors (like crizotinib and ceritinib), respectively is associated with substantial improvements in progression-free survival (PFS), objective response rates (RRs) and possibly overall survival (OS) also.[<xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref6" ref-type="bibr">6</xref>] Moreover, with such a molecularly targeted management approach, improvements in these ‘physician-centred’ outcomes (OS, PFS, RR) are achieved with lesser toxicity (as compared to standard chemotherapy regimens) and are associated with improvement in health-related quality of life (QOL) and symptom control which are important ‘patient-centred’ outcomes.[<xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref8" ref-type="bibr">8</xref>] Another important change related to management of advanced/metastatic NSCLC that has occurred in the recent past has been the need to distinguish squamous and non-squamous histological subtypes in view of the differences in treatment approaches to these two subgroups.[<xref rid="ref9" ref-type="bibr">9</xref>] Apart from the availability of specific molecularly targeted therapies for adenocarcinoma, pemetrexed, an anti-folate drug, has been proven to be the most effective and preferred third-generation agent for use in this histological subtype as first-line chemotherapy (in combination with a platinum agent) and subsequently as maintenance treatment (in the absence of disease progression with first-line chemotherapy).[<xref rid="ref10" ref-type="bibr">10</xref>]</p><p>For treatment naïve patients with stage IV NSCLC and good performance status (PS), histology guided platinum-based doublet chemotherapy for 4-6 cycles remains the standard of care when predictive molecular biomarkers for targeted therapy (namely sensitizing mutations in the EGFR gene for oral EGFR-TKIs and rearrangements in the ALK gene for crizotinib) have either not been assessed or are absent. Although radiation therapy plays an important role in management of medically inoperable stage I-II NSCLC and as part of concurrent chemoradiotherapy for locally advanced stage III NSCLC, its use (upfront or after initial systemic therapy) in stage IV NSCLC is limited to palliation of specific situations, like presence of superior vena caval (SVC) obstruction, symptomatic brain metastases or spinal cord compression, and severe pain that is uncontrolled or poorly controlled with analgesics prescribed as per the WHO stepladder algorithm for cancer pain management.[<xref rid="ref11" ref-type="bibr">11</xref>] Even amongst radiation oncologists, considerable variations exist related to the timing and dosage schedule for use of radiation therapy in stage IV NSCLC as opposed to a near consensus on its usage in early and locally advanced disease.[<xref rid="ref12" ref-type="bibr">12</xref>] The randomized trial by Sou and colleagues that appears in the current issue of the journal is an attempt to compare different dosage and fractionation schedules of thoracic external beam radiation therapy (EBRT) for palliation of pain in treatment naοve stage IV NSCLC. Randomization of 156 patients was done into three different schedules namely 17 Gy delivered in 2 fractions; 20 Gy delivered in 5 fractions and the standard 30 Gy delivered in 10 fractions. The first two schedules both led to completion of EBRT in one week while the third required two weeks. The authors reported similar magnitude of pain relief in all groups when assessed at three different time points (2, 6 and 12 weeks) after completion of thoracic EBRT. The three groups also had similar OS and improvements in QOL. Both OS and QOL are known to improve with chemotherapy and the lack of information in this study about percentage of patients who received subsequent chemotherapy (including regimens and number of cycles administered) remains a potential confounder.[<xref rid="ref13" ref-type="bibr">13</xref><xref rid="ref14" ref-type="bibr">14</xref>] We would have also expected the authors to have discussed the factors that led this population of newly diagnosed stage IV NSCLC patients to being treated upfront with radiation rather than with systemic therapy (chemotherapy/targeted therapy). The above-mentioned limitations notwithstanding, the important message from this study is that lesser fractions of thoracic EBRT (hypofractionation) are associated with similar results as are conventional radiation schedules although it would have helpful if the profile of radiation therapy-associated toxicity (especially esophageal) observed in the three groups had been reported. Data from previously published studies and trials also indicate that in advanced NSCLC, protracted palliative thoracic EBRT offers no significant benefit for either symptom relief, improvement in QOL or OS when compared with short-term hypofractionated treatment schedules.[<xref rid="ref15" ref-type="bibr">15</xref><xref rid="ref16" ref-type="bibr">16</xref><xref rid="ref17" ref-type="bibr">17</xref>] A systematic review that compared use of higher versus lower dose EBRT for symptom control in advanced lung cancer found similar results for control of individual symptoms (cough, chest pain, hemoptysis) although improvement in overall symptom burden was significantly better with higher dose EBRT.[<xref rid="ref18" ref-type="bibr">18</xref>] One year (but not 2 years) OS rates have been observed to be better with higher dose EBRT.[<xref rid="ref18" ref-type="bibr">18</xref><xref rid="ref19" ref-type="bibr">19</xref>] In addition, the cost of therapy is significantly lower with shorter hypofractionation schedules.[<xref rid="ref20" ref-type="bibr">20</xref>] The American Society for Radiation Oncology (ASTRO) in its evidence-based clinical practice guideline for use of palliative thoracic radiotherapy in lung cancer also states that lesser EBRT dose/fractionation schedules provide good symptomatic relief and may be used for patients requesting a shorter treatment course and/or in those with poor PS.[<xref rid="ref21" ref-type="bibr">21</xref>] In resource-constrained settings as ours, wherein health care facilities tend to be overburdened including those wherein lung cancer patients are treated, use of hypofractionated schedule offers several potential advantages.[<xref rid="ref22" ref-type="bibr">22</xref><xref rid="ref23" ref-type="bibr">23</xref>] First, it reduces the number of hospital visits that patients are required to undertake while undergoing radiation therapy. Second, it reduces the number of sessions that treating radiation oncologists need to schedule for a given patient. Third, it reduces the overall length (duration) of radiation therapy (faster completion of treatment from time of initiation). Fourth, it also reduces the direct costs related to radiation therapy and possibly even the indirect costs by reducing radiation therapy-related toxicity.</p><p>It is important to realize that palliative interventions are not merely those which target the disease <italic>per se</italic> (e.g. chemotherapy or radiation therapy) but are also those in which symptom-based management is undertaken e.g. use of medical thoracoscopy for malignant pleural effusions and use of interventional pulmonology procedures for airway obstruction.[<xref rid="ref24" ref-type="bibr">24</xref><xref rid="ref25" ref-type="bibr">25</xref>] Since the publication of the randomized trial by Temel and colleagues involving metastatic NSCLC patients, there has been renewed interest and enthusiasm in the concept of early integration of palliative care with standard oncological care.[<xref rid="ref26" ref-type="bibr">26</xref>] Traditional versus early palliative care models differ from each other substantially in the sense that the former approach is focused on institution of palliative care only after life-prolonging or curative treatment is no longer being administered while in the latter approach (integrated model), both palliative care and life-prolonging care are provided throughout the course of disease.[<xref rid="ref27" ref-type="bibr">27</xref>] This is important because palliative care is frequently misinterpreted as being synonymous with end-of-life care while in true terms, it aims to relieve suffering, in all of its dimensions, throughout the course of a patient's illness. In the trial by Temel <italic>et al</italic>., the intervention arm (early integrated palliative care with standard oncological treatment) showed significant improvement in symptoms, QOL (spiritual and end-of-life domains) and even OS as compared to the conventional arm (standard oncological treatment only).[<xref rid="ref26" ref-type="bibr">26</xref>] These gains were achieved with a simultaneous reduction in the utilization of health care services and in the overall cost of cancer care. Some of these improvements in patient outcomes have been replicated subsequently in a larger trial as well.[<xref rid="ref28" ref-type="bibr">28</xref>] These results have led to generation of a consensus opinion that all metastatic NSCLC patients should be offered concurrent palliative care and standard oncological care at initial diagnosis in view of its immense potential for improving ‘patient-centred’ outcomes and reducing burden on caregivers (typically immediate family members).[<xref rid="ref29" ref-type="bibr">29</xref>]</p><p>There have been suggestions that for Asia, wherein countries can range from being resource-poor (resource constrained) to resource-rich, the concept of integrating palliative care with standard oncological care could range from <italic>basic</italic> to <italic>maximal</italic> depending upon the level of resources available.[<xref rid="ref30" ref-type="bibr">30</xref>] Even at the <italic>basic</italic> level, pain should be routinely assessed - ‘<italic>fifth vital sign</italic>’ - and managed with appropriate analgesic prescriptions. In a comprehensive approach, the key interventions in palliative care (integrated with standard oncological treatment) actually encompass five major domains: (1) Illness understanding, (2) Symptom management, (3) Treatment decision making, (4) Illness coping (patient and family caregiver), and (5) Care planning (referrals and drug prescriptions).[<xref rid="ref31" ref-type="bibr">31</xref>]</p><p>The American Society of Clinical Oncology (ASCO; <ext-link ext-link-type="uri" xlink:href="http://www.asco.org">www.asco.org</ext-link>) and the European Society for Medical Oncology (ESMO; <ext-link ext-link-type="uri" xlink:href="http://www.esmo.org">www.esmo.org</ext-link>) which are two of the most renowned professional oncology bodies in the world offer opportunities to physicians and oncologists in developing countries to enhance their knowledge about palliative care. The Conquer Cancer Foundation of the ASCO offers the International Development and Education Award (IDEA) (<ext-link ext-link-type="uri" xlink:href="http://www.conquercancerfoundation.org/cancer-professionals/grants-awards/international-development-and-education-awards">http://www.conquercancerfoundation.org/cancer-professionals/grants-awards/international-development-and-education-awards</ext-link>) and its associated IDEA-Palliative Care. The ESMO offers the Palliative Care Fellowship (<ext-link ext-link-type="uri" xlink:href="http://www.esmo.org/Career-Development/Oncology-Fellowships/Fellowship-Offers/Palliative-Care">http://www.esmo.org/Career-Development/Oncology-Fellowships/Fellowship-Offers/Palliative-Care</ext-link>). Our personal experience with both of the above has been very encouraging in terms of the insight into evidence based clinical approach and potential for improvement in cancer management that such experiences provide. Despite being highly competitive globally, we recommend that individuals who have a keen interest in oncological palliative care should consider applying for these annual awards/fellowships.</p> |
Laying the ground for research of interstitial lung disease in our country: ILD India registry | Could not extract abstract | <contrib contrib-type="author"><name><surname>Singh</surname><given-names>Virendra</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Sharma</surname><given-names>Bharat Bhushan</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib> | Lung India : Official Organ of Indian Chest Society | <p>India is a country with great diversity where dresses, dialects and diets vary drastically from place to place. But, above all there is a vast universality in thinking and emotions throughout India. Likewise, many chronic diseases have identical presentations, but with subtle and yet important regional differences.</p><p>Interstitial lung disease (ILD) represents a group of about 200 distinct disorders involving lung parenchyma. ILDs are often referred to as diffuse parenchymal lung disease (DPLD). We have used both these terms in this editorial. Uniform approach to diagnosis and treatment of ILD is buttressed from time to time with the help of standard guidelines. In 2002, the American Thoracic Society/European Respiratory Society (ATS/ERS) guidelines classified idiopathic interstitial pneumonias (IIPs) into seven specific entities and offered standardized terminology and diagnostic criteria. The “gold standard” need of a histological diagnosis was changed to a multidisciplinary approach.[<xref rid="ref1" ref-type="bibr">1</xref>]</p><p>New information about ILD is regularly available after 2002 guidelines. The 2013 update is a supplement to the previous 2002 guidelines.[<xref rid="ref2" ref-type="bibr">2</xref>] There was a need to provide a better clinical algorithm for diagnosis and management of IIPs. Idiopathic nonspecific interstitial pneumonia (NSIP) is now accepted as a specific disease entity and in smokers respiratory bronchiolitis-ILD (RB-ILD) is increasingly diagnosed without surgical lung biopsy. Pleuroparenchymal fibroelastosis as well as bronchiolocentric inflammation and fibrosis are recognized as specific rare entities.</p><p>According to 2013 update, there are three broad categories: First, <italic>major</italic> idiopathic interstitial pneumonias comprising of idiopathic pulmonary fibrosis (IPF), nonspecific interstitial pneumonia, RB-ILD, desquamative interstitial pneumonia, cryptogenic organizing pneumonia and acute interstitial pneumonia. Second, <italic>rare</italic> idiopathic interstitial pneumonias are pleuroparenchymal fibroelastosis and lymphoid interstitial pneumonia. The third and final category is <italic>unclassifiable</italic> idiopathic interstitial pneumonias.[<xref rid="ref2" ref-type="bibr">2</xref>]</p><p>Incorporation of genetic and molecular studies may revolutionize the approach to diagnosis and classification of the IIPs. Some potential biomarkers useful in the differential diagnosis and prognosis of ILD are chemokine ligand (CCL); intercellular adhesion molecule (ICAM); Krebs von den Lungen-6 (KL-6); matrix metalloproteinase (MMP); protein encoded by S100-A12 gene (S100-A12); surfactant protein (SP); vascular cell adhesion protein (VCAM).[<xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref3" ref-type="bibr">3</xref>]</p><p>Usually, there are multiple sources of data collection for ILD or any other similar disease of great public health importance.[<xref rid="ref4" ref-type="bibr">4</xref>] Studies based on questionnaires for pulmonary physicians, use of pre-existing databases, such as hospital database and death register, or small case-series are usual source of data. In these studies, there are major methodological limitations and heterogeneity which make the comparison of epidemiological parameters virtually impossible. The data available from national registries, on the other hand, can provide more homogeneous information.</p><p>The pioneer ILD registry by Coultas <italic>et al</italic>. was from a county of New Mexico in the United States.[<xref rid="ref5" ref-type="bibr">5</xref>] According to this registry, the prevalence of IPF was about 20 per lakh in males as against about 13 per lakh in females. According to Thomeer <italic>et al</italic>. a lower proportion of IPF was reported from Belgium.[<xref rid="ref6" ref-type="bibr">6</xref>] On the other hand, an Italian registry reported IPF in 27% of cases.[<xref rid="ref7" ref-type="bibr">7</xref>]</p><p>According to a recent review of 15 studies, prevalence of IPF in the United States is between 14 and 63 cases per lakh population and an incidence of 7% to17% as against Europian incidence of 0.2% to7%.[<xref rid="ref8" ref-type="bibr">8</xref>] It can be assumed that with introduction of better diagnostic amenities and superior overall life expectancy, the incidence of ILD will further increase. Some newer IPF registries are being set up which are likely to yield important data on IPF after the revision of diagnostic criteria in 2002.[<xref rid="ref9" ref-type="bibr">9</xref><xref rid="ref10" ref-type="bibr">10</xref>]</p><p>There is a paucity of literature on the pattern, determinants, distribution and response of treatment of ILD in India. Small regional studies have contributed a lot in our understanding of the disease. Though no true epidemiological study on prevalence of ILDs and its different subgroups from India is available; according to the studies available proportion of IPF may vary between approximately 30% and 45% [<xref ref-type="table" rid="T1">Table 1</xref>]. In all Indian studies, CTD associated ILD, hypersensitivity pneumonitis and sarcoidosis are present in significant proportions.</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Studies reporting pattern of ILD in India</p></caption><graphic xlink:href="LI-31-320-g001"/></table-wrap><p>In 1979, Jindal <italic>et al</italic>. published their data on cases of DPLD seen over a period of five years and among them 46% of cases were having IPF.[<xref rid="ref11" ref-type="bibr">11</xref>] In 1984, Sharma <italic>et al</italic>. reported IPF to be present in 28.6% of their patients with DPLD.[<xref rid="ref12" ref-type="bibr">12</xref>] In 2004, Maheshwari <italic>et al</italic>. showed female preponderance of IPF and mean age of presentation about 50 years.[<xref rid="ref13" ref-type="bibr">13</xref>] In the same year a group of investigators from south India supported the fact that secondary DPLD (55%) was more common than IPF.[<xref rid="ref14" ref-type="bibr">14</xref>] In addition subjective improvement with steroids was more in secondary DPLD as compared to IPF. In 2010, Sen <italic>et al</italic>. in their retrospective analysis reported that besides IPF, sarcoidosis, ILDs secondary to CTD and hypersensitivity pneumonitis were the main diagnoses.[<xref rid="ref15" ref-type="bibr">15</xref>] After 2011 ATS/ERS guidelines, there is a drastic change in our algorithm to approach to IIPs. Unfortunately, no large Indian study worth reporting is available in more recent period.</p><p>ILD India registry is step toward knowing these facts about these diseases especially the IPF.[<xref rid="ref16" ref-type="bibr">16</xref>]</p><p>ILD India registry was started in 2011. Inclusion criteria are respiratory symptoms such as shortness of breath and cough and bilateral abnormalities in x-ray/high-resolution computed tomography (HRCT) scan of thorax. History, physical examination, spirometry, and HRCT chest are essential. Six-minute walk test is optional. If patient has symptoms consistent with a collagen vascular disease then rheumatoid factor and ANA are done. If either test is positive a full panel is done. If a patient with an initial diagnosis of ILD develops pulmonary tuberculosis over the course of treatment, he/she is included in the ILD India registry. A patient having history of AFB smear positive tuberculosis without prior ILD is not included. However, patients with no history of AFB positivity and a doubtful radiographic pattern are included as this may be due to sarcoidosis or other similar ILDs. In patients with no clear cut pattern of ILD bronchoalveolar lavage, transbronchial lung biopsy and open lung biopsy are required in order to try to establish a diagnosis. Any infectious or malignant diseases are criteria of exclusion in the registry. Detailed history of exposure and past medications is incorporated in registry to include all possible factors linked to the disease. We recently encountered a series of cases of hypersensitivity pneumonitis after very unusual exposure to dug-well which was not previously reported from India or rest of the world.[<xref rid="ref17" ref-type="bibr">17</xref>] The environmental assessment of dug-well as well as detailed work-up of patients suggested toward <italic>Aspergillus</italic> species as the main culprit.</p><p>There is a simple procedure for submitting data of individual patients. After taking informed consent the investigator fills the proforma. Relevant investigations are done (spirometry and HRCT scan thorax are mandatory). Investigator makes a diagnosis and submits the proforma online. Investigator then sends the following data in electronic format to the National Co-ordinator: Spirometry tracings with report (scanned or good photocopy), X-ray chest report and photo, HRCT scan (preferably a DICOM CD) and histopathology slides along with a copy of biopsy report. Both inspiratory/expiratory and supine/prone films are preferable. A slice thickness of 1 to 1.5 mm is required. If clinical features and chest radiograph suggest hilar enlargement, then contrast is required to delineate mediastinal structures. Confirmation of diagnoses is done by expert panel (two radiologists, two physicians and two histopathologists) at national and international levels as a two-step process. There is a facility of data entry on follow up visits and any unforeseen event (e.g. death) is informed to the registry.</p><p>This issue of Lung India contains an article on the pattern of ILD in eastern India.[<xref rid="ref18" ref-type="bibr">18</xref>] Kundu <italic>et al</italic>. have studied clinical, laboratory and imaging parameters of patients with ILD and compared the presentations of IPF with Connective Tissue Disease Associated ILD (CTD-ILD). They found IPF to be present in 38.04% of their study participants and CTD-ILD in 31.5%. In western literature, the incidence of IPF exceeds far beyond the other groups. As far as the frequency of individual ILD is concerned, hypersensitivity pneumonitis and sarcoidosis are close competitors with CTD-ILD throughout world. They also found that IPF and CTD-ILD vary considerably with respect to certain demographical, clinical, physiological and radiographic parameters. In this study, comparison of serum markers of CTD between IPF and CTD-ILD, in our opinion, is superfluous. Comparisons of biomarkers between these would have been more informative. Another variance in this study was exclusion of many cases that could have been included. As stated above, we include upper lobe fibrotic patterns where there is no history of AFB positivity. NSIP patterns are also included in the registry as an isolated group of IIP other than that associated with CTD-ILD even if no biopsy is available. A confident diagnosis of NSIP can be made based on clearly defined HRCT features.[<xref rid="ref19" ref-type="bibr">19</xref><xref rid="ref20" ref-type="bibr">20</xref><xref rid="ref21" ref-type="bibr">21</xref>] The follow-up of the cases shown in the study is too short to be of significant importance for a disease with a variable long term course such as ILD.[<xref rid="ref22" ref-type="bibr">22</xref>] Nevertheless, this study documents pattern of ILD from eastern part of India and is a commendable effort directed toward workup of patients with ILD in their region.</p><p>All the premium Indian journals of pulmonary diseases are regularly publishing scientific material on ILD but it is much less as compared to the fast growing body of word literature on ILD. Moreover, most of the Indian literature on ILD is in the form of interesting case reports or case series on individual ILDs. We advocate for more focused research on this disease especially on IPF by our own indigenous people.</p> |
Utility of FDG-PET-CT scanning in assessing the extent of disease activity and response to treatment in sarcoidosis | <sec id="st1"><title>Background:</title><p>Radionuclide imaging modalities have increasingly been evaluated in the assessment of organ involvement in sarcoidosis. Fluoro-deoxyglucose positron emission tomography–computed tomography (FDG–PET–CT) scanning has received increasing attention in the recent years. The aim of our study was to evaluate the utility of FDG–PET–CT in determining the extent of organ involvement and disease activity in patients of sarcoidosis and to assess its utility in the evaluation of response to therapy. The secondary objective was to compare the agreement between clinical, radiological (HRCT) and metabolic indices (FDG–PET–CT) of disease activity.</p></sec><sec id="st2"><title>Materials and Methods:</title><p>This was a prospective observational study conducted between March 2007 and December 2008 at a tertiary care referral center in north India. Twenty-five symptomatic and histopathologically proven cases of sarcoidosis underwent FDG–PET–CT scanning at baseline and a follow-up scan in 21 patients at 6-9 months post-treatment with glucocorticoids.</p></sec><sec id="st3"><title>Results:</title><p>FDG–PET–CT scan detected metabolic disease activity in 24 of the 25 patients with clinically active sarcoidosis. It also demonstrated many clinically inapparent sites of disease activity. Complete or partial metabolic response was seen in 17 of the 21 patients in whom a follow-up scan was available. Substantial degree of agreement was found between the metabolic response and the radiological response, whereas moderate agreement was found between clinical and metabolic responses.</p></sec><sec id="st4"><title>Conclusions:</title><p>FDG–PET–CT scanning is a useful imaging modality to assess disease activity, extent of disease involvement and response to treatment in clinically active sarcoidosis. There is substantial agreement between the HRCT and metabolic parameters of disease activity. Further, large sample size studies are proposed in order to identify the subset of patients who are likely to benefit the most from this sensitive modality of imaging, especially in developing countries where the cost of the procedure is an important concern.</p></sec> | <contrib contrib-type="author"><name><surname>Guleria</surname><given-names>Randeep</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Jyothidasan</surname><given-names>Amudhan</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Madan</surname><given-names>Karan</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Mohan</surname><given-names>Anant</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Kumar</surname><given-names>Rakesh</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib><contrib contrib-type="author"><name><surname>Bhalla</surname><given-names>Ashu Seith</given-names></name><xref ref-type="aff" rid="aff3">2</xref></contrib><contrib contrib-type="author"><name><surname>Malhotra</surname><given-names>Arun</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib> | Lung India : Official Organ of Indian Chest Society | <sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Sarcoidosis is a chronic inflammatory multisystem disease that usually affects middle-aged adults and frequently presents with bilateral hilar lymphadenopathy, pulmonary parenchymal involvement and ocular and skin lesions. Diagnosis is established by a clinico-radiological picture consistent with sarcoidosis along with tissue confirmation of non-caseating granulomas and exclusion of other granulomatous conditions.</p><p>The term “activity” in sarcoidosis refers to ongoing inflammation that necessitates appropriate therapy. Several biochemical and radiological markers have been used as indices of disease activity, but there is often a lack of concordance in these markers and no gold standard is available to validate these markers. Treatment of sarcoidosis remains controversial and there is little consensus on the optimum duration of therapy and assessment of disease activity.</p><p>The role of fluoro-deoxy glucose–positron emission tomography (FDG–PET) scanning in assessing the extent of disease spread or metastasis and its utility in assessing response to treatment in the form of chemotherapy or radiotherapy is well defined in many neoplastic conditions, and its utility has also been recognized in certain inflammatory conditions. FDG–PET and a combination of this procedure with computed tomography scanning (FDG–PET–CT) has gained prominent attention in patients with sarcoidosis over the last two decades as a means to assess disease activity and response to therapy. Its role has also been evaluated in patients with cardiac sarcoidosis. Herein, we present our preliminary experience on the utility of 18F-FDG–PET–CT scanning in assessing the extent of disease activity and response to treatment in sarcoidosis patients from a tertiary care referral hospital in north India.</p></sec><sec sec-type="materials|methods" id="sec1-2"><title>MATERIALS AND METHODS</title><p>The aim of our study was to evaluate the utility of FDG–PET–CT scan in determining the extent of organ involvement and disease activity in patients of sarcoidosis and to assess its utility in the evaluation of response to therapy. The secondary objective was to compare the agreement between clinical, radiological (HRCT) and metabolic indices (FDG–PET–CT) of disease activity.</p><p>Newly diagnosed symptomatic and treatment-naïve patients of sarcoidosis were included in the study. The study design was prospective with one baseline FDG–PET–CT scan and a follow-up scan at 6-9 months duration following treatment. Patients willing to participate in the study and satisfying the American Thoracic Society (ATS) diagnostic criteria,[<xref rid="ref1" ref-type="bibr">1</xref>] i.e. compatible clinico-radiological features and tissue biopsy showing non-caseating granulomas, were included in the study. Exclusion criteria were asymptomatic disease, any obvious associated comorbid illness that may cause false-positive FDG uptake (like active malignancy/other inflammatory conditions, uncontrolled diabetes mellitus) and pregnancy. The study protocol was approved by the ethics committee of the institute and written informed consent was obtained from each of the participants.</p><p>Baseline investigations included routine biochemical investigations and complete hemogram. Levels of serum calcium, serum angiotensin-converting enzyme (ACE) and erythrocyte sedimentation rate were measured. Chest radiograph, tuberculin skin test, spirometry with pulmonary diffusing capacity for carbon monoxide (DLCO) using single breath technique, ophthalmological evaluation for uveitis and a contrast-enhanced CT examination of the thorax (including high-resolution scan images of the lung) were also performed. As part of diagnostic evaluation, all patients underwent a flexible bronchoscopy examination wherein samples of transbronchial lung biopsy and bronchial biopsy were obtained. Diffusion capacity for carbon monoxide was performed using the single breath technique. High-resolution images of the lungs were obtained in all the patients at the time of the CT examination. Enrolled participants subsequently underwent a whole body FDG–PET–CT scan.</p><sec id="sec2-1"><title>Whole body FDG–PET–CT scan procedure</title><p>Patients were kept fasting for 4 h before the injection of 18F-FDG. FDG–PET–CT scans were performed on the dedicated scanners (SiemensWashington, USA, [Biograph 64]). The blood glucose level was checked to ensure that it was less than 150 mg/dL before the injection of 18F-FDG. 18F-FDG was administered in a dose of 5.2 MBq (0.14 mCi)/kg through a peripheral vein 1 h before imaging. Initial CT acquisition was performed without oral or intravenous contrast injection, followed by PET scan. During the uptake phase, patients sat quietly in a dimly lit room and were asked to refrain from talking, walking and any other muscular activity to prevent non-specific FDG uptake in the skeletal muscles. Sequential overlapping emission scans of the neck, chest, abdomen and pelvis were acquired 60 min after the injection of the radiotracer.</p><p>Data interpretation was performed by an experienced nuclear medicine physician who was blinded to the clinical findings. PET images were looked for area of increased radiotracer uptake. Corresponding area in the CT images and fused PET–CT images were corroborated. Lesions with increased tracer uptake in the PET scan were considered to be metabolically active disease sites. The extent of disease was determined by the presence of tracer uptake in the involved organs, such as the thoracic and extrathoracic lymph nodes, lungs, spleen, liver, bone, muscle, skin, parotid glands, etc. Tracer uptake was quantified in terms of standardized uptake value (SUV). SUV was taken as an index of disease activity. The maximum SUV (SUVmax) in the pathological sites of uptake in whole-body PET was taken as the pre-treatment SUVmax.</p></sec><sec id="sec2-2"><title>Treatment and follow-up</title><p>Patients were started on treatment based on the treatment protocol of ATS (1999). For pulmonary sarcoidosis, the initial dosage was 0.5-1 mg/kg/d of prednisone. The dose was then slowly tapered to 5-10 mg daily or an every other day regimen over 6 months. Treatment was continued for a minimum of 12 months in most patients. Patients were on a regular follow-up. After 6-9 months of therapy, the patients were reassessed with all the parameters studied earlier, including a repeat FDG–PET–CT scan for disease activity.</p></sec><sec id="sec2-3"><title>Response assessment</title><p>Clinical response was assessed as complete, partial and no response based on the complete resolution of symptoms, persistence of one or more symptoms and clinical worsening, respectively. Radiological response was assessed based on the degree of resolution of lymphadenopathy and parenchymal changes.</p><sec id="sec3-1"><title>Complete radiological response</title><p>Total resolution of mediastinal and peripheral lymphadenopathy to less than 1 cm and >90% resolution of parenchymal changes excluding chronic changes such as bullae, emphysema, traction bronchiectasis, bronchial distortion, parenchymal bands and fibrosis.</p></sec><sec id="sec3-2"><title>Incomplete radiological response</title><p>Decrease in the size of lymph nodes but the residual nodes were still significant, i.e. more than 1 cm in size, as well as a 10-90% decrease in lung parenchymal abnormalities.</p></sec><sec id="sec3-3"><title>No response or worsening radiologically</title><p>Increase in the size of existing lymph nodes and/or presence of new groups of lymph nodes or worsening of parenchymal lesions with or without the appearance of new lesions.</p><p>Metabolically active disease was said to be present on the basis of abnormal tracer uptake in the involved tissue on FDG–PET–CT. Metabolic response was assessed by the change in the uptake quantified in terms of SUV. Non-physiological tracer uptake in the various organs was documented.</p></sec><sec id="sec3-4"><title>Complete metabolic response</title><p>If there was more than 70% fall in post-treatment SUV over pre-treatment SUV.</p></sec><sec id="sec3-5"><title>Partial metabolic response</title><p>Twenty percent to 70% fall in post-treatment SUV over pre-treatment SUV.</p></sec><sec id="sec3-6"><title>No response or disease progression</title><p>If there was less than 20% fall or an increase in post-treatment SUV over pre-treatment SUV.</p></sec></sec><sec id="sec2-4"><title>Statistical analysis</title><p>Results of the FDG–PET–CT scan were compared with the clinical, biochemical and radiological response. Non-parametric tests were used in most comparisons in view of the small sample size. The Wilcoxon signed rank test was used for comparison of continuous variables before and after treatment. The Kappa test was used as a measure of agreement among clinical, radiological, metabolic and biochemical responses. The Chi-square test was used to determine the association between various CT findings and metabolic activity in PET scan. Correlation between the initial SUVmax and clinical response to therapy was performed using the Spearman correlation.</p></sec></sec><sec sec-type="results" id="sec1-3"><title>RESULTS</title><p>Thirty-three patients of sarcoidosis who were satisfying the inclusion criteria were screened between the period March 2007 and December 2008. Four patients were excluded as they did not consent to participate. Five patients were excluded as they were clinically asymptomatic. Twenty-five patients were finally recruited for the study. All the patients were histopathologically proven cases of sarcoidosis. Histologic evidence was established by flexible bronchoscopy and transbronchial lung biopsy in 24 patients and by endomyocardial biopsy in one patient. Twenty-one patients were successfully followed-up after 6-9 months of steroid treatment.</p><p>Baseline clinical characteristics of the patients are summarized in <xref ref-type="table" rid="T1">Table 1</xref>. Constitutional symptoms like fatigue, weight loss, loss of appetite with or without fever along with respiratory complaints like cough and shortness of breath were the most common presenting complaints (60%). One patient presented with nephrolithiasis secondary to hypercalcemia. Another patient presented with ventricular arrhythmias with bradycardia in whom endomyocardial biopsy showed evidence of non-caseating granulomas and a diagnosis of cardiac sarcoidosis was made. One patient presented with hip pain secondary to bone sarcoidosis. Nine of the 25 patients had received anti-tuberculous medications prior to the diagnosis of sarcoidosis and were referred to our center for non-response to therapy.</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Baseline clinical characteristics of the study subjects</p></caption><graphic xlink:href="LI-31-323-g001"/></table-wrap><sec id="sec2-5"><title>Baseline FDG–PET–CT scan findings</title><p>Of the 25 study patients, PET–CT scan showed tracer uptake in 24 patients. Uptake was quantified in terms of SUVmax for each patient. SUVmax of a given patient is the maximum non-physiological uptake in the whole body PET–CT scan irrespective of the tissue or organ involved. However, the site of maximum uptake was also noted in order to compare the same organ or tissue in the post-treatment scan. Extent of involvement as detected by pathological tracer uptake in various organs was also noted. The most common site of increased metabolic activity was the mediastinal lymph nodes, followed by lung parenchyma. Intraabdominal and peripheral lymph nodes, spleen, liver, muscle, bone and skin were the other organs that showed an increased metabolic activity as detected by the PET–CT scan. One patient had uptake in thyroid gland while another in the stomach. The findings are summarized in <xref ref-type="fig" rid="F1">Figure 1</xref>. Median SUVmax is found to be 6.4 (range 1.1-37). The mean SUVmax was 9.36 ± 7.40.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Positron emission tomography–computed tomography tracer uptake of involved organs at the baseline in 25 study subjects</p></caption><graphic xlink:href="LI-31-323-g002"/></fig></sec><sec id="sec2-6"><title>Follow-up data</title><p>Four patients were lost to follow-up and follow-up data were available for 21 patients. All patients received treatment with oral prednisolone and clinical response was assessed after 2 months, after which the steroid doses were tapered accordingly. After 6-9 months of therapy, the patients were reassessed for clinical symptoms and relevant hematological and biochemical investigations along with spirometry, CT thorax and whole body PET–CT scan were repeated.</p></sec><sec id="sec2-7"><title>Response to therapy</title><sec id="sec3-7"><title>Clinical response</title><p>Complete clinical response was noted in nine patients and partial response was seen in 11 patients [<xref ref-type="fig" rid="F2">Figure 2</xref>]. One patient with cardiac sarcoidosis had recurrence of arrhythmias and congestive cardiac failure on tapering of steroid doses and was considered as having clinical worsening. Joint pain was the most common symptom that persisted at the end of the follow-up period. ESR (baseline levels 39.4 ± 16.5, follow-up levels 20.66 ± 12.48; <italic>P</italic> < 0.001) and serum ACE levels (baseline levels 80.2 ± 38.7, follow-up levels 33.7 ± 16.8; <italic>P</italic> < 0.001) reduced significantly post-treatment in the 21 patients who could be followed-up. There was no significant change in serum calcium levels, FEV<sub>1</sub> and DLCO post-treatment.</p><fig id="F2" position="float"><label>Figure 2</label><caption><p>Clinical response on follow-up in 21 study subjects</p></caption><graphic xlink:href="LI-31-323-g003"/></fig></sec><sec id="sec3-8"><title>Radiological response</title><p>Complete radiological response was seen in eight patients while partial response was noted in nine patients [<xref ref-type="fig" rid="F3">Figure 3</xref>]. Four patients did not show any radiological signs of improvement.</p><fig id="F3" position="float"><label>Figure 3</label><caption><p>Radiological response on follow-up in 21 study subjects</p></caption><graphic xlink:href="LI-31-323-g004"/></fig></sec><sec id="sec3-9"><title>Metabolic response</title><p>Complete metabolic response as seen by total resolution of pathological tracer uptake is seen in eight patients [<xref ref-type="fig" rid="F4">Figure 4</xref>]. Partial response was seen in nine patients. Therefore, cumulatively, 17 of 21 patients showed evidence of improvement following steroid therapy, with a decrease or total absence of metabolic activity. Four patients had an apparent absence of response or disease progression in terms of persistence of metabolic activity to pre-treatment levels or an increase in SUVmax in comparison with the pre-treatment values. The patterns of complete, partial and no metabolic response are shown in Figures <xref ref-type="fig" rid="F5">5</xref>-<xref ref-type="fig" rid="F7">7</xref>.</p><fig id="F4" position="float"><label>Figure 4</label><caption><p>Metabolic response on follow-up in 21 study subjects</p></caption><graphic xlink:href="LI-31-323-g005"/></fig><fig id="F5" position="float"><label>Figure 5</label><caption><p>Baseline (left panel) and follow-up (right panel) positron emission tomography images of patient with complete metabolic response</p></caption><graphic xlink:href="LI-31-323-g006"/></fig><fig id="F6" position="float"><label>Figure 6</label><caption><p>Baseline (left panel) and follow-up (right panel) positron emission tomography images of patient with partial metabolic response</p></caption><graphic xlink:href="LI-31-323-g007"/></fig><fig id="F7" position="float"><label>Figure 7</label><caption><p>Baseline (left panel) and follow-up (right panel) positron emission tomography images of patient with no metabolic response</p></caption><graphic xlink:href="LI-31-323-g008"/></fig></sec></sec><sec id="sec2-8"><title>Comparison of clinical response, radiological response and metabolic response</title><p>In patients who had a complete clinical response, 66% showed a complete metabolic response as well while 22% still had evidence of residual disease activity (partial metabolic response) in PET–CT. One patient apparently had a persistence of metabolic activity (pre-treatment SUVmax 5.6 and post-treatment 7.1) in the involved sites. In patients who had a partial or incomplete clinical response, majority of them showed an incomplete metabolic response as well and a few showed an increase in metabolic activity. One patient with cardiac sarcoidosis had a relapse of disease and developed recurrence of ventricular arrhythmias. The PET–CT scan showed an increase in metabolic activity.</p><p>Among the 14 patients who had elevated serum ACE levels at the time of diagnosis, 11 showed normalization of levels during follow-up. Of the patients who had normalization of serum ACE levels on follow-up, complete metabolic response was seen in four patients and partial metabolic response was seen in four patients, while the other three had no response or worsening in the PET–CT scan. Serum ACE levels increased in two patients on follow-up while the PET–CT scan in them showed a decrease in metabolic activity on follow-up. The agreement of PET–CT response with other indices of disease activity is shown in <xref ref-type="fig" rid="F8">Figure 8</xref>.</p><fig id="F8" position="float"><label>Figure 8</label><caption><p>Agreement of positron emission tomography -computed tomography response with other indices of disease activity</p></caption><graphic xlink:href="LI-31-323-g009"/></fig></sec></sec><sec sec-type="discussion" id="sec1-4"><title>DISCUSSION</title><p>There is no consensus on the optimal duration of immunosuppressive treatment in patients with sarcoidosis, and the accurate assessment of disease activity still remains an area of active interest. Disease activity in sarcoidosis is not precisely defined and there is no general consensus on its meaning. Chest radiographs are insensitive in differentiation between active disease and inactive fibrotic disease.[<xref rid="ref2" ref-type="bibr">2</xref>] HRCT is a very useful modality for the assessment of lung parenchymal involvement in sarcoidosis patients; however, correlates of HRCT findings with the extent of active/inactive disease are also not well defined. Also, large sample patient data on the utility of serial HRCT scanning for follow-up assessment of pulmonary sarcoidosis patients and their correlation with change in lung function parameters is also not available.[<xref rid="ref3" ref-type="bibr">3</xref>] In patients with a predominance of fibrotic opacities on the chest radiographs/CT scans, assessment of disease activity is very difficult but nonetheless extremely important. Identification of active disease in this subset of patients can allow institution of appropriate treatment, which can possibly halt the decline in pulmonary function.</p><p>Nuclear medicine imaging modalities that have been studied in patients with sarcoidosis include Gallium-67 scinitgraphy, somatostatin receptor scintigraphy and PET imaging. Gallium scintigraphy is now used sparingly at many centers. Compared with FDG–PET scanning, the sensitivity of Gallium scintigraphy is poorer (97% vs. 88%) and the radiation dose is nearly three times higher.[<xref rid="ref4" ref-type="bibr">4</xref>] Another disadvantage of the technique is prolonged procedure time as image acquisition is delayed after 24 h of agent administration. However, a negative gallium scan combined with normal levels of serum ACE has a high negative predictive value for ruling out the disease.[<xref rid="ref5" ref-type="bibr">5</xref>] Somatostatin receptor scintigraphy is performed most commonly using the radiopharmaceutical, Indium-111 pentetreotide and has been found to be a useful modality for diagnosis of thoracic involvement with sarcoidosis. However, a disadvantage of this technique is that it can miss many sites of extrathoracic disease involvement.[<xref rid="ref6" ref-type="bibr">6</xref>]</p><p>Over the last decade, FDG–PET scanning has emerged as a potentially useful modality of imaging in patients with sarcoidosis. FDG–PET is a metabolic imaging technique. It relies on the principle of increased accumulation and metabolism of glucose by the malignant or inflammatory areas. FDG is a radioactive analogue of glucose that enters cells via the same receptors that are involved in glucose uptake and gets converted into FDG 6 phosphate by the enzyme hexokinase, similar to glucose metabolism by the glycolytic pathway. FDG 6 phosphate is not metabolized further and gets entrapped in the cell. Therefore, the level of FDG uptake is proportional to the level of glycolysis is the tissue. This explains the mechanism of increased uptake of FDG in malignancy, inflammatory and infectious processes.[<xref rid="ref3" ref-type="bibr">3</xref>] A combined modality using FDG–PET and CT scanning (FDG–PET–CT), which was used in our study, has been found to be more sensitive than PET in isolation.</p><p>In a study by Teirstein <italic>et al</italic>., 137 patients with sarcoidosis underwent 188 whole body FDG–PET examinations.[<xref rid="ref7" ref-type="bibr">7</xref>] The SUVmax ranged between 2 and 15.8, and most patients had an SUV of more than 3. The median SUVmax in our study was 6.4 (range 1.1-37). The most common FDG avid sites were mediastinal lymph nodes (54 scans), extrathoracic lymph nodes (30 scans) and lung (24 scans). There were 31 positive scan findings in six other organs. Forty-eight of the repeat scans were performed to follow the results of therapy, primarily in patients with cardiac sarcoidosis. Of the repeat scans, 11 exhibited therapy-related decreased SUVs. In most patients, symptoms, conventional imaging findings and physiologic data paralleled the improvement seen on the PET scans, as was also seen in our study. It was found that PET adequately demonstrates sites of active disease that can be used to obtain biopsies in apparently occult disease.</p><p>Braun <italic>et al</italic>. described 20 patients of sarcoidosis (13 thoracic and seven cases of extrathoracic) in whom FDG–PET was performed.[<xref rid="ref8" ref-type="bibr">8</xref>] To evaluate the response to treatment, five enrolled patients underwent a second 18F-FDG–PET–CT. Complete regression of all foci of pathological tracer uptake was shown in two cases, permitting corticosteroid withdrawal after 2 and 6 months. Improvement but incomplete regression of mediastinal and pulmonary disease occurred in two patients treated with corticosteroids for 19 and 21 months. Disease progression was detected in one patient.</p><p>PET scanning has also been reported to have a prognostic role in patients of sarcoidosis as uptake on PET correlates with BAL fluid neutrophil count. A high number of neutrophils in the BAL has been demonstrated to be a poor prognostic marker in patients with sarcoidosis.[<xref rid="ref9" ref-type="bibr">9</xref>] Recent studies have also highlighted the possible clinical utility of PET–CT in influencing clinical management decisions in patients with sarcoidosis.[<xref rid="ref10" ref-type="bibr">10</xref>]</p><p>A number of questions have emerged after the utilization of PET scanning in patients with sarcoidosis. In the study by Teirstein <italic>et al</italic>., of the 11 patients who had a partial clinical response, no decrease in SUV was observed in two patients. Similar observations were seen in our study, wherein nearly one-fifth of the patients who had complete clinical response had evidence of active tracer uptake on their follow-up PET scans. On the contrary, treatment response as evidenced by total resolution of pathological tracer uptake in the published literature has been noted to occur as early as 2 months of steroid therapy in some cases.[<xref rid="ref11" ref-type="bibr">11</xref>] These observations raise an important issue as to whether PET–CT scanning could be used to guide the optimum duration of therapy in these patients. The first scenario suggests that clinical response occurs earlier than metabolic response and taking clinical response alone as the criterion to stop therapy might not be appropriate and disease activity may flare. The second suggests that in some patients the resolution of metabolic activity can be pretty dramatic; hence, a shorter duration of treatment might be equally effective rather that prolonged treatment. Large sample follow-up data in this regard may be able to answer these questions more appropriately in the future.</p><p>Another issue is as to when to perform a repeat PET scanning for the assessment of disease activity in sarcoidosis. Repeating the PET scan at 2-3 months is unlikely to help in therapeutic decision in patients with partial clinical response as most of these patients may not show a complete metabolic response. For clinically unresponsive patients, it may be useful to perform the scan early as an increase in the uptake would point toward a need for possible relooking at the diagnosis and/or optimization or change of treatment regimen. However, the optimum duration of follow-up scanning in this regard still needs to be determined.</p><p>In our study, there was a good agreement between radiological and metabolic response. Of the three patients in whom there were discordant findings, two showed a partial resolution of radiological abnormalities while the PET–CT showed a modest increase in the uptake. In one patient, there was persistent mediastinal lymph nodal enlargement on follow-up CT suggesting no response; however, PET showed reduction in tracer uptake. These findings suggest that CT still remains an extremely useful and cost-effective modality for follow-up response assessment. It would be important to identify the subgroup of patients who would be likely to benefit from the addition of PET-CT imaging for response assessment, which may be useful for predicting the outcome. In our opinion, this group of patients is likely to comprise of patients in whom the radiological picture predominantly consists of irreversible changes like fibrosis and bronchiectasis and the physician is considering the possibility of disease activity within those lesions. Future studies targeted at this group of patients will likely answer this issue more appropriately.</p><p>There are inherent limitations of our study owing to a smaller sample size as it was a preliminary study at our center. However, our study lays the ground for further larger sample-sized studies on the utility of FDG–PET–CT in Indian patients with sarcoidosis. Use of SUVmax for surrogate of disease activity as has been used in previous studies needs to be considered in the light of the pattern of disease involvement. As disease involvement in sarcoidosis is diffuse in contrast to solid organ tumors, which usually have a primary large focal lesion, quantified uptake of FDG might be more relevant than SUVmax estimation at a focal area. However, studies are not yet available that have tried to correlate quantified FDG uptake with clinical parameters.[<xref rid="ref3" ref-type="bibr">3</xref>] It is difficult to precisely quantify clinical response; therefore, a comparison with objective measures like SUV and metabolic response cannot be accurate. Most patients in our study had stage 1 or stage 2 disease patterns. It has been proposed in previous studies that PET scan may be useful in patients with stage 3 and 4 disease to differentiate reversible component needing steroid treatment from irreversible fibrotic changes. Hence, our results may not reflect the results that will be obtained in such a population. The extent of involvement as a prognostic factor becomes important with the presence of cardiac or neurologic sarcoidosis. The protocol used for the whole body PET–CT scan in this study could have missed asymptomatic cardiac or neurosarcoidosis. Histological proof of the involvement in all the organs showing uptake is not possible to obtain, and the same has been the case with previous studies. Another issue is differentiatingf non-specific uptake or uptake due to intercurrent infection from sarcoidosis disease activity.</p><p>The results of our study show that FDG–PET–CT scanning is a sensitive nuclear imaging modality that adequately demonstrates sites of disease activity in patients with sarcoidosis. There is a good correlation of PET–CT findings with conventional markers of disease activity, like the clinical response and CT findings. In difficult clinical scenarios, it can also guide in planning the site of biopsy in order to establish a histopathological diagnosis. However, important issues exist regarding the evaluation of this investigation modality in resource-constrained settings. As the procedure is costly (nearly five times the cost of a contrast-enhanced CT examination of the thorax in an Indian setting), it is extremely important to use this modality judiciously and to identify those patients who are most likely to benefit from its performance, and it makes a difference to their overall outcome. The answer to these questions will require the conductance of large, well-conducted studies with follow-up assessment corroborated with histopathological measures to better delineate the utility of FDG–PET–CT imaging in patients of sarcoidosis.</p></sec> |
Utility of noninvasive ventilation in high-risk patients during endoscopic retrograde cholangiopancreatography | <sec id="st1"><title>Background:</title><p>There is little evidence on noninvasive ventilation (NIV) preventing respiratory complications in high-risk patients undergoing endoscopy procedures.</p></sec><sec id="st2"><title>Objectives:</title><p>The objective of this study is to demonstrate that the application of NIV through a nasal interface can prevent the appearance of ventilatory alterations during endoscopic retrograde cholangiopancreatography (ERCP) in patients with risk factors associated with the development of hypoventilation.</p></sec><sec id="st3"><title>Patients and Methods:</title><p>A non-randomized interventional study was performed on 37 consecutive high-risk patients undergoing ERCP. During the procedure, 21 patients received oxygen by nasal cannula (3 L/minute) and sixteen received NIV through a nasal mask. Arterial blood gas analyses were conducted before and immediately after the ERCP. An Acute Physiology and Chronic Health Evaluation (APACHE) score pre-ERCP was recorded. The complications during the procedure were recorded.</p></sec><sec id="st4"><title>Results:</title><p>The groups with and without NIV were comparable. A post-ERCP pH of <7.35 was found in eight patients, who did not receive ventilatory support (38.1%) compared to zero patients in the NIV group (<italic>P</italic> = 0.006). A post-ERCP pCO<sub>2</sub> >45 mmHg was found in one case (6.3%) in the NIV-group and in nine cases in the nasal cannula group (42.9%; <italic>P</italic> = 0.01). The median pCO<sub>2</sub> post-ERCP was lower (36.5 ± 6.2 vs. 44.5 ± 6.8 mmHg) (<italic>P</italic> = 0.001) and median pH post-ERCP was higher (7.41 ± 0.4 vs. 7.34 ± 0.5) (<italic>P</italic> = 0.001) in patients treated with NIV. In the multivariate analysis, after adjusting for gender, the APACHE score, pH and pCO<sub>2</sub> pre-ERCP, age, propofol doses, and procedure duration, the following differences were maintained (pCO<sub>2</sub> difference = 5.54, 95% Confidence Interval (CI) =2.3 – 8.7, pH difference = 0.047, and 95% CI = 0.013 – 0.081). Among the 37 procedures, four complications occurred: One in the NIV group and three in the nasal cannula group. None of them was related to NIV.</p></sec><sec id="st5"><title>Conclusions:</title><p>Our preliminary results demonstrate that in high-risk patients undergoing ERCP, hypercapnia and respiratory acidosis are frequent. NIV prevents the appearance of these complications.</p></sec> | <contrib contrib-type="author"><name><surname>Folgado</surname><given-names>Miguel Angel</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>De la Serna</surname><given-names>Carlos</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib><contrib contrib-type="author"><name><surname>Llorente</surname><given-names>Alfonso</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Rodríguez</surname><given-names>SJ.</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib><contrib contrib-type="author"><name><surname>Ochoa</surname><given-names>Carlos</given-names></name><xref ref-type="aff" rid="aff3">2</xref></contrib><contrib contrib-type="author"><name><surname>Díaz-Lobato</surname><given-names>Salvador</given-names></name><xref ref-type="aff" rid="aff4">3</xref><xref ref-type="corresp" rid="cor1"/></contrib> | Lung India : Official Organ of Indian Chest Society | <sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Endoscopic retrograde cholangiopancreatography (ERCP) with sedation and analgesia, performed by well-trained endoscopists, has proved to be a safe procedure, although significant complications may occur as a result of instrumentation or due to the effects of sedation and analgesia.[<xref rid="ref1" ref-type="bibr">1</xref><xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref3" ref-type="bibr">3</xref><xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref7" ref-type="bibr">7</xref>] These complications are more frequent when patients are deeply sedated, undergo complex or prolonged explorations, or undergo therapeutic procedures.[<xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref6" ref-type="bibr">6</xref>] Respiratory complications are very significant in high-risk patients. Respiratory insufficiency and clinical hypoventilation are common during the realization of ERCP for patients with hypoventilation risk factors, such as, elderly patients with chronic heart diseases, patients with a history of respiratory failure, patients who are morbidly obese, and patients with obesity-hypoventilation syndrome (OHS).[<xref rid="ref8" ref-type="bibr">8</xref><xref rid="ref9" ref-type="bibr">9</xref><xref rid="ref10" ref-type="bibr">10</xref>] Supplementary oxygen is usually indicated in these cases. Oxygen is also provided to patients with severe anemia and to patients in whom desaturation events are observed during exploration.[<xref rid="ref11" ref-type="bibr">11</xref><xref rid="ref12" ref-type="bibr">12</xref>] There is little evidence of noninvasive ventilation (NIV) preventing respiratory complications in high-risk patients undergoing endoscopy procedures, mainly percutaneous endoscopic gastrostomy.[<xref rid="ref13" ref-type="bibr">13</xref><xref rid="ref14" ref-type="bibr">14</xref><xref rid="ref15" ref-type="bibr">15</xref>] The purpose of our study is to assess the efficacy and safety of NIV through a nasal mask to prevent gasometric alterations in high-risk patients undergoing ERCP.</p></sec><sec sec-type="methods" id="sec1-2"><title>PATIENTS AND METHODS</title><p>Starting with the hypothesis that the application of NIV through a nasal interface could prevent the appearance of ventilatory alterations during ERCP in patients with risk factors associated with the development of hypoventilation, a non-randomized interventional study in 37 consecutive patients scheduled to undergo ERCP was proposed. The study was approved by the Ethical Committee of our institution and informed consent was obtained from all the study participants.</p><p>Patients who presented with at least one of the several previously defined risk factors believed to predispose the patient to ventilatory alterations in this context were included. An advanced age (>70 years), a body mass index (BMI) >35, a New York Heart Association (NYHA) functional class II–IV for congestive heart failure (CHF), prior ischemic heart disease history, and chronic obstructive pulmonary disease stage III–IV according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria (FEV<sub>1</sub> < 50%), were considered as risk factors.</p><p>The control group, comprising 21 patients, received oxygen through the conventional procedure at the flow rate usually used in our practice (nasal prongs, 3 L/minute) (Group 1). Subsequently, the other sixteen patients (experimental group) received oxygen plus NIV with a BiPAP-Synchrony Ventilatory Support System (Philips-Respironics<sup>®</sup>) ventilator through a Contour Deluxe nasal mask (Philips-Respironics<sup>®</sup>) (Group 2).</p><p>For sedation, in all cases, propofol was administered intravenously, with an initial dose of 0.5 mg/kg and subsequent bolus doses of 0.25 mg/kg, to maintain an adequate sedation level (grade 3-4 on the Ramsay scale). Simultaneously, meperidine was administered as an analgesic in a 50 mg intravenous dose.</p><p>Without disconnecting the patient from the oxygen source, two arterial blood gas analyses were performed, one before and the other immediately after the ERCP. The heart rate, respiratory rate, arterial blood pressure, oxygen saturation, and electrocardiographic trace were continuously monitored during the procedure.</p><p>Prior to the start of the exploration, based on the data from the medical history, each patient was assigned a score on the APACHE II scale in order to create groups with equal levels of severity. The duration of exploration was documented for patients of both groups.</p><p>Once the patient was placed on the ERCP table, a base arterial blood gas sample was obtained. Next, oxygen was initiated in the control group, and oxygen plus NIV through a nasal mask in the experimental group. In all cases the ventilator was programmed at 7 cm/H<sub>2</sub>O inspiratory pressure (IPAP) and 5 cm/H<sub>2</sub>O expiratory pressure (EPAP), with good tolerance to these pressures in all patients.</p><p>Heart rate and saturation (SaO<sub>2</sub>) were continuously monitored and noninvasive measurement of arterial pressure, every five minutes, was begun. Once the monitoring had started and after the patient received positive pressure, he/she was then placed in the left lateral decubitus position to start the procedure. Before introducing the duodenoscope, 50 mg meperidine and 0.5 mg/kg propofol was given. The endoscope was inserted through a glove finger fitted into a mouth guard. The system worked as a valve and did not affect the performance of the bronchoscopy procedure or the pressures administered during noninvasive ventilation.</p><p>Once the exploration started, the ventilator settings were reprogrammed to Average Volume Assured Pressure Support (AVAPS) ventilation, to ensure a minimum tidal volume of 500 ml, obtained by means of automatic adjustment of the ventilator's inspiratory positive airway pressure (IPAP). The IPAP range was set at a minimum of 7 and a maximum of 30 cm/H<sub>2</sub>O. The expiratory positive airway pressure (EPAP) was set at 5 cm/H<sub>2</sub>O. The respiratory rate was determined by the patient with a backup rate of 15. The AVAPS mode was chosen to guarantee the contribution of a volume minute with a backup rate of 15 bpm during the procedure, of about 7.5 liters. Every five minutes, simultaneously with the arterial pressure measurement, the level of sedation was assessed by means of the clinical Ramsay scale. Similarly, the tidal volume, propofol dose used, duration of the procedure, and potential adverse effects were documented.</p><p>Once the procedure was completed, a new arterial blood gas sample was obtained, while the ventilatory support continued. Once the ventilatory support was withdrawn, the patient's respiratory capacity and oxygen saturation level were assessed, together with the patient's capacity to respond to verbal, tactile, and pain stimulation. If the patient was capable of breathing by himself/herself and kept at >90% oxygen saturation, with the nasal cannula at 3 L/minute, together with a good response to tactile or verbal stimulation, the patient was transferred to a Recovery Room under a nurse's supervision.</p><sec id="sec2-1"><title>Statistical analysis</title><p>For the analysis of the results the statistical package SPSS 11.5 for Windows was used. The absolute and relative frequencies of discrete variables were calculated. Continuous variables were described in terms of mean, median, standard deviation, and interquartile range. Proportions of patients with pH < 7.35, pCO2 >45 or adverse events in each group were compared using the Chi-square or exact tests. Continuous variables were contrasted with the Student's <italic>t</italic>-test or Mann-Whitney test. Adjusted mean differences of pH and pCO2 between groups and their 95% confidence intervals were estimated by multiple linear regression by using, sex, APACHE score, pre-CPR pH, pCO2, age, propofol doses, and length of the procedure as covariates. The level of statistical significance was <italic>P</italic> < 0.05.</p></sec></sec><sec sec-type="results" id="sec1-3"><title>RESULTS</title><p>The two groups were comparable before the study in terms of age, APACHE score, and presence of hypoventilation risk factors. They presented baseline pH and pCO<sub>2</sub> values within the normal parameters. Both groups received comparable median propofol doses. However, the NIV group had a larger proportion of men than the nasal cannula group. The patient and pre-procedure characteristics are shown in <xref ref-type="table" rid="T1">Table 1</xref>.</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Baseline characteristics of the patients included in the study</p></caption><graphic xlink:href="LI-31-331-g001"/></table-wrap><p>Endoscopic retrograde cholangiopancreatography could be performed in 87.5 and in 95.2% of the cases, with and without NIV. The procedures lasted longer in the NIV group. Sphincterotomy was performed in 93.7 and 90.4% in each group, respectively. In the NIV group, the median pCO<sub>2</sub> post-ERCP was lower (36.5 vs. 44.5 mmHg; <italic>P</italic> = 0.001) and median pH post-ERCP was higher than in the control group (7.41 vs. 7.34, <italic>P</italic> = 0.001). These differences were maintained after performing a multivariate adjustment by gender, APACHE score, pH and pCO<sub>2</sub> pre-ERCP, age, propofol dose, and duration of procedure (difference of pCO<sub>2</sub> = 5.54, CI 95% =2.3-8.7, difference of pH = 0.047, CI 95% =0.013-0.081). The procedure outcomes are shown in <xref ref-type="table" rid="T2">Table 2</xref>.</p><table-wrap id="T2" position="float"><label>Table 2</label><caption><p>Outcomes related to the procedure. The procedures lasted longer in the NIV group. The propofol doses were similar in both groups. The main technique performed was sphincterotomy</p></caption><graphic xlink:href="LI-31-331-g002"/></table-wrap><p>A post-ERCP pH of <7.35 was found in eight patients who did not receive ventilatory support (38.1%) compared to zero patients in the NIV group (<italic>P</italic> = 0.006). A post-ERCP pCO<sub>2</sub> > 45 mmHg was found in one case (6.3%) in the nasal intermittent mandatory ventilation (NIMV) group and in nine cases in the nasal cannula group (42.9%; <italic>P</italic> = 0.01).</p><p>No significant differences were noted in terms of the number of complications associated with ERCP in the two groups: In the NIV group, one patient showed hypotension, while in the nasal cannula group there were two cases of cardiac disturbances: One case of first-degree atrioventricular block and one case of atrial fibrillation. A patient in this group presented with a post-sphincterotomy hemorrhage, which was controlled after sclerosis with 1/10.000 adrenaline. There were no complications related to NIV during the procedure.</p></sec><sec sec-type="discussion" id="sec1-4"><title>DISCUSSION</title><p>Our study has shown how in high-risk patients undergoing ERCP, hypercapnia and respiratory acidosis are common problems. In the control group 38.1% of patients presented with respiratory acidosis and a 42.9% had hypercapnia after ERCP procedure. Several studies have demonstrated that the use of sedation gives rise to a higher degree of success and a better acceptance of the exploration by the patient, as well as a global increase of the percentage of completed explorations and comfort for the endoscopist.[<xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref16" ref-type="bibr">16</xref><xref rid="ref17" ref-type="bibr">17</xref><xref rid="ref18" ref-type="bibr">18</xref>] However, the increasing use of sedation and analgesia techniques involves an increase in costs as well as morbidity and mortality rates. It is estimated that cardiopulmonary complications currently account for over 50% of all the complications associated with digestive endoscopy and are mainly attributable to the effects of sedation and secondary hypoventilation. They range from mild, temporary hypoxemic events to severe and potentially fatal cardiopulmonary disorders.[<xref rid="ref10" ref-type="bibr">10</xref><xref rid="ref19" ref-type="bibr">19</xref><xref rid="ref20" ref-type="bibr">20</xref>] In several recent series,[<xref rid="ref19" ref-type="bibr">19</xref><xref rid="ref20" ref-type="bibr">20</xref>] global morbidity associated with sedation in this context has been estimated between 6 and 54/10,000 patients, with a mortality between 0 and 5/10,000 patients.</p><p>Patients with a risk-profile (elderly, obese, chronic cardiac or bronchial disease, and III and IV ASA Grades) may suffer more respiratory or hemodynamic alterations, as we have seen in our study. Our patients were selected according to the above-mentioned risk criteria, and all of them were administered deep sedation (level 4-5 on the Ramsay scale) with propofol and meperidine. In the NIV group, this treatment was administered and supervised by two physicians from the Emergency Department of our hospital, well acquainted with the management of both drugs and experts in noninvasive ventilatory support and in cardiopulmonary resuscitation techniques.</p><p>As we have found in our study, NIV through a nasal mask plus oxygen prevented gasometric alterations in high-risk patients undergoing ERCP compared to the control group. Although they were considered high risk, all patients were normocapnic when they entred of the study. However, there was only one hypercapnic patient in the NIV group compared to nine hypercapnic patients in the control group after the procedure. Similar results were seen in relation to the number of patients with respiratory acidosis. Moreover, the NIV group presented no heart conduction disturbances, while in the nasal cannula group, two patients presented a first degree atrioventricular block and one presented with atrial fibrillation. These data suggest the NIV could be effective regardless of the values of PCO<sub>2</sub>. As the number of patients is too small, more studies are necessary to clarify these preliminary results.</p><p>Technological advances in NIV over the last decade have produced masks, with an exclusive nasal interface, to ensure efficient ventilation, which allows oral endoscopic explorations to be performed simultaneously. In this context, the application of preset and constant levels of positive pressure on the airway during inspiration and expiration could prevent the occurrence of blood gas alterations. Likewise, CO2 retention secondary to hypoventilation events could be prevented. There are technical issues related to oral air leaks during an endoscopic procedure. The efficacy of endoscopic procedure performed through the mouth with the aid of a mouth guard has been described. Similar to Chiner <italic>et al</italic>.,[<xref rid="ref21" ref-type="bibr">21</xref>] we placed the mouth guard inside a latex glove, which was then tied off using the conventional suture material around the outer surface of the guard, and a finger of the glove was left protruding from the central part. Once the glove had been tied off, the excess material was cut away and a small incision was made in the glove finger. Endoscopy was performed through this incision. Like other authors's experience, NIV was effective in our patients. As we have mentioned earlier, the system works as a valve and does not affect the performance of the bronchoscopy procedure or the pressures administered during noninvasive ventilation.</p><p>Current guidelines (2) recommend the administration of supplementary oxygen during prolonged procedures (ERCP, endoscopic ultrasonography) in those patients in whom hypoxemia could cause or exacerbate myocardial injuries (elderly, severe anemia, previous cardiopathy), or in patients with arterial oxygen desaturation (Grade of Recommendation A, Level of Evidence 1). We think this approach has to change. The results of our pilot study provide encouraging information about the efficacy and safety of NIV applied to deep sedation during ERCP. These data suggest the utilization of NIV associated with oxygen therapy in high-risk patients in order to prevent hypoventilation and appearance of respiratory acidosis.</p><p>This study has many limitations. First, it is not a randomized study and this clearly implies a selection bias. Nevertheless, we have studied 37 consecutive high-risk patients scheduled to undergo ERCP, and the group was homogeneous. Second, we have only analyzed the blood gas parameters. There is a doubt if the changes in these levels in the control group will likely result in complications during the procedure. Third, we do not know the real impact of NIV on a patient's morbidity or mortality. A surrogate outcome marker of arterial blood gases post the procedure does not predict decreased complications and it may have been interesting to repeat this after cessation of NIV, to see if applying ventilatory support during ERCP has any lasting effects. Anyhow, the main aim of this study was for it to be a pilot study that would open the door to larger studies.</p><p>In conclusion, the present, prospective, interventional study has shown that in high-risk patients undergoing ERCP, hypercapnia and respiratory acidosis are common problems. It provides evidence that NIV with bi-level ventilation can be an efficient technique to prevent cardiopulmonary complications during complex endoscopic procedures under deep sedation. It significantly reduces the occurrence of blood gas alterations in treated patients. The results of our pilot study provide encouraging information about the efficacy and safety of NIV applied to deep sedation during ERCP. Further studies are needed in order to confirm these preliminary results and obtain definitive conclusions on this subject.</p></sec> |
Multidrug-resistant-tuberculosis treatment in the Indian private sector: Results from a tertiary referral private hospital in Mumbai | <sec id="st1"><title>Background:</title><p>There is very limited data on the experience and outcome of multidrug-resistant tuberculosis (MDR-TB) patients treated privately out of the DOTS plus program. Goal of this study is to provide characteristics and treatment outcomes of a prospective cohort of MDR-TB patients managed at a private tertiary referral institute.</p></sec><sec id="st2"><title>Materials and Methods:</title><p>A prospective analysis of a cohort of MDR-TB patients treated in a tertiary private hospital, with the back-up of a Level 2 mycobacterial laboratory, which has recently received recognition by the Revised National Tuberculosis Control Program (RNTCP) for second-line drug susceptibility (DST). All patients received an individualized MDR regimen on an ambulatory basis.</p></sec><sec id="st3"><title>Results:</title><p>Our 68% success rates are respectable and show that given the right laboratory backing, MDR-TB can be managed successfully in selected private practice settings.</p></sec> | <contrib contrib-type="author"><name><surname>Udwadia</surname><given-names>Zarir F.</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Moharil</surname><given-names>Gautam</given-names></name><xref ref-type="aff" rid="aff1"/></contrib> | Lung India : Official Organ of Indian Chest Society | <sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Tuberculosis (TB) is a major threat to world health. After HIV/AIDS, it is the most common cause of death from an infectious disease worldwide. A major obstacle to TB control is the emergence of mycobacterial resistance to anti-tuberculous chemotherapy.[<xref rid="ref1" ref-type="bibr">1</xref>] Multidrug-resistant tuberculosis (MDR-TB), is caused by strains of <italic>Mycobacterium tuberculosis</italic> that are resistant to Isoniazid and Rifampicin. The outcome results of MDR-TB patients remain suboptimal. In a recent meta-analysis, the cure rate for MDR-TB patients was reported at about 54% to 64%.[<xref rid="ref2" ref-type="bibr">2</xref>]</p><p>Drug-resistant <italic>M. tuberculosis</italic> strains may account for 10% of the 8 million new cases of tuberculosis occurring annually worldwide. The latest WHO global resistance report, released in Oct. 2013, estimated that there were 79,000 cases of MDR-TB from India in 2012.[<xref rid="ref3" ref-type="bibr">3</xref>] This accounted for 17.6% of the world's MDR-TB burden. Between them, India and China accounted for 50% of global MDR-TB.</p><p>There is a paucity of information on the management of Indian MDR-TB patients from the private sector. In this study we present data regarding the epidemiology, clinical characteristics, treatment strategies and outcome of a cohort of MDR-TB patients from a tertiary care private hospital in the city of Mumbai.</p></sec><sec sec-type="materials|methods" id="sec1-2"><title>MATERIALS AND METHODS</title><p>The study was conducted in Mumbai in a tertiary referral center. Mumbai has a high MDR-TB burden.[<xref rid="ref4" ref-type="bibr">4</xref>]</p><p>This was a prospective observational descriptive study. We included consecutive patients having a microbiological diagnosis of MDR-TB presenting for consultation to a tertiary care private hospital in Mumbai from May 2006 to 2010. All patients had presented to a single chest consultant with a special interest in MDR-TB. Patients were only included for the study when cultures done at the mycobacterial lab of the hospital reconfirmed at least isoniazid and rifampicin resistance.</p><p>On their first visit, sputum culture and drug-sensitivity testing (DST) to first- and second-line drugs were sent off to the microbiology laboratory of our hospital. This is a level 2 mycobacterial laboratory which has recently received WHO recognition as an Intermediate Reference Laboratory (IRL). TB culture is performed by the liquid culture i.e. Mycobacterial Growth Indicator Tube (MGIT) method with DST also being performed via the same methods.[<xref rid="ref5" ref-type="bibr">5</xref>] The critical concentrations used for the MGIT DST of the anti-TB drugs tested (13 in all) are as follows: Streptomycin 1 mcg/ml; Isoniazid 0.1 mcg/ml; Rifampicin 1 mcg/ml; Ethambutol 5 mcg/ml; Kanamycin 2 mcg/ml; Ethionamide 5 mcg/ml; PAS 4 mcg/ml; Ofloxacin 2 mcg/ml; Moxifloxacin 0.25 mcg/ml; Amikacin 1 mcg/ml; Clofazamine 0.5 mcg/ml; Capreomycin 2.5 mcg/ml and Pyrazinamide 100 mcg/ml.</p><p>The patients were analyzed on their initial visit with regard to their age, sex, occupation, income and prior treatment history. They were all initially prescribed an empirical drug regimen containing at least four drugs they had not previously received while awaiting their sensitivity report. The patients returned for their second consult with the DST (usually 6 to 10 weeks later) and were then prescribed a final individualized regimen based on this report and a knowledge of their past drug history. Patients were not on DOTS as these were private patients.</p><p>Whenever possible, an average of 5 new/sensitive drugs was chosen. On subsequent visits, at 2-3 monthly intervals, the patients weight, side effects due to drugs and changes made to the therapy were recorded. Sputum smears were done on every visit and sputum cultures at 3 monthly intervals. The radiological status was assessed with a chest X ray on each visit with a CT scan being performed if surgery was being contemplated. All the patients included in the study gave written informed consent and the hospital IRB gave approval for the study on 29 April 2006.</p><p>Data was collected at every patient visit from the hospital records by a research registrar.</p><p>For qualitative data Pearson's Chi-square test was used to test the relationship of categorized dependent and independent variables. Normality of the data was checked by the Kolmogorov Smirnov test. The Mann Whitney U test was used to compare number of drugs with outcome. Statistics were all analyzed using SPSS 15.0 to analyze the data.</p></sec><sec sec-type="results" id="sec1-3"><title>RESULTS</title><p>A total 78 patients with MDR TB were included. The mean age was 29.7 years ranging from 15 to 70 years. Forty-four (56.4%) were females and 34 (43.6%) were males. The patients were from areas in and around Mumbai but cases were referred from other states of India including Gujarat, Assam and Uttar Pradesh. Fever was the most common symptom occurring in 77 (98.7%) patients. Cough was seen in 71 (91%) patients. Hemoptysis was recorded in 18 (23.1%) patients. Radiologically, 33 (42.3%) patients had unilateral disease while 42 (53.8%) had bilateral and advanced disease. This was a heavily pre-treated population of TB patients. The average duration of anti-tuberculous treatment received by the group as a whole prior to referral to us was 20 months (range 2-120 months). Thirteen (16.6%) patients had received anti-tuberculosis drugs for 3 or more years. The average duration of second-line drug therapy in patients who had previously received these drugs was 22 months (range 3-96 months).</p><p>While, by definition, 100% of patients were resistant to isoniazid and rifampicin, sensitivity to pyrazinamide was retained in 67 (87%) of patients. Several patients were also resistant to multiple second-line drugs, with 50% of patients being fluoroquinolone resistant (pre-XDR). 7% of patients met the revised WHO diagnosis of XDR-TB.[<xref rid="ref6" ref-type="bibr">6</xref>] Overall, the cohort was resistant to a mean of 4.6 drugs. The resistance pattern has been summarized in Figures <xref ref-type="fig" rid="F1">1</xref> and <xref ref-type="fig" rid="F2">2</xref>. A wide range of side effects was noted during therapy, some requiring discontinuation of the culprit drug as shown in <xref ref-type="table" rid="T1">Table 1</xref>. Surgical resection of the infected lobe or lung was carried out in eight (10.2%) patients. Two underwent lobectomies and six underwent pneumonectomy. Of the 78 patients included in this study, 53 (68%) completed treatment and were declared cured while 12 (15%) failed treatment and 13 (16%) defaulted. The statistical analysis of those variables that are historically considered significant enough to alter outcome was made. These are shown in <xref ref-type="table" rid="T2">Table 2</xref>.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Drug-specific resistance pattern</p></caption><graphic xlink:href="LI-31-336-g001"/></fig><fig id="F2" position="float"><label>Figure 2</label><caption><p>Number of drugs to which the patients were resistant: Patients had a mean resistance to 4.3 drugs</p></caption><graphic xlink:href="LI-31-336-g002"/></fig><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Frequency of drug usage and their side effects</p></caption><graphic xlink:href="LI-31-336-g003"/></table-wrap><table-wrap id="T2" position="float"><label>Table 2</label><caption><p>Statistical analysis of the variables associated with adverse outcome in the study</p></caption><graphic xlink:href="LI-31-336-g004"/></table-wrap></sec><sec sec-type="discussion" id="sec1-4"><title>DISCUSSION</title><p>India has a large and expanding population of MDR-TB patients. A DOTS-plus strategy has just been introduced in a phased manner in India and the seven year pilot experience has been encouraging.[<xref rid="ref7" ref-type="bibr">7</xref>] However, this still leaves uncovered the large numbers of MDR-TB patients who seek out private practitioners in an attempt to cure their MDR-TB. These private practitioners often are of varying standards, many of whom are inadequately qualified to deal with this problem. Their prescribing practice has received widespread censure and multiple treatment errors often served only to amplify drug resistance from MDR to XDR and beyond.[<xref rid="ref8" ref-type="bibr">8</xref>] There are no clear national guidelines or microbiological lab facilities at their disposal. A careful literature search revealed almost no data on how Indian private practitioners manage their MDR-TB patients and what this study attempts to provide such data from a tertiary referral private hospital, with strong microbiology laboratory back up where a good sized cohort were treated on an ambulatory basis.</p><p>We observed a high prevalence in patients in a younger age group. The mean age of our MDR-TB cohort was 29.1 years. Females were more represented in our study. People from the lower socioeconomic strata were more likely to be affected by the disease but no stratum of society was exempt. Our patients had more severe and extensive disease, usually bilateral. We also observed that bilateral disease was the most significant marker of treatment failure in our group of patients (<italic>P</italic> value 0.092). A past history of tuberculosis was seen in almost 75% of our patients. This was expected as ours being a tertiary care centre most of the patients were referred. Our patients were a heavily pre-treated group of chronic MDR-TB patients. They had consulted an average of 2.6 doctors prior to visiting our center. In many ways they are representative of the majority of MDR-TB patients in this country who see multiple private practitioners with poor compliance rates. Physician-related treatment errors lead to development of MDR-TB.[<xref rid="ref9" ref-type="bibr">9</xref>] Poorly prescribed second-line drugs only serve to amplify resistance and contribute to development of XDR-TB.</p><p>We feel access to a detailed DST from a reliable laboratory greatly contributed to our good outcomes. The use of individualized treatment, based on reliable DST is thus essential if these patents are to be successfully cured. Despite their poor resistance patterns we felt it was essential to offer each patient five to six new drugs in their regimen. Thus, Group 4 and newer drugs like linezolid were frequently resorted to in order to make up the required number of drugs essential for an effective regimen.</p><p>The recommended number of drugs used to treat MDR-TB is a subject of controversy. The ATS recommend four to six drugs for the treatment of MDR-TB.[<xref rid="ref10" ref-type="bibr">10</xref>] The WHO however recommends at least four drugs.[<xref rid="ref11" ref-type="bibr">11</xref>] The consensus is that more drugs may be required for more serious patients with previous use of second-line drugs or whose DST profile is adverse. Our patients were mainly referred, pre-treated patients and belonged to this category. The average number of drugs used in our patients was 5.5. We believe including more drugs may have contributed to our success rates.[<xref rid="ref12" ref-type="bibr">12</xref>]</p><p>Our study found seven patients to have extensively drug-resistant tuberculosis (XDR-TB) defined as cases where TB isolates were resistant to isoniazid, rifampicin as well as to any quinolone and any injectable second-line drugs (Kanamycin, capreomycin, amikacin).[<xref rid="ref6" ref-type="bibr">6</xref>] Thus, about 10% of our MDR-TB patients have XDR-TB, which is in keeping with prior data on XDR-TB from the country.[<xref rid="ref13" ref-type="bibr">13</xref>] The treatment principles applied to XDR-TB patients in our study were no different from MDR-TB and these patients had similar cure rates.</p><p>We observed high rates of resistance to streptomycin (75%) and fluoroquinolones (50%) in our patient population. Quinolones are broad-spectrum antibacterial agents which are the most widely prescribed group of antibiotics across India. Their widespread and indiscriminate use contributes to emergence of fluoroquinolone-resistant TB.[<xref rid="ref14" ref-type="bibr">14</xref>] Fluoroquinolone resistance in MDR-TB is an emerging problem globally[<xref rid="ref15" ref-type="bibr">15</xref>] and has recently been reported from our center in India.[<xref rid="ref16" ref-type="bibr">16</xref>] The indiscriminate use of fluoroquinolones needs to be checked to control the spread of MDR-TB and XDR-TB.[<xref rid="ref17" ref-type="bibr">17</xref>]</p><p>Every attempt was made to incorporate a quinolone in our treatment regimen as quinolone use has been identified to be a good marker of successful outcome.[<xref rid="ref18" ref-type="bibr">18</xref>] Among the quinolones, moxifloxacin was used the most frequently. The pharmacological profile of moxifloxacin with its relatively long half life and high area under the time-concentration curve suggests that this agent may be an ideal anti-TB drug.[<xref rid="ref19" ref-type="bibr">19</xref>] All our XDR-TB patients also received this drug despite (by definition) having ofloxacin resistance in their DST reports.</p><p>Susceptibility to PZA, PAS, Kanamycin and Ethionamide was retained in larger number of our TB isolates. Thus, if an empirical treatment regimen is planned (where culture and sensitivity is not available) these drugs should ideally be incorporated.</p><p>Linezolid has emerged as a useful drug for treatment of MDR TB with good <italic>in vitro</italic> susceptibility.[<xref rid="ref20" ref-type="bibr">20</xref>] We used linezolid in 18 of our patients. The use of linezolid did not make any significant difference to the outcome. We observed severe side effects in seven of these patients requiring discontinuation of the drug. One case of bilateral optic neuropathy and six cases of severe peripheral neuropathy were seen.</p><p>The use of amino glycosides has also been recommended as injectable therapy is known to be a predictor of positive outcome. We attempted to continue the aminoglycoside for an average of 6 months in our patient cohort. Surgery was recommended in only 8 patients (10.5%) The majority of our patients had extensive bilateral disease at presentation. The cost of surgery was also a constraining factor in a private hospital. Some studies have shown the positive impact surgery can have in the management of MDR-TB.[<xref rid="ref21" ref-type="bibr">21</xref>]</p><p>Our MDR-TB cohort was treated exclusively on an out-patient basis. The cost of hospitalizing patients for MDR-TB as practiced in the West would be prohibitive. Several studies have shown that MDR-TB can be successfully treated on an outpatient basis.[<xref rid="ref22" ref-type="bibr">22</xref>]</p><p>The CDC, ATS and WHO all recommend a direct observational therapy for treatment of MDR-TB. Whilst DOTS has had a tremendous positive impact on tuberculosis in general, applying DOTS with its daily, complex regimens extending to periods of up to 24 months would not be practical in a resource poor country like India. Hence, while every effort was made to stress and ensure good compliance, and as much as a DOT approach would have been preferred, our private patients took self-supervised treatment. Wherever possible we recruited a responsible and motivated family member to help supervise the regular administration of the drugs advised.</p><p>There is meager data on the treatment outcome of MDR TB patients from India. Almost all available data is from the public sector. Ours is the first large-sized study from the private sector. Subhash <italic>et al</italic>.[<xref rid="ref23" ref-type="bibr">23</xref>] analyzed data in 100 patients retrospectively. They found a high default rate (45 patients) with 31 patents showing clinical and 13 showing radiological response. Another study from TRC Chennai[<xref rid="ref24" ref-type="bibr">24</xref>] showed that with an individualized treatment strategy in 66 patients’ successful outcome was seen in 37% patients while 26% failed and 24% defaulted. A pilot study from the public sector has just been reported from New Delhi.[<xref rid="ref7" ref-type="bibr">7</xref>] This looked at DOTS-plus in an urban resource poor setting using DOTS-plus services under the existing RNTCP. This provided respectable cure rates of 61% in 126 MDR-TB patients.</p><p>A prospective study by Joseph <italic>et al</italic>. from Tamil Nadu (India) looked at the outcome of standardized treatment (RNTCP) for patients with MDR-TB, in 2007, included 38 MDR-confirmed patients and showed successful outcome in 66% (25/38).[<xref rid="ref25" ref-type="bibr">25</xref>]</p><p>A retrospective review of medical records of 11 adolescents enrolled between July 2007 and January 2013 was undertaken, in a Médecins Sans Frontières (MSF) project in Mumbai, India. Patients were initiated on either empirical or individualized second-line ambulatory anti-TB treatment under direct observation. Favorable results were seen in four (36.5%) patients: One was cured and three were still on treatment with negative culture results. Seven patients (64%) had poor outcomes: Four (36.5%) died and three (27%) defaulted.[<xref rid="ref26" ref-type="bibr">26</xref>]</p><p>Another retrospective study by Arora <italic>et al</italic>. analyzed the records of 66 patients with MDR-TB treated with a fully supervised standardized treatment regimen. Among 28 patients completing 2 years of treatment, 67.9% were cured, 14.3% died, 17.9% defaulted.[<xref rid="ref27" ref-type="bibr">27</xref>]</p><p>In a prospective study by Dhingra <italic>et al</italic>., 27 MDR-TB patients were included, from August 2002 to December 2004 at New Delhi Tuberculosis Centre and according to DST individualized treatment regimens were tailored. Of the 27 patients, 13 were cured and 10 defaulted. Radiological improvement was observed in two third of cases.[<xref rid="ref28" ref-type="bibr">28</xref>]</p><p>In a cross-sectional study, conducted by Jana <italic>et al</italic>., in West Bengal, between January 2003 and January 2008, out of 1487 TB patients, 31 MDR-TB patients were identified and treated. Successful outcome was seen in 64.51%, relapse in 12.90%, treatment failure was seen in 19.35%.[<xref rid="ref29" ref-type="bibr">29</xref>]</p><p>Despite the absence of DOT the number of patients who dropped out of our study was no more than had been reported in other studies. Our treatment success rate of almost 70% was equivalent to other Indian studies and is comparable to other international studies. We feel the two possible reasons behind our better success rates could be that all patients were under the care of a single physician with a special interest in MDR-TB and that our hospital mycobacterial laboratory has been reliably performing DST with good quality control over the last decade. Indeed it has recently been recognized as the sole Intermediate Reference Laboratory (IRL) for the city of Mumbai by WHO.</p></sec><sec sec-type="conclusion" id="sec1-5"><title>CONCLUSION</title><p>India houses one of the largest MDR-TB populations in the world. Till the government sponsored DOTS-plus programme expands, these unfortunate patients have very limited treatment options. Most Indian MDR-TB patients are compelled to visit private practitioners of varying standards in desperate attempts to cure their disease. This study shows that treatment of MDR-TB can be successful on an outpatient basis even in a resource-limited country like India. If managed outside the national programme, it should be undertaken in an experienced private center with sound mycobacterial laboratory back-up. There is a paucity of data available on the treatment offered in the private sector to Indian MDR-TB patients. This study is an attempt to fill that gap. Our patient cohort was managed under optimal Indian conditions in a private hospital with an excellent laboratory backup. We hope these results inspire other Indian centers to take on the challenge of treating this disease and reporting their results in large numbers.</p></sec> |
Correlation of exhaled nitric oxide, nasal nitric oxide and atopic status: A cross-sectional study in bronchial asthma and allergic rhinitis | <sec id="st1"><title>Objective:</title><p>Exhaled nitric oxide (FE<sub>NO</sub>) and nasal nitric oxide (n NO) measurement is an area of ongoing research in the study of airway inflammation. The atopic status is known to influence the levels of FE<sub>NO</sub> and n NO. This study was undertaken to study the relationship between nitric oxide measurements in bronchial asthma and allergic rhinitis along with their correlation with atopic profile of Indian population.</p></sec><sec id="st2"><title>Materials and Methods:</title><p>Ninety subjects were recruited for the study comprising of 25 each of bronchial asthma (BA), allergic rhinitis (AR), bronchial asthma with allergic rhinitis (BA-AR) and 15 healthy controls. These were assessed for atopy and exhaled breath analysis of nitric oxide. The measurements of FE<sub>NO</sub> and n NO levels were done using NIOX chemiluminescence analyzer. Atopy was assessed by skin prick testing (SPT) against 58 common aero-allergens and subjects with ≥1 positive SPT were labeled as atopic.</p></sec><sec id="st3"><title>Results:</title><p>The BA-AR and BA groups had higher FE<sub>NO</sub> levels in comparison to the control (<italic>P</italic> < 0.05) and AR group (<italic>P</italic> < 0.05). The AR and BA-AR groups had higher n NO levels compared to the control group (<italic>P</italic> < 0.05) and BA group (<italic>P</italic> < 0.05). The increasing FE<sub>NO</sub> levels significantly correlated with the increase in the number of allergen sensitization in patients suffering from BA-AR (<italic>P</italic> < 0.05). However, the BA group showed a weaker positive correlation (<italic>P</italic> = 0.07).</p></sec><sec id="st5"><title>Conclusion:</title><p>FE<sub>NO</sub> is a non-invasive marker of airway inflammation. Also, FE<sub>NO</sub> levels correlate with presence and degree of atopy in BA and AR. Simultaneously, n NO could be a surrogate marker of rhinitis.</p></sec> | <contrib contrib-type="author"><name><surname>Gupta</surname><given-names>Nitesh</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Goel</surname><given-names>Nitin</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Kumar</surname><given-names>Raj</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib> | Lung India : Official Organ of Indian Chest Society | <sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>The nitric oxide (NO) in the lung/airways has a key role as a vasodilator, bronchodilator, neurotransmitter and inflammatory mediator.[<xref rid="ref1" ref-type="bibr">1</xref><xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref3" ref-type="bibr">3</xref><xref rid="ref4" ref-type="bibr">4</xref>] Exhaled nitric oxide (FE<sub>NO</sub>) and nasal nitric oxide (n NO) measurement is an area of ongoing research in the study of airway inflammation.</p><p>The FE<sub>NO</sub> level measurement has been validated and standardized for supporting the diagnosis in cases of eosinophilic inflammation of airways, bronchial hyperreactivity and asthma.[<xref rid="ref5" ref-type="bibr">5</xref>]</p><p>FE<sub>NO</sub> levels have been higher in atopic than non-atopic bronchial asthma patients and some studies also reported that healthy atopic subjects without symptoms or signs of airway disorders have higher FE<sub>NO</sub> levels than non-atopic subjects.[<xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref7" ref-type="bibr">7</xref>] Similarly the effect of clinical atopy, with atopic cases having higher levels as compared to non-atopic cases.[<xref rid="ref4" ref-type="bibr">4</xref>]</p><p>To the best of our knowledge, the literature on exhaled breath and nasal nitric oxide from India is lacking. Hence, this study was undertaken to answer the question about the relationship between the noninvasive methods of nitric oxide measurements in bronchial asthma and allergic rhinitis and their correlation with atopic profile of Indian population.</p></sec><sec sec-type="materials|methods" id="sec1-2"><title>MATERIALS AND METHODS</title><sec id="sec2-1"><title>Study design and demographics</title><p>The diagnosed patients of bronchial asthma (BA), allergic rhinitis (AR) and bronchial asthma with allergic rhinitis (BA-AR) were enrolled for the study from the outpatient clinics. A total of 90 subjects (36 females and 54 males) aged between 6-38 years were evaluated and they were divided into 4 groups: Group A - 25 Bronchial asthma (without allergic rhinitis); Group B - 25 Allergic rhinitis (without bronchial asthma); Group C - 25 Bronchial asthma with allergic rhinitis and Group D - 15 controls. Patients from all the 4 groups were further subdivided into atopic and non-atopic subgroups based on the skin prick test results for the purpose of analysis. The diagnosis of asthma and allergic rhinitis were based on Global Initiative for Asthma (GINA)[<xref rid="ref8" ref-type="bibr">8</xref>] and Allergic Rhinitis and its Impact on Asthma (ARIA) [2008][<xref rid="ref9" ref-type="bibr">9</xref>] respectively. The subjects with inability to satisfactorily perform the nitric oxide maneuver were excluded. Other exclusion criterion were 1) Smoker (Former and current smokers) 2) Inhaled/nasal/oral steroid intake in preceding one month 3) Episode of upper or lower respiratory tract infection in the preceding one month and 4) History of urticaria/eczema. All the 90 subjects underwent a battery of investigations including baseline spirometry, FE<sub>NO</sub> and n NO measurements, skin prick tests and blood sampling, absolute eosinophil counts and serum total IgE levels.</p><p>Written informed consent was obtained from all subjects/parents (in case of subject's age <18 years) to participate in the study. The study protocol was approved by institutional ethical committee.</p></sec><sec id="sec2-2"><title>Measurement of FE<sub>NO</sub> and n NO</title><p>The measurements of exhaled nitric oxide and nasal nitric oxide was performed using NIOX chemiluminescence analyzer (Aerocrine AB, Solna, Sweden) in accordance with the 2005 ATS/ERS recommendations.[<xref rid="ref10" ref-type="bibr">10</xref>]</p><p>The patient was inserted the mouthpiece, inhaled through mouth to total lung capacity (TLC) and then immediately exhaled at a constant flow rate (50 mL/s) to residual volume without breath-holding. The duration of exhalation had to be sufficient (>4 seconds in subjects <12 years and >6 seconds in subjects >12 years). Repeated, reproducible exhalations were performed to obtain at least two NO plateau values that agreed with in 10% of each other. The mean level of two reproducible recordings was used as the result value. The n NO measurement was done using the same analyzer by the nasal aspiration method. The measurements were always performed at the same place and the sampling timing was between 10-11 am.</p></sec><sec id="sec2-3"><title>Skin prick testing</title><p>Skin prick testing (SPT) to 58 common aeroallergens was performed in all the patients as per standard guidelines.[<xref rid="ref11" ref-type="bibr">11</xref>] These are the commonest aeroallergens in clinical practice of allergy in India as per the study by Singh <italic>et al</italic>.[<xref rid="ref12" ref-type="bibr">12</xref>] Atopy was defined as a positive skin prick test (wheal diameter of >3 mm as compared to buffer saline as control) for at least ≥1 aeroallergen.[<xref rid="ref11" ref-type="bibr">11</xref>]</p></sec><sec id="sec2-4"><title>Measurement of serum total IgE and absolute eosinophil counts</title><p>Serum Total IgE was estimated by ELISA method using MINILYSER - TECAN, Austria, Calbiotech kit as per manufacturer's instructions. The number of peripheral blood eosinophils was counted in EDTA containing blood samples using an automated analyzer.</p></sec><sec id="sec2-5"><title>Spirometry with reversibility</title><p>Spirometry was performed on a dry, rolling-seal spirometer of the Benchmark model lung function machine (P.K. Morgan, Kent, UK). Maximal expiratory flow volume curves were obtained as per the ATS recommendations.[<xref rid="ref13" ref-type="bibr">13</xref>]</p></sec><sec id="sec2-6"><title>Statistical analysis</title><p>All data analysis was performed using SPSS statistical package version 16.0 for windows (SPSS, Chicago, IL, USA). The data was examined for distribution and homogeneity of variances was checked before applying parametric tests. The data on FE<sub>NO</sub> and n NO were expressed as mean ± SD. The variables were compared between the four groups using one-way analysis of variance (ANOVA) and the <italic>post hoc</italic> Bonferroni alpha significant difference test for multiple comparisons. The univariate analyses of factors associated with FE<sub>NO</sub> was done using Pearson correlation. The conventional 5% level (<italic>P</italic> < 0.05) was considered to be statistically significant.</p></sec></sec><sec sec-type="results" id="sec1-3"><title>RESULTS</title><p>The demographic characteristics of all the four groups are shown in <xref ref-type="table" rid="T1">Table 1</xref>. Of the total 90 subjects, 54 were males and rests of 36 subjects were females. Overall there were 54 atopic and 36 non-atopic patients in the study. The mean ages, age distribution, anthropometric variables (height, weight, BMI) were normally distributed amongst all the groups.</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Patient demographics and laboratory investigations</p></caption><graphic xlink:href="LI-31-342-g001"/></table-wrap><sec id="sec2-7"><title>Exhaled nitric oxide (FE<sub>NO</sub>)</title><p>The mean levels of FE<sub>NO</sub> in all the four groups are depicted in <xref ref-type="table" rid="T1">Table 1</xref>. All the three groups had higher FE<sub>NO</sub> levels compared to the control group [<xref ref-type="table" rid="T1">Table 1</xref>]. The BA and BA-AR groups had significantly higher levels than the AR group [<xref ref-type="fig" rid="F1">Figure 1a</xref>]. Also, the BA-AR group had higher FE<sub>NO</sub> levels when compared to the BA group although the comparison failed to reach statistical significance.</p><fig id="F1" position="float"><label>Figure 1a</label><caption><p>Intergroup comparison of FENO levels ppb (mean ± SD); <italic>P</italic> < 0.05 – statistically significant; NS- Not significant; BA- Bronchial asthma; AR- Allergic rhinitis: BA-AR- Bronchial asthma with allergic rhinitis</p></caption><graphic xlink:href="LI-31-342-g002"/></fig></sec><sec id="sec2-8"><title>Nasal nitric oxide (n NO)</title><p>The mean levels of n NO in all the four groups are shown in <xref ref-type="table" rid="T1">Table 1</xref>. The AR group and BA-AR group had significantly higher n NO levels as compared to the control and BA groups [<xref ref-type="fig" rid="F2">Figure 1b</xref>]. However, the BA group had n NO levels lower than the control group.</p><fig id="F2" position="float"><label>Figure 1b</label><caption><p>Intergroup comparison of n NO levels ppb (mean ± SD); <italic>P</italic> < 0.05 – statistically significant; NS- Not significant; BA- Bronchial asthma; AR- Allergic rhinitis: BA-AR- Bronchial asthma with allergic rhinitis</p></caption><graphic xlink:href="LI-31-342-g003"/></fig></sec><sec id="sec2-9"><title>Atopic profile and nitric oxide levels</title><p>The control group had 8 atopic subjects and 7 non-atopic subjects. The FE<sub>NO</sub> levels between the atopic and non-atopic control subjects did not differ significantly (<italic>P</italic> = 0.771). The atopic patients suffering from BA, AR and BA-AR had statistically significant higher levels of FE<sub>NO</sub> in comparison to non-atopic patients of same group [<xref ref-type="fig" rid="F3">Figure 2</xref>]. However, in comparison the mean n NO levels did not differ significantly; between the atopic and non-atopic patients in all the four groups.</p><fig id="F3" position="float"><label>Figure 2</label><caption><p>Intragroup comparison of FENO levels between atopic and non-atopic patients; <italic>P</italic> < 0.05 - statistically significant BA- Bronchial asthma; AR- Allergic rhinitis: BA-AR- Bronchial asthma with allergic rhinitis</p></caption><graphic xlink:href="LI-31-342-g004"/></fig></sec><sec id="sec2-10"><title>Number of positive responses on skin prick test and nitric oxide levels</title><p>The patients in all the four groups were further subdivided into 3 sub-groups on the basis of number of positive responses on SPT; 0, 1-3 and >3. The FE<sub>NO</sub> and n NO levels were analyzed in these subgroups [<xref ref-type="table" rid="T2">Table 2</xref>]. The FE<sub>NO</sub> levels positively and significantly correlated with the number of positive responses on SPT in patients suffering from BA-AR (<italic>P</italic> < 0.05). The BA group and AR group also showed a positive correlation albeit a weaker one (<italic>P</italic> = 0.07). However, no significant correlation of n NO levels and number of positive response on SPT were found in any of the four groups.</p><table-wrap id="T2" position="float"><label>Table 2</label><caption><p>Correlation of FE<sub>NO</sub> and n NO with SPT positivity of number of allergens</p></caption><graphic xlink:href="LI-31-342-g005"/></table-wrap></sec><sec id="sec2-11"><title>Serum total IgE, AEC, spirometry and nitric oxide levels</title><p>The serum total IgE and AEC levels were significantly higher in BA, AR and BA-AR groups when compared to the control group. However, no significant difference was found in their levels in atopic and non-atopic patients in each of the four groups. In the study, overall FE<sub>NO</sub> levels correlated significantly with serum total IgE levels (<italic>P</italic> = 0.002), however n NO levels were not significantly correlated with serum total IgE levels (<italic>P</italic> = 0.254). The AEC levels also correlated positively and significantly with FE<sub>NO</sub> levels (<italic>P</italic> = 0.004) and n NO levels (<italic>P</italic> = 0.01). The PFT parameters FEV<sub>1</sub>, FVC and FEV<sub>1</sub>/FVC did not show a significant correlation with either FE<sub>NO</sub> or n NO levels.</p></sec></sec><sec sec-type="discussion" id="sec1-4"><title>DISCUSSION</title><p>Exhaled nitric oxide (FE<sub>NO</sub>) measurement is non-invasive, simple and well tolerated method and is now used as a clinical biomarker for assessment of airway inflammation.[<xref rid="ref5" ref-type="bibr">5</xref>] Similarly, nasal nitric oxide (n NO) measurement is useful in the diagnosis, treatment and follow-up of patients with nasal pathology, especially because as it is noninvasive.[<xref rid="ref14" ref-type="bibr">14</xref>]</p><p>In the present study we have demonstrated that healthy individuals had mean FE<sub>NO</sub> levels of 12.73 ± 7.8 ppb whereas other studies on Chinese, African population and data from asthma and allergy research group had mean FE<sub>NO</sub> levels ranging from 20-39ppb.[<xref rid="ref15" ref-type="bibr">15</xref><xref rid="ref16" ref-type="bibr">16</xref><xref rid="ref17" ref-type="bibr">17</xref><xref rid="ref18" ref-type="bibr">18</xref>] In a study by Ciprandi <italic>et al</italic>.,[<xref rid="ref19" ref-type="bibr">19</xref>] the children suffering from asthma and rhinitis had mean FE<sub>NO</sub> levels of 41 ppb, with asthma had 37 ppb and those with rhinitis had 31 ppb (<italic>P</italic> < 0.001). We also found that FE<sub>NO</sub> levels were highest in BA-AR group (41.44 ± 29.9 ppb) followed by BA group (34.96 ± 17.2 ppb) and AR group (16.40 ± 10.3 ppb). In agreement with the literature, we observed higher FE<sub>NO</sub> levels in BA and BA-AR groups in comparison to the control group (<italic>P</italic> < 0.05) and AR group (<italic>P</italic> < 0.05). No statistical difference was found among any other groups for FE<sub>NO</sub> levels. The findings are consistent with previous studies.[<xref rid="ref19" ref-type="bibr">19</xref><xref rid="ref20" ref-type="bibr">20</xref>]</p><p>The measurement of nasal inflammation in AR would rely on detecting increased levels of nasal NO resulting from increased expression of iNOS (inducible nitric oxide synthase), akin to the increase of exhaled nitric oxide levels in bronchial asthma.[<xref rid="ref21" ref-type="bibr">21</xref>] In a study by Stewart <italic>et al</italic>.,[<xref rid="ref17" ref-type="bibr">17</xref>] the mean n NO levels of 853.3 ppb in AR and 763.4 ppb in BA-AR groups were higher in comparison to 674.1 ppb levels in BA group. In our study also the n NO levels in AR (271.44 ± 120.3 ppb) and BA-AR groups (336.42 ± 124.6 ppb) were significantly higher in comparison to BA group (100.58 ± 111.2 ppb) and control group (114.50 ± 76.0 ppb). The above findings are attributed to increase in local production of nitric oxide in nasal epithelium in patients suffering from allergic rhinitis.[<xref rid="ref17" ref-type="bibr">17</xref><xref rid="ref21" ref-type="bibr">21</xref>] We did not find any statistical difference among any other groups for n NO levels. These results were in agreement with the previous studies by Kharitonov <italic>et al</italic>.,[<xref rid="ref22" ref-type="bibr">22</xref>] and Lee <italic>et al</italic>.[<xref rid="ref23" ref-type="bibr">23</xref>]</p><p>The effect of presence and degree of clinical atopic conditions on FE<sub>NO</sub> levels has been addressed in the literature. The higher FE<sub>NO</sub> levels in atopic asthmatics has been documented, however the effect of atopy on FE<sub>NO</sub> levels of healthy subjects requires further validation.[<xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref24" ref-type="bibr">24</xref>] Rouhos <italic>et al</italic>.,[<xref rid="ref25" ref-type="bibr">25</xref>] in his study showed atopic constitution, defined as positive skin prick test results,which does not increase FE<sub>NO</sub> levels in healthy nonsmoking adults with no signs or symptoms of airway disorders. Similar to this study, we also found no significant difference in FE<sub>NO</sub> levels with respect to atopic status in the control group.</p><p>In the present study the atopic BA, AR and BA-AR subjects had significantly higher FE<sub>NO</sub> levels in comparison to non-atopic subjects of the same group. Previous studies by Jouvaville <italic>et al</italic>.[<xref rid="ref18" ref-type="bibr">18</xref>] and Gratziou <italic>et al</italic>.[<xref rid="ref26" ref-type="bibr">26</xref>] have also found the similar result. In a study by Kumar <italic>et al</italic>.[<xref rid="ref27" ref-type="bibr">27</xref>] the atopic allergic rhinitis subjects had statistically significant higher FE<sub>NO</sub> levels when compared to non-atopic subjects. Similar result was observed in the present study and this supports the hypothesis of a presence of subclinical inflammation of lower airways, and may predict development of asthma in future.</p><p>We found higher levels of nNO in atopic subjects of BA, AR and BA-AR in comparison to non-atopic subjects; though the relationship was statistically not significant. Olin <italic>et al</italic>.[<xref rid="ref6" ref-type="bibr">6</xref>] and Kharitonov <italic>et al</italic>.[<xref rid="ref4" ref-type="bibr">4</xref>] in their studies observed similar results.</p><p>The association between the degree of atopy and FE<sub>NO</sub> levels has been studied by Ho <italic>et al</italic>.[<xref rid="ref28" ref-type="bibr">28</xref>] and Strunk <italic>et al</italic>.,[<xref rid="ref29" ref-type="bibr">29</xref>] and they concluded that as number of positive responses on skin prick test increases, the FE<sub>NO</sub> levels also increases. We also found the similar positive correlation between FE<sub>NO</sub> levels and number of positive responses on SPT in BA-AR group (<italic>P</italic> < 0.05). Similarly, atopic BA subjects also showed a positive correlation albeit a weaker one (<italic>P</italic> = 0.07). The plausible explanation for this finding has been attributed to difference of inflammatory cells recruitment in atopics (eosinophilic) and non-atopic asthmatics (neutrophilic) as well as to cell activity of NO producing cells.[<xref rid="ref18" ref-type="bibr">18</xref>] However, in a study by Moore <italic>et al</italic>.,[<xref rid="ref30" ref-type="bibr">30</xref>] in asthmatics FE<sub>NO</sub> was not always associated with the number of positive skin prick test response. Thus, there exists a complex relationship between atopic profile and FE<sub>NO</sub> levels which requires further evaluation. Also, we found no significant association between n NO levels and the number of allergens positive on SPT in any of the groups of our study.</p><p>The higher FE<sub>NO</sub> and n NO levels in atopic subjects could be attributed to induction of iNOS enzyme. iNOS is the characteristic enzyme found in association with mucosal mast cells, eosinophils, and T-lymphocyte activation as described in cases of allergic rhinitis and bronchial asthma.[<xref rid="ref31" ref-type="bibr">31</xref><xref rid="ref32" ref-type="bibr">32</xref>] In bronchial biopsies of asthmatics an up regulation of expression of iNOS has been observed and immuno-cytochemical studies have also demonstrated expression of NOSs in human nasal mucosa.[<xref rid="ref33" ref-type="bibr">33</xref><xref rid="ref34" ref-type="bibr">34</xref><xref rid="ref35" ref-type="bibr">35</xref>]</p><p>The FE<sub>NO</sub> and n NO levels are known to be influenced by age, sex, height, smoking and atopy status of the individual.[<xref rid="ref5" ref-type="bibr">5</xref>] In current study, all the groups (AR, BA, BA with AR and Controls) were comparable with respect to all these factors except the atopic status. Hence, we could evaluate correlation of atopic status with FE<sub>NO</sub> and n NO levels. This was the strength of our study.</p><p>The work is limited by the small number of subjects enrolled. Hence, a further large-scale population based study is required for assessing applicability of FE<sub>NO</sub> and n NO levels in evaluation of BA and/or AR patients.</p><p>FeNO and nNo may serve as non-invasive marker of airway inflammation and atopy. Hence, they can be used for early recognition of airway inflammation and also as a guide for follow-up and management of BA and AR. This requires further large scale studies to validate the levels of FE<sub>NO</sub> and n NO and simultaneously confirm the correlation with atopic status.</p></sec><sec sec-type="conclusions" id="sec1-5"><title>CONCLUSIONS</title><p>In conclusion, the FE<sub>NO</sub> levels reflect the inflammatory activity of the airway epithelium and is associated with presence as well as degree of atopy in a sensitized patient, determined by positive skin prick test results to common aeroallergens. The study also highlights the need for evaluation of patient for the existence of allergic rhinitis or bronchial asthma in cases with higher FE<sub>NO</sub> levels. The n NO levels can be used as an inflammatory marker for supporting the diagnosis of coexisting allergic rhinitis in bronchial asthma patients. Further, large scale studies are required to develop reference equations and cutoff values for FE<sub>NO</sub> and n NO levels to aid in diagnosis and follow-up in Indian population.</p></sec> |
A comparative study of different dose fractionations schedule of thoracic radiotherapy for pain palliation and health-related quality of life in metastatic NSCLC | <sec id="st1"><title>Introduction:</title><p>To investigate the effect of different hypo fractionated thoracic radiotherapy schedules in relation to thoracic pain relief, overall survival and post radiotherapy HRQOL in metastatic NSCLC.</p></sec><sec id="st2"><title>Material and methods:</title><p>Stage IV NSCLC and had intra-thoracic symptoms, included in the study. Patients were randomly assigned to three treatments arms. (i) 17 Gy in 2 fractions in one week (ii) 20 Gy in five fractions in one week. (iii) 30 Gy in 10 fractions in two weeks. BPI module was used to assess pain score before and after the thoracic radiotherapy. Functional assessment of cancer therapy-G (FACT-G) used to investigate changes in HRQOL. Clinicians’ assessment of symptom improvement were recorded at 2<sup>nd</sup>, 6<sup>th</sup> and 12<sup>th</sup> weeks after completion of TRT.</p></sec><sec id="st3"><title>Results:</title><p>Pain relief, HRQOL and OS were equivalent in all the three arms. The median OS were 6 months, 5 months, 6 months in arm A, B and arm C, respectively.</p></sec><sec id="st4"><title>Conclusion:</title><p>Protracted palliative thoracic radiotherapy renders no added advantage of relief of symptoms, HRQOL and overall survival compared to short course palliative TRT in metastatic NSCLC.</p></sec> | <contrib contrib-type="author"><name><surname>Sau</surname><given-names>Sourav</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Sau</surname><given-names>Saikat</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib><contrib contrib-type="author"><name><surname>Dutta</surname><given-names>Premnath</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Gayen</surname><given-names>Ganesh Chandra</given-names></name><xref ref-type="aff" rid="aff3">2</xref></contrib><contrib contrib-type="author"><name><surname>Banerjee</surname><given-names>Sanatan</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Basu</surname><given-names>Avijit</given-names></name><xref ref-type="aff" rid="aff4">3</xref></contrib> | Lung India : Official Organ of Indian Chest Society | <sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Metastatic non-small cell lung carcinoma (NSCLC) usually present with symptoms from the primary intra-thoracic tumor including dyspnea, chest pain, cough, and hemoptysis.[<xref rid="ref1" ref-type="bibr">1</xref>] Thoracic palliative radiotherapy is an effective modality of treatment in relieving symptoms.[<xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref3" ref-type="bibr">3</xref>] Patients with metastatic NSCLC are not suitable for curative treatment and yield a poor prognosis with limited survival. Consequently, a limited treatment period is usually advocated, if adequate palliative effect is attained. Hypo fractioned thoracic radiotherapy (TRT) may fulfill this criterion. Multiple randomized studies favor hypo fractionated treatment policy[<xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref7" ref-type="bibr">7</xref>] and others advise against hypo fractionated treatment because of increased toxicity.[<xref rid="ref8" ref-type="bibr">8</xref><xref rid="ref9" ref-type="bibr">9</xref><xref rid="ref10" ref-type="bibr">10</xref><xref rid="ref11" ref-type="bibr">11</xref>]</p></sec><sec sec-type="materials|methods" id="sec1-2"><title>MATERIALS AND METHODS</title><sec id="sec2-1"><title>Patients</title><p>182 eligible patients were included in this trial between July 2007 and March 2012. Patients were recruited from the department of radiation oncology, Medical College and Hospitals, Kolkata and the department of radiation oncology, Burdwan Medical College and Hospital, Burdwan.</p><p>Inclusion criteria of the eligible patients were:</p><p>
<list list-type="bullet"><list-item><p>Histologicaly or cytologically confirmed NSCLC</p></list-item><list-item><p>Aged over 18 years</p></list-item><list-item><p>Stage IV</p></list-item><list-item><p>Performance status (PS) ≤ Eastern Cooperative Oncology Group (ECOG) 3</p></list-item><list-item><p>Expected survival more than 3 months</p></list-item><list-item><p>Patients had intra-thoracic symptoms</p></list-item><list-item><p>No history of previous TRT.</p></list-item></list>
</p><p>Written informed consent was taken from all the patients before random assignment to palliative TRT.</p><p>Eligible patients were assigned randomly to one of the three treatment arms with the help of blind envelope method. Twelve patients were ineligible because of incorrect stage and patient's refusal/terminated their palliative radiotherapy prematurely. Significantly, higher number of patients in arm C terminated their planned palliative TRT. All the patients were assessed on an intention to treat basis. Baseline patients’ characteristics are presented in <xref ref-type="table" rid="T1">Table 1</xref>. Fourteen patients died within 3 months of post radiotherapy. All these patients were excluded from statistical analysis.</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Baseline characteristic of all eligible patients according to assigned treatment arms</p></caption><graphic xlink:href="LI-31-348-g001"/></table-wrap><p>All the patients were subjected to the following diagnostic work up: Clinical examination, radiological examination (chest X-rays, CECT thorax, and abdomen), and blood chemistry profile. ECOG PS was recorded for every patient before thoracic palliative radiotherapy. CECT or magnetic resonance imaging (MRI) brain and whole body bone scan were performed whenever indicated by symptoms.</p></sec><sec id="sec2-2"><title>Thoracic radiotherapy</title><p>TRT administered with parallel oppose AP/PA fields encompassing the primary tumor with 1.5-2 cm margin using Co<sup>60</sup> beams. Mediastinal and supraclavicular region were not routinely included in the portals unless enlarged node caused symptoms. Radiotherapy fractionated scheme are as follows:</p><p>
<list list-type="bullet"><list-item><p>Arm A: 17 Gy in 2 fractions in one week</p></list-item><list-item><p>Arm B: 20 Gy in 5 fractions in one week</p></list-item><list-item><p>Arm C: 30 Gy in 10 fractions in two weeks.</p></list-item></list>
</p><p>To prevent possible side effects, patients were prophylactically treated with prednisolone 50 mg once daily as indicated.</p><p>After completion of TRT, significant percentage of patients received palliative systemic therapy according to their PS and histopathology. Only six patients in arm A and two patients in arm B were re-irradiated to the chest because of bone metastasis. Forty-five patients received palliative radiotherapy outside the chest because of bone and brain metastasis.</p></sec><sec id="sec2-3"><title>Health-related quality of life</title><p>Consecutive patients requiring palliative TRT for metastatic NSCLC enrolled in longitudinal assessment of health-related quality of life (HRQoL) study at their first visit in the department of radiation oncology in Medical College and Hospitals, Kolkata and Burdwan Medical College and Hospital, Burdwan. Patients were followed up and HRQoL was assessed with the FACT-G questionnaire (FACT-G, Bengali format) before the radiotherapy, 2<sup>nd</sup>, 6<sup>th</sup>, and 12<sup>th</sup> week after completion of palliative TRT.[<xref rid="ref12" ref-type="bibr">12</xref>]</p><p>FACT-G, a 27-item general module and is grouped into the four domains: Physical, functional, social well-being (each with seven items), emotional well-being (six items).[<xref rid="ref13" ref-type="bibr">13</xref>]</p><p>The response scales are Likert type, with scoring ranges from 0 to 4. Domain score were obtained by summing the score of their item, ranging from 0 to 28 in the physical, functional, and social well-being and from 0 to 24 in the emotional well-being domains. All the domains score were added, so that a higher score indicates better HRQoL. The FACT-G is reliable and validated instrument for measuring HRQoL in cancer patients.</p></sec><sec id="sec2-4"><title>Pain relief evaluation</title><p>Brief pain inventory (BPI) is a self-administered assessment tool used in pain management. The basic concept of BPI is to provide the information on the intensity of pain and the degree to which it interferes with the daily activity. The BPI is a brief and easy to use tool for assessment of pain in clinical research. The long version of the BPI has the front and back body diagrams, the four pain severity items and seven pain interference items rated on 0-10 scale and the question about percentage of pain relief by analgesic. The long version uses 7 days recall period. The interference items are presented with 0-10 scale, with ‘0’ = no interference and ‘10’ = interference completely. The BPI measures how much pain has interfered with the seven daily activities including general activity, walking, work, mood, enjoyment of life, relation with others, and sleep. The BPI pain interference typically scored as the mean of the seven interference items. We chose Bengali translation of validated BPI for the interference items for statistical analysis.[<xref rid="ref14" ref-type="bibr">14</xref>] Clinically significant change was defined as at least one-step change on the symptom scale.</p><p>Clinical follow up after 12<sup>th</sup> week was optional according to the individual needs. Subsequent palliative treatment allowed, such as chemotherapy, targeted therapy or best supportive care, were according to the requirements and PS. All the patients were followed up every 3 months thereafter.</p></sec><sec id="sec2-5"><title>Statistical analysis</title><p>The primary objective of the study was the patient-reported pain relief. Overall survival (OS) and the HRQoL among the three treatment arms were considered as the secondary objectives.</p><p>Categorical variables were analyzed using the Pearson's test or Chi-square test. Continuous variables were analyzed using Spearman's test and Kruskal-Wallis test. The log-rank was applied for comparison of OS.</p></sec></sec><sec sec-type="results" id="sec1-3"><title>RESULTS</title><sec id="sec2-6"><title>Pain relief evaluation</title><p>There were no differences among the treatment arms with respect to the relief of chest pain. A total of 83.3% in arm A, 79.6% in arm B, and 87% in arm C (<italic>P</italic> > 0.05) had reported significant relief of chest pain within 2 weeks from the start of palliative TRT [<xref ref-type="fig" rid="F1">Figure 1</xref>, <xref ref-type="table" rid="T2">Table 2</xref>]. Furthermore, the palliative effect of pain relief lasted throughout the primary follow up periods.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Pain relief (a) and health-related quality of life score (week 0-12) for FACT - G physical (b), functional (c), social (d), emotional (e)</p></caption><graphic xlink:href="LI-31-348-g002"/></fig><table-wrap id="T2" position="float"><label>Table 2</label><caption><p>Mean scores for BPI and all FACT-G items module in arm A, arm B, and arm C at baseline and at 2<sup>nd</sup>, 6<sup>th</sup>, and 12<sup>th</sup> week after of palliative thoracic radiotherapy</p></caption><graphic xlink:href="LI-31-348-g003"/></table-wrap></sec><sec id="sec2-7"><title>HRQoL analysis</title><p>Mean scores of all the domains of FACT-G questionnaires at baseline and during follow up are listed in <xref ref-type="table" rid="T2">Table 2</xref>. At 2<sup>nd</sup> week of follow up, patients in all the three arms reported reduced physical and functional well-being scores compared with baseline scores, thereafter increased and reached its highest value at 6<sup>th</sup> week of post-TRT. Patients in arm A reported reduced social and emotional well-being scores compared with baseline scores, whereas patients in arm B and C reported increased mean social and emotional well-being scores during post palliative TRT [<xref ref-type="fig" rid="F1">Figure 1</xref>].</p></sec><sec id="sec2-8"><title>Overall survival</title><p>The median OS was 5 months for the entire cohort as a whole. Different hypo fractionated palliative TRT schedule did not <italic>per se</italic> contribute to any significant difference in median OS [<xref ref-type="fig" rid="F2">Figure 2</xref>]. The median OS were 6, 5, and 6 months in arm A, B and arm C, respectively [<xref ref-type="table" rid="T3">Table 3</xref>]. Statistically significant association (<italic>P</italic> < 0.005) was observed between the PS and the median OS of the patients. Median OS being 7 months in patients with good PS (ECOG0-1) as compared with 5 months in those with poor PS (ECOG 2-3) [<xref ref-type="fig" rid="F3">Figure 3</xref>]. Subgroup of patients (arm C) with good PS and those treated with more fractionated (30 Gy in 10 fractions) palliative RT had better median OS compared with patients with poor PS [<xref ref-type="fig" rid="F4">Figure 4</xref>]. Tumor histology did not <italic>per se</italic> contribute to any significant difference in median OS (<italic>P</italic> = 0.911).</p><fig id="F2" position="float"><label>Figure 2</label><caption><p>Overall survival of patients according to different dose fractionation schedule</p></caption><graphic xlink:href="LI-31-348-g004"/></fig><table-wrap id="T3" position="float"><label>Table 3</label><caption><p>Median overall survival of the patients analyzed with respect to clinical variables</p></caption><graphic xlink:href="LI-31-348-g005"/></table-wrap><fig id="F3" position="float"><label>Figure 3</label><caption><p>Overall survival of the patients according to performance status</p></caption><graphic xlink:href="LI-31-348-g006"/></fig><fig id="F4" position="float"><label>Figure 4</label><caption><p>(a) Overall survival of patients (17 Gy per 2 F) according to performance status; (b) overall survival of patients (20 Gy per 5 F) according to performance status; (c) overall survival of patients (30 Gy per 10 F) according to performance status</p></caption><graphic xlink:href="LI-31-348-g007"/></fig><p>On univariate analysis, variables such as gender, religion, histo-pathological type, radiation dose and fractionations and treatment portal size had no significant effect on median OS. However, PS had shown significant difference in median OS on univariate analysis [<xref ref-type="table" rid="T4">Table 4</xref>]. On multivariate analysis using Cox regression model, only PS had significant influence on OS [<xref ref-type="table" rid="T5">Table 5</xref>].</p><table-wrap id="T4" position="float"><label>Table 4</label><caption><p>Univariate analysis: Correlation between overall survival and clinical or demographic variables</p></caption><graphic xlink:href="LI-31-348-g008"/></table-wrap><table-wrap id="T5" position="float"><label>Table 5</label><caption><p>Multivariate analysis by Cox regression model for overall survival and clinical or demographic variables</p></caption><graphic xlink:href="LI-31-348-g009"/></table-wrap></sec></sec><sec sec-type="discussion" id="sec1-4"><title>DISCUSSION</title><p>Metastatic NSCLC is one of the leading causes of cancer-related deaths in India. Although treatment outcomes in advanced disease remain modest, with the paradigm shifts in the approach to treatments, they are steadily improving. Customized treatments based on the tumor histology and genetic profiles have becomes the standard of care.</p><p>Systemic chemotherapy with the platinum-based doublets is the standard of care in metastatic or advanced NSCLC.[<xref rid="ref15" ref-type="bibr">15</xref><xref rid="ref16" ref-type="bibr">16</xref><xref rid="ref17" ref-type="bibr">17</xref>] This leads to modest survival advantage and improve QoL. TRT remains an important treatment modality for patients with symptom from intra-thoracic disease. There is no consensus which fractionation scheme should be used for palliative TRT. Different palliative TRT fractionation schedules, ranging from 10 Gy in single fractionation to 30 Gy in 10 fractions are used in clinical practice considering the patients preference, PS, and institutional protocol. In 2002, Cochrane meta-analysis of 10 randomized palliative radiotherapy trials in NSCLC was published.[<xref rid="ref3" ref-type="bibr">3</xref>] The palliative effect on disease-related symptoms were equivalent in all the fractionation schedules regardless of total radiotherapy dose.</p><p>Considering the poor prognosis for our patients, the primary objective of the study was the pain relief. This result was consistent with the previous studies in metastatic NSCLC.[<xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref7" ref-type="bibr">7</xref>] HRQoL data did not reveal any significant difference among the study groups except social well-being. There is slight decrease in physical and functional well-being mean score of FACT G at 2<sup>nd</sup> week post-TRT in all the arms. It indicates a slow deterioration of HRQoL in the physical and functional domains just after palliative hypo fractionated TRT. However, social well-being mean score increased over time up to 12 weeks post treatment except in the arm A (17 Gy in 2 fractions). Whereas, social well-being score decreased just after post radiotherapy and continued to decrease throughout the follow up periods.</p><p>The TRT side effects were mild and temporary. Dysphasia, a symptom related to radiation oesophagitis, increased during and shortly after the radiotherapy compared with the pretreatment level. It appears early in arms A (17 Gy in 2 fractions) and B (20 Gy in 5 fractions) than arm C (30 Gy in 10 fractions) and subsides within few weeks after completion of radiotherapy.</p><p>PS is the most important prognostic factor for median OS. In our study, we observed longer survival period in patients with good PS, as expected. However, there was no significant difference detected in OS among the three different palliative radiotherapy arms. In addition, subgroup analyses of patients with poor PS, the hypo fractionated (17 Gy in 2 fractions) TRT was equally effective as more fractionated high dose TRT (30 Gy in 10 fractions). We observed a non-significant increase in median survival in patients with good PS and those treated with more fractionated high dose TRT (30 Gy in 10 fractions). Bezjak et a l.[<xref rid="ref11" ref-type="bibr">11</xref>] reported a study (10 Gy in fractions vs. 20 Gy in 5 fractions) and found a significantly superior median survival in favor of higher dose TRT (6.0 vs. 4.2 months, <italic>P</italic> = 0.03). This difference was restricted to the good PS, which is similar to our results.</p></sec><sec sec-type="conclusion" id="sec1-5"><title>CONCLUSION</title><p>The efficacy of hypo fractionated TRT with respect to symptom palliation and OS in metastatic NSCLC is equivalent to that of more protracted or higher dose TRT. However, stage IV NSCLC with good PS may be benefited from more protracted TRT schedule. The side effect of hypo fractionated TRT was tolerable and comparable with protracted and higher dose TRT. Short-term hypo fractionated treatment is convenient for patients with limited expected survival.</p></sec> |
Spectrum of diffuse parenchymal lung diseases with special reference to idiopathic pulmonary fibrosis and connective tissue disease: An eastern India experience | <sec id="st1"><title>Objective:</title><p>To evaluate the clinical spectrum of diffuse parenchymal lung diseases (DPLD) encountered in the Indian setting and to compare idiopathic pulmonary fibrosis (IPF) and connective tissue disease associated DPLD (CTD-DPLD), the two commonest aetiologies.</p></sec><sec id="st2"><title>Materials and Methods:</title><p>A prospective study of clinical, imaging and laboratory parameters of patients diagnosed as DPLD and followed up in the Pulmonary Medicine Department of a tertiary-care teaching institution in eastern India was conducted over a period of one year.</p></sec><sec id="st3"><title>Results:</title><p>92 patients of DPLD were diagnosed in the study period with IPF (<italic>n</italic> = 35, 38.04%), CTD-DPLD (<italic>n</italic> = 29, 31.5%), hypersensitivity pneumonitis (<italic>n</italic> = 10, 10.9%), sarcoidosis (<italic>n</italic> = 5, 5.4%) and silicosis (<italic>n</italic> = 5, 5.4%) being the common causes. The CTD-DPLD group had a lower mean age (39.5 ± 1.86 vs 56.9 ± 1.12 years), a longer duration of symptoms (3.5 ± 0.27 vs 2.5 ± 0.26 years), more extra pulmonary manifestations, significantly more base line FVC and 6-minute-walk-distance than the IPF patients. 19 patients of IPF (54%) opted for treatment. All the IPF patients had a significant fall in FVC after six months (mean change -0.203 ± 0.01 litres) compared to the CTD-DPLD group (mean change - 0.05 ± 0.04 litres.)</p></sec><sec id="st4"><title>Conclusion:</title><p>CTD-DPLD patients belong to a younger age group, with longer duration of symptoms, more extrapulmonary features, better physiological parameters and better response to therapy than IPF patients. Larger prospective epidemiological studies and enrolment in clinical trials are necessary for better understanding of the spectrum of diffuse parenchymal lung disorders and their therapeutic options.</p></sec> | <contrib contrib-type="author"><name><surname>Kundu</surname><given-names>Somenath</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Mitra</surname><given-names>Subhra</given-names></name><xref ref-type="aff" rid="aff2">1</xref><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Ganguly</surname><given-names>Joydeep</given-names></name><xref ref-type="aff" rid="aff3">2</xref></contrib><contrib contrib-type="author"><name><surname>Mukherjee</surname><given-names>Subhasis</given-names></name><xref ref-type="aff" rid="aff4">3</xref></contrib><contrib contrib-type="author"><name><surname>Ray</surname><given-names>Souvik</given-names></name><xref ref-type="aff" rid="aff5">4</xref></contrib><contrib contrib-type="author"><name><surname>Mitra</surname><given-names>Ritabrata</given-names></name><xref ref-type="aff" rid="aff1"/></contrib> | Lung India : Official Organ of Indian Chest Society | <sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Diffuse parenchymal lung diseases (DPLD) comprise a wide spectrum of disorders, with varied presentations and prognosis. Foremost among the known causes is the connective tissue disease (CTD), the others being occupational or environmental exposure and drug-induced pneumopathies. Among the disorders of unknown cause, idiopathic pulmonary fibrosis (IPF) has the worst outcome.[<xref rid="ref1" ref-type="bibr">1</xref>] Most publications in the 1980s and 1990s from this country dealt with CTD-associated DPLD (CTD-DPLD)[<xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref3" ref-type="bibr">3</xref>] and there were some early reports on fibrosing alveolitis.[<xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref5" ref-type="bibr">5</xref>] With subsequent characterization of idiopathic interstitial pneumonias (IIPs),[<xref rid="ref1" ref-type="bibr">1</xref><xref rid="ref6" ref-type="bibr">6</xref>] an increase in the burden of IPF is being reported from various centers in India.[<xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref8" ref-type="bibr">8</xref><xref rid="ref9" ref-type="bibr">9</xref>] Although high-resolution computed tomography scan (HRCT) pattern and pathological findings in IIPs are now being applied to CTD-DPLD, there are differences in the treatment and prognosis, making it imperative to differentiate the two conditions.[<xref rid="ref10" ref-type="bibr">10</xref>]</p><p>In this study, we prospectively evaluated the clinical spectrum of DPLDs encountered in the Indian setting and compared the two leading causes of DPLD namely IPF and CTD-DPLD.</p></sec><sec sec-type="materials|methods" id="sec1-2"><title>MATERIALS AND METHODS</title><sec id="sec2-1"><title>Study design</title><p>A prospective observational study was conducted inthe Pulmonary Medicine Department of a tertiary care teaching institution in eastern India over a period of one year, on cases of diffuse parenchymal lung disease. Written informed consent was taken from all the patients and the study was cleared by the institute's Ethics Committee.</p><p>The inclusion criteria were:</p><p>
<list list-type="order"><list-item><p>Patients having clinical features/pulmonary function test suggestive of, and HRCT consistent with DPLD</p></list-item><list-item><p>Age 12 years and above, either sex.</p></list-item></list>
</p><p>The exclusion criteria were:</p><p>
<list list-type="order"><list-item><p>Patients with prior corticosteroid therapy, for more than one month</p></list-item><list-item><p>Patients not willing to follow the study protocol</p></list-item><list-item><p>DPLD cases diagnosed to have tuberculosis were excluded from the final analysis.</p></list-item></list>
</p><p>Detailed demographical and clinical parameters including age, smoking history, environmental, occupational, and drug exposure, duration and severity of breathlessness, clubbing, end-inspiratory crepitations, and extrapulmonary features (rashes, arthritis, Raynaud's phenomenon, dysphagia, oral ulcers), were assessed. HRCT of the thorax was evaluated for ground-glass opacities, reticular shadows, subpleural involvement, septal thickening, nodular lesions, and honeycombing/traction bronchiectasis. Contrast was given to patients suspected to have mediastinal lymphadenopathy. Spirometry, six-minute walk test (6MWT), according to the ATS guidelines,[<xref rid="ref11" ref-type="bibr">11</xref>] and echocardiography was done in all cases at the baseline. Tricuspid jet velocity >3.4 m/s and estimated pulmonary artery systolic pressure >50 mmHg[<xref rid="ref12" ref-type="bibr">12</xref>] were considered to be pulmonary hypertension. The antinuclear antibody (ANA) profile, Rheumatoid factor (RF), anti-cyclic citrullinated peptide (anti-CCP), antinuclear cytoplasmic antiboby (ANCA), and serum angiotensin converting enzyme (SACE) were estimated. Bronchoalveolar lavage (BAL) study, transbronchial lung biopsy (TBLB), and biopsies of lymph node, skin lesions or kidney were done as needed.</p></sec><sec id="sec2-2"><title>Diagnosis</title><p>A multidisciplinary approach involving a radiologist, pathologist, and rheumatologist was taken, to ensure accuracy of the diagnosis. A case was labeled as IPF as per the current guidelines of the American Thoracic Society/the European Respiratory Society/the Japanese Respiratory Society/the Latin American Thoracic Association (ATS/ERS/JRS/ALAT),[<xref rid="ref13" ref-type="bibr">13</xref>] that is, exclusion of known causes and the usual interstitial pneumonia (UIP) pattern on HRCT. Diagnosis of CTD and sarcoidosis was based on their respective diagnostic criteria, while a history of exposure, HRCT patterns, and BAL fluid lymphocytosis, were taken into account for diagnosing hypersensitivity pneumonitis.[<xref rid="ref14" ref-type="bibr">14</xref>]</p></sec><sec id="sec2-3"><title>Management</title><p>Cases of IPF were counseled and those consenting for treatment were prescribed a regimen of oral steroids (prednisolone 0.5 mg/kg/day tapering over three months to 0.125mg/kg/day), azathioprine (2 mg/kg/day to a maximum of 150 mg/day) + N-acetylcysteine (600 mg, orally, thrice a day) with modification, if necessary, due to comorbidities.[<xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref13" ref-type="bibr">13</xref><xref rid="ref15" ref-type="bibr">15</xref>]</p><p>Scleroderma patients were put on low-dose corticosteroid (10 mg) plus cyclophosphamide (2 mg/kg body weight),[<xref rid="ref16" ref-type="bibr">16</xref>] rheumatoid arthritis cases on disease modifying drugs including methotrexate and corticosteroids.[<xref rid="ref17" ref-type="bibr">17</xref>] Symptomatic sarcoidosis patients with stage 2 or 3 disease or disabling extrapulmonary features were prescribed corticosteroids.[<xref rid="ref18" ref-type="bibr">18</xref>] The vasculitis group was treated with prednisolone (1 mg/kg) and cyclophosphamide (2 mg/kg),[<xref rid="ref19" ref-type="bibr">19</xref>] the hypersensitivity pneumonitis patients were treated chiefly by environmental management, and in the severely symptomatic patients a short course of corticosteroids was used.[<xref rid="ref14" ref-type="bibr">14</xref>]</p></sec><sec id="sec2-4"><title>Follow-up</title><p>These patients were followed-up at the DPLD clinic based on clinical, spirometry, and 6MWT tests, every six months. Those on immunosuppressives were monitored monthly for hemogram and renal parameters. A patient with a fall in the forced vital capacity (FVC) of 10% or more from the baseline and/or a fall in the oxygen saturation below 88% before or during the 6MWT, with reduction in the six-minute walk distance, at six months, was considered to be with unstable disease.[<xref rid="ref13" ref-type="bibr">13</xref>]</p></sec><sec id="sec2-5"><title>Statistical analysis</title><p>The statistical analysis was performed using the SPSS version 10.0 (SPSS Inc, Chicago, IL) software for MS-Windows. Cases of IPF and CTD–DPLD were compared with respect to the clinical, radiological, and physiological parameters. Descriptive frequencies were expressed in terms of mean ± standard error of mean (SEM). The <italic>P</italic> value was calculated using the Fisher's exact test for categorical variables and a student t-test was used for continuous variables and a <italic>P</italic> < 0.05 was considered to be significant.</p></sec></sec><sec sec-type="results" id="sec1-3"><title>RESULTS</title><sec id="sec2-6"><title>Pattern of diagnosis</title><p>In this study, a total number of 112 patients with diffuse parenchymal lung diseases were enrolled initially. In 13 patients, a diagnosis of tuberculosis was made, and subsequently in two, lung carcinoma. Another five patients had clinicoradiological features suggestive of idiopathic non-specific interstitial pneumonia (idiopathic-NSIP), but they did not consent to a lung biopsy. These cases were excluded from the study, bringing the final population to 92.</p><p>Idiopathic pulmonary fibrosis (IPF) was found to be the most common variety (<italic>n</italic> = 35, 38.04%) followed by CTD (<italic>n</italic> = 29, 31.5%) [<xref ref-type="table" rid="T1">Table 1</xref>]. Of the 29 cases of CTD, systemic sclerosis (<italic>n</italic> = 17, 58.6%) and rheumatoid arthritis (RA) (<italic>n</italic> = 8, 27.5%) were the most common followed by systemic lupus erythematosus (SLE) (two cases), polymyositis, and ankylosing spondylitis (one case each). Small vessel vasculitis was diagnosed in four cases, of which two were granulomatous polyangitis, one was Churg-Strauss syndrome, and one microscopic polyangitis.</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Etiological distribution of diffuse parenchymal lung disease cases</p></caption><graphic xlink:href="LI-31-354-g001"/></table-wrap></sec><sec id="sec2-7"><title>Clinical parameters</title><p>In contrast to IPF, a majority of patients of CTD–DPLD belonged to a younger age group (<italic>n</italic> = 25, 86.2% in 20-50 years) [<xref ref-type="table" rid="T2">Table 2</xref>]. Although there was not much difference in the pulmonary symptoms and signs between IPF and CTD–DPLD [<xref ref-type="table" rid="T3">Table 3</xref>] among the extrapulmonary features, clubbing was distinctly more common in IPF (92%), while the Raynaud's phenomenon, dysphagia, and digital ulceration were seen in systemic sclerosis in 82, 58, and 35% of the cases, respectively. Joint deformity was noted in seven out of eight patients of RA, skin rashes and oliguria were seen in the SLE- and ANCA-associated vasculitis group, peripheral neuropathy in those with the Churg-Strauss syndrome, and one case of Wegener's granulomatosis.</p><table-wrap id="T2" position="float"><label>Table 2</label><caption><p>Demographic parameters</p></caption><graphic xlink:href="LI-31-354-g002"/></table-wrap><table-wrap id="T3" position="float"><label>Table 3</label><caption><p>Clinical parameters</p></caption><graphic xlink:href="LI-31-354-g003"/></table-wrap></sec><sec id="sec2-8"><title>Clinicophysiological parameters</title><p>Compared to CTD–DPLD, IPF patients had significantly less baseline FVC and six-minute walk distance [<xref ref-type="table" rid="T4">Table 4</xref>]. Pulmonary hypertension was seen in eight cases (47%) of systemic sclerosis (SSc), two cases of RA and one case of polymyositis.</p><table-wrap id="T4" position="float"><label>Table 4</label><caption><p>Physiological parameters</p></caption><graphic xlink:href="LI-31-354-g004"/></table-wrap></sec><sec id="sec2-9"><title>Imaging</title><p>All cases of IPF (<italic>n</italic> = 35, 100%) showed a classical UIP pattern on the HRCT thorax [<xref ref-type="table" rid="T5">Table 5</xref>]. The HRCT findings differed among the various subgroups of CTD–DPLDs. A majority of RA (<italic>n</italic> = 8) patients had a UIP pattern with predominant lower lobe (87%) subpleural (75%) involvement, with septal thickening (75%), and a minority had an NSIP pattern (12%). The SSc patients (<italic>n</italic> = 17) showed an NSIP pattern in 58.8% and a UIP pattern in 47%. One patient with SLE-DPLD showed a lower lobe, subpleural, reticular shadow, with honeycombing, and the other, bilateral diffuse ground-glass opacity, suggestive of diffuse alveolar hemorrhage. Polymyositis-interstitial pneumonia showed an NSIP pattern, while the case of ankylosing spondylitis had bilateral upper lobe fibrosis.</p><table-wrap id="T5" position="float"><label>Table 5</label><caption><p>High-resolution computed tomography thorax</p></caption><graphic xlink:href="LI-31-354-g005"/></table-wrap></sec><sec id="sec2-10"><title>Serum markers</title><p>Serum autoantibodies were helpful in differentiating CTD–DPLD from the IPF [<xref ref-type="table" rid="T6">Table 6</xref>]. In RA, The rheumatoid factor and anti-CCP antibody were found to be positive in 100 and 88% of the cases respectively, while in SSc, the anti Scl-70 and anti-centromere antibodies were positive in 88 and 12% of the cases.</p><table-wrap id="T6" position="float"><label>Table 6</label><caption><p>Serum markers</p></caption><graphic xlink:href="LI-31-354-g006"/></table-wrap></sec><sec id="sec2-11"><title>Spectrum of diffuse parenchymal lung diseases</title><p>Besides IPF and CTD–DPLD, ten cases were diagnosed as hypersensitivity pneumonitis (HP). Nine of these cases had exposure to pigeons and presented with chronic symptoms. Ground-glass opacities in the upper lobes with cystic changes and BAL fluid lymphocytosis (>30% of the total cell count) were seen in all the ten patients of HP.</p><p>Serum ACE was elevated in all the five patients of sarcoidosis (mean value of 98 U/L). The serum calcium and 24-hour urine calcium were elevated in three patients. The Mantoux test was negative with 10 TU in all five cases. The chest X-ray showed Stage 2 disease in three and Stage 3 disease in two. HRCT showed upper lobar involvement, with peribronchovascular thickening (100%), centrilobular nodules (60%), septal thickening, and ground-glass opacities (40%). Transbronchial lung biopsy (TBLB) showed non-caseating granuloma consistent with sarcoidosis in four cases. Cervical lymph nodes biopsy demonstrated non-caseating granuloma in the other case.</p><p>All five cases of silicosis were of workers involved in sandblasting, with upper zone involvement and mediastinal adenopathy (80%) including egg-shell calcification in one case. A case of pulmonary Langerhans cell histiocytosis (PLCH) presented with pneumothorax and characteristic HRCT features [<xref ref-type="fig" rid="F1">Figure 1</xref>], while the case of alveolar microlithiasis had its unique imaging features [<xref ref-type="fig" rid="F2">Figure 2</xref>].</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Pulmonary Langerhans cell histiocytosis presenting with pneumothorax</p></caption><graphic xlink:href="LI-31-354-g007"/></fig><fig id="F2" position="float"><label>Figure 2</label><caption><p>Alveolar microlithiasis</p></caption><graphic xlink:href="LI-31-354-g008"/></fig></sec><sec id="sec2-12"><title>Follow up</title><p>All cases were counseled for treatment options and followed up in the DPLD clinic. Nineteen out of 35 patients in the IPF group voluntarily agreed to treatment with the standardized triple drug regimen. Cases of CTD–DPLDs were put on appropriate therapy. Although 47% of the patients in the IPF group receiving the therapy experienced subjective improvement of dyspnea, on spirometry, all cases showed more than a 10% fall in FVC at six months [<xref ref-type="table" rid="T7">Table 7</xref>]. Eighty-four percent of the treated patients developed systemic hypertension, 63% diabetes, and 52% had at least one exacerbation (mainly infection) requiring hospitalization. Of the remaining 16 patients of IPF, who did not opt for triple drug therapy, after six months, 75% of them expressed subjective improvement of dyspnea, but all had a significant fall in FVC and an oxygen desaturation of <88% after the six-minute walk test. Improvement in the 6MWD, after six months, was seen in three patients (15.7%) in the treatment group and in seven patients (43.7%), who did not receive the triple drug therapy. Therefore, the triple drug therapy for the IPF group did not show any positive outcome. Two patients (on therapy) died within six months due to respiratory failure during exacerbation. Both patients had advanced disease, with a baseline dyspnea of grade 4 MMRC, FVC <1.25 liters, post six-minute walk test, and oxygen saturation <88%. On the contrary, in the CTD–DPLD group, 62.1% of the patients showed subjective improvement of dyspnea, 13 (44.8%) patients showed improvement in 6MWD, with 31% of the cases showing 6MWD of more than 300 m. Mean (± SEM) value of 6MWD and FVC after six months of treatment were 209.14 ± 9.72 m and 1.01 ± 0.03 l in the IPF group, and 267.5 ± 12.7 m and 1.17 ± 0.05 l, respectively, in the CTD–DPLD group.</p><table-wrap id="T7" position="float"><label>Table 7</label><caption><p>Treatment outcome after six months</p></caption><graphic xlink:href="LI-31-354-g009"/></table-wrap></sec></sec><sec sec-type="discussion" id="sec1-4"><title>DISCUSSION</title><p>Our study showed that IPF was the most common entity (38.04%) among DPLD cases [<xref ref-type="table" rid="T1">Table 1</xref>] followed by CTD–DPLD (31.5%), hypersensitivity pneumonitis (10.9%), sarcoidosis (5.4%), and silicosis (5.4%). The frequency of IPF varied from 28.6 to 46% in two studies from north India[<xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref5" ref-type="bibr">5</xref>] and from 43 to 45% in two studies from the south,[<xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref9" ref-type="bibr">9</xref>] while CTD–DPLD ranged from 18 to 50.8%.[<xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref7" ref-type="bibr">7</xref>] Sarcoidosis presenting with DPLD was reported in 9.6 and 22% among the DPLD cases.[<xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref9" ref-type="bibr">9</xref>]</p><p>On account of high prevalence of tuberculosis in India, many patients with interstitial lung disorders are misdiagnosed to be with tuberculosis, and receive antitubercular drugs (ATDs).[<xref rid="ref20" ref-type="bibr">20</xref>] In our study, 64 patients had a prior history of intake of ATDs for more than one month, although none had tuberculosis. Ten percent of the cases of cryptogenic organizing pneumonia[<xref rid="ref21" ref-type="bibr">21</xref>] and 37% of sarcoid patients[<xref rid="ref22" ref-type="bibr">22</xref>] from Mumbai were initially diagnosed with TB and received ATDs. On the contrary, tuberculosis may mimic DPLDs, as 13 cases in our study, initially suspected to be DPLD, were subsequently found to be suffering from tuberculosis with the help of a BAL/TBLB study. This was also the finding of others.[<xref rid="ref7" ref-type="bibr">7</xref>] Awareness about a varied DPLD spectrum and differentiation from tuberculosis is a very important issue in the Indian context.</p><p>The mean age of IPF cases [<xref ref-type="table" rid="T2">Table 2</xref>] in our series was 56.8 ± 8.3 years, similar to other Indian studies (53 ± 10[<xref rid="ref9" ref-type="bibr">9</xref>] and 50.6 ± 11.9 years[<xref rid="ref8" ref-type="bibr">8</xref>]), but less than that quoted in western literature (two-third of the cases over 60 and mean age at diagnosis of 66 years).[<xref rid="ref6" ref-type="bibr">6</xref>] The preponderance of males and smokers in the IPF group in this study is similar to the Indian and western literature.[<xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref13" ref-type="bibr">13</xref>] The median time between the onset of symptoms and diagnosis in the IPF group, studied by Johnston <italic>et al</italic>.,[<xref rid="ref23" ref-type="bibr">23</xref>] was 12 months. Mean duration of illness before diagnosis was 2.5 years in our IPF group and 2.25 years in the group studied by Subhash <italic>et al</italic>.[<xref rid="ref9" ref-type="bibr">9</xref>] Low awareness of the disease, over-reliance on the chest radiograph,[<xref rid="ref7" ref-type="bibr">7</xref>] and high TB burden is probably responsible for the delayed diagnosis compared to western literature.</p><p>Pulmonary symptoms and signs among CTD-DPLD were similar to that of IPF, except clubbing, which was uncommon in this group. The observations of Rajasekaran <italic>et al</italic>.[<xref rid="ref24" ref-type="bibr">24</xref>] were similar. Pulmonary hypertension was demonstrated in 22% of our IPF cases and 34.5% of the CTD-DPLD cases [<xref ref-type="table" rid="T4">Table 4</xref>]. It had been reported in 30% among DPLD cases by Subhash <italic>et al</italic>.[<xref rid="ref9" ref-type="bibr">9</xref>] and in a third of the patients with advanced IPF (right heart catheterization) in western literature.[<xref rid="ref25" ref-type="bibr">25</xref>] The prevalence of isolated pulmonary arterial hypertension and pulmonary hypertension with interstitial lung disease in systemic sclerosis were similar and ranged between 18 and 22% in various studies.[<xref rid="ref26" ref-type="bibr">26</xref>]</p><p>Previously the diagnosis of IPF was based on fulfilling the major and minor criteria in the absence of surgical lung biopsy (SLB).[<xref rid="ref6" ref-type="bibr">6</xref>] According to the current guidelines, a classical UIP pattern on HRCT, in an appropriate clinical setting, is sufficient to make a diagnosis of IPF.[<xref rid="ref13" ref-type="bibr">13</xref>]</p><p>The HRCT patterns in patients with CTD-DPLD mostly correlate with the lung pathology.[<xref rid="ref27" ref-type="bibr">27</xref>] Of the four usual patterns described in RA-associated DPLD[<xref rid="ref17" ref-type="bibr">17</xref>] our cases predominantly showed UIP followed by NSIP, but none had an organizing pneumonia or bronchiolitis pattern. Although, as with western literature, NSIP was the most common radiographic subtype among SSc-associated DPLD,[<xref rid="ref17" ref-type="bibr">17</xref>] there was also a significant number of our SSc patients with a UIP pattern. Therefore, in our series, CTD associated DPLD had more of a UIP pattern (<italic>n</italic> = 16, 55.2%) than NSIP (<italic>n</italic> = 12, 41.4%), although traditionally NSIP was known to be more frequent.</p><p>Decline in the FVC of 10% or more at six months and decreased 6MWD with desaturation have been shown to be measure of disease progression and surrogate markers of mortality.[<xref rid="ref13" ref-type="bibr">13</xref><xref rid="ref28" ref-type="bibr">28</xref><xref rid="ref29" ref-type="bibr">29</xref><xref rid="ref30" ref-type="bibr">30</xref>] In our study, 54% (<italic>n</italic> = 19) of the cases of IPF, who opted for the standardized triple drug regimen, with prednisolone, azathioprine, and acetylcysteine, showed no benefit (in fact, there were more drug side effects) compared to the 46% (<italic>n</italic> = 16) who opted for only supportive treatment. The triple therapy (prednisolone, azathioprine, and NAC) arm had to be discontinued due to excess morbidity and mortality compared to the placebo arm and the NAC arm in PANTHER-IPF study.[<xref rid="ref31" ref-type="bibr">31</xref><xref rid="ref32" ref-type="bibr">32</xref>]</p><p>The SSc-associated DPLD patients treated with oral cyclophosphamidewere found to have less change in FVC and functional ability compared to the placebo-treated patients in the North American Scleroderma Lung study.[<xref rid="ref16" ref-type="bibr">16</xref>] However, a recent meta-analysis found no significant improvement of pulmonary function with cyclophosphamide treatment.[<xref rid="ref36" ref-type="bibr">36</xref>] Unlike in the IIPs, there appears to be no difference in survival between those with an NSIP and those with a UIP pattern.[<xref rid="ref37" ref-type="bibr">37</xref>]</p><p>Treatment of RA-DPLD is essentially empirical in the absence of randomized controlled trials, and the best response has been reported with RA-associated organizing pneumonia.[<xref rid="ref17" ref-type="bibr">17</xref>] In their study, Rajasekaran <italic>et al</italic>.,[<xref rid="ref24" ref-type="bibr">24</xref>] found patients with RA-DPLD to have a better prognosis than those with IPF, with median survival rate of 60 months versus 27 months, respectively. However, no distinction regarding prognosis was made between the UIP and NSIP patterns in their RA-DPLD group.</p><p>Park <italic>et al</italic>.[<xref rid="ref35" ref-type="bibr">35</xref>] reported a better prognosis of the CTD-DPLD group, not only due to the higher prevalence of NSIP in this group, but also due to a better prognosis of CTD–DPLD patients, even those with a histological pattern of UIP, compared with patients with IPF. However, some studies quote a poor prognosis and similar mortality rates between the two groups.[<xref rid="ref33" ref-type="bibr">33</xref><xref rid="ref34" ref-type="bibr">34</xref>]</p><p>In this study, as a whole, the CTD–DPLD group fared better than the IPF group (both the treated and observation arms) at six months of therapy [<xref ref-type="table" rid="T7">Table 7</xref>].</p><p>Our study is probably one of the few studies from India that has looked at the spectrum of DPLD prospectively and has attempted a comparison between two of its largest contributors. However, the study has certain limitations, in that, the diffusing capacity of the lung could not be done for monitoring the disease progress; triple drug therapy versus supportive therapy in the IPF group was not randomized, the CTD–DPLD groups were not age- or sex-matched, and surgical biopsy was not feasible.</p><p>To conclude, the picture of diffuse parenchymal lung disease revealed in our study is similar to some of the retrospective studies from India. IPF seems to have presented a decade earlier in our country compared to the West. Both the burden of tuberculosis and its role as a ‘mimicker’ of DPLD caused a significant delay in the diagnosis of IPF in our country. Compared to IPF, the CTD–DPLD patients belonged to a younger age group, with a longer duration of symptoms, more extrapulmonary features, better physiological parameters, a mix of NSIP and UIP patterns on HRCT, and a better response to therapy. Larger prospective epidemiological studies and enrollment in clinical trials are necessary for a better understanding of the spectrum of diffuse parenchymal lung disorders and their therapeutic options.</p></sec> |
Evaluation of the effect of pulmonary hydatid cyst location on the surgical technique approaches | <sec id="st1"><title>Purpose:</title><p>A hydatid cyst is the most common lung parasitic disease and is endemic in Iran. A hydatid cyst is more common in the right lung and lower lobes.</p></sec><sec id="st2"><title>Objective:</title><p>The aim of this study was to assess surgical treatment of pulmonary hydatid cysts and whether the location of cyst affects surgical technique approaches.</p></sec><sec id="st3"><title>Materials and Methods:</title><p>This study was performed on 87 patients with a pulmonary hydatid cyst who were referred to Qaem Hospital from 2010 to 2012. Selection of surgical technique was according to size, location, and the number of cysts. Patients were divided into two groups: (1) surgery with preserving lung parenchyma and (2) lobectomy. Afterward, the relationship between the location of cyst and surgical technique approaches was evaluated. Data was analyzed by Chi-square and Fisher exact tests.</p></sec><sec id="st4"><title>Results:</title><p>In this study, no significant relation was found between the size (<italic>P</italic> = 0.682) and number (<italic>P</italic> = 0.344) of cysts and lobectomy necessity. But, there was a significant relation between the occurrence of cyst in middle lobe and lobectomy necessity (<italic>P</italic> = 0.016).</p></sec><sec id="st5"><title>Conclusions:</title><p>According to the results, type of surgical technique does not depend on the size and number of cysts, but it may be related to the location of the cyst and the ratio of lung destruction.</p></sec> | <contrib contrib-type="author"><name><surname>Sadrizadeh</surname><given-names>Ali</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Haghi</surname><given-names>Seyed Ziaollah</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Masuom</surname><given-names>Seyed Hossein Fattahi</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib><contrib contrib-type="author"><name><surname>Bagheri</surname><given-names>Reza</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Dalouee</surname><given-names>Marziyeh Nouri</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib> | Lung India : Official Organ of Indian Chest Society | <sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Hydatid disease had been recognized since the Galen and Hipocrates, and in 1808 Rudolphi used the term hydatid cyst to describe echinococcosis in humans. A hydatid cyst is a parasitic infestation caused most commonly by echinococcal granulosus. The most common location of a hydatid cyst is liver and lungs at the second place (10-40%).[<xref rid="ref1" ref-type="bibr">1</xref>] Generally, a pulmonary hydatid cyst is diagnosed by radiological imaging, physical examination and history findings.</p><p>The preferable treatment for pulmonary hydatid cysts is operation. Various surgical procedures are described in the literature. The surgical procedures may be conservative (cystostomy, enucleation of intact cysts, removal of the cyst after needle aspiration with or without pericystectomy, with maximal preservation of lung parenchyma) or radical (lung parenchyma resection with cystotomy and pericystectomy, wedge resection, segmentectomy and lobectomy).[<xref rid="ref2" ref-type="bibr">2</xref>] However, the selection of surgical technique depends on the conditions throughout surgery. While hydatid disease is endemic in Iran and the highest rate is reported for Khorasan Province and few studies have evaluated the relation between location of the cysts and type of surgical technique, we aimed to perform this study to assess surgical treatment of pulmonary hydatid cysts and evaluate whether the location of a cyst affects the choice of surgical technique.</p></sec><sec sec-type="materials|methods" id="sec1-2"><title>MATERIALS AND METHODS</title><p>In this study, the patients with a pulmonary hydatid cyst who had been referred to thorax surgery section of Qaem Hospital related to Mashhad University of Medical Sciences from 2010 to 2012 were evaluated. Among 99 patients, 12 cases were excluded because 6 patients had multiple bilateral lung hydatid cysts and they treated medically, 4 patients were inoperable due to other medical problem and 2 patients rejected surgical treatment.</p><p>Sampling method was non-probable and simple sampling. Initially, all conditions of the study were completely described for the patients and in case of their assent; they would sign a written informed consent. Moreover, the study was approved by the Regional Ethics Committee of Mashhad University of Medical Sciences (project number: 900415).</p><p>The studied variables included age, gender, clinical symptoms, type of surgery, postoperative complications, number of hydatid cysts in lungs and their size and location, hospitalization, bilateral lung hydatid cyst and mortality. A questionnaire was prepared and the information was recorded.</p><p>The patients were admitted as elective or emergency. Diagnosis was definite in the patients referred as elective. For the patients who had been referred as emergency, after obtaining history and physical examination, chest x-ray and CT scan were performed. For the cases with unsure diagnosis, indirect hemaglutinin serology test was done. The surgical technique approach was based on the location, size and number of cysts.</p><p>Preoperative evaluation was performed. Among 87 patients, for 14 cases with over 40 years old we did an electrocardiography and in 13 patients with history of chronic lung disease, lung function tests were performed. According to the results of these tests and having no contraindication for surgery, the patients were candidates for surgery.</p><sec id="sec2-1"><title>Surgical technique</title><p>Before the operation, the anesthetist was asked to start corticosteroids to prevent anaphylactic shock. All the cases were put in decubitus lateral position and posterolateral thoracotomy was performed through the fifth, sixth or seventh (depending on the location of the cyst) intercostal spaces. To prevent contamination of thoracotomy site and the surrounding tissue with hydatid cyst fluid, the surrounding of cyst was trapped with gas containing hypertonic sodium chloride solution 20%. During the operation, two suctions were ready; one inserted into trocar and the other in terminal suction.</p><p>According to the location, number and size of cysts, we selected each of the following operation techniques: Cystotomy and bronchial closure and capitonnage, cystotomy and bronchial closure, segmentectomy, enucleation, and lobectomy. Then, the patients were evaluated for the relation between the location of a cyst and the choice of surgery. The patients were followed up for 1 month in terms of postoperative complications.</p><p>To describe data, statistical charts and tables were used and data analysis was performed by SPSS version 19. The Chi-square test was used to compare treatment outcome and morbidity in the different methods of surgery and variance analysis was used to compare the duration of hospitalization in the different methods of surgery. <italic>P</italic> ≤ 0.05 was considered statistically significant.</p></sec></sec><sec sec-type="results" id="sec1-3"><title>RESULTS</title><p>A total of 87 patients with diagnosis of a pulmonary hydatid cyst were evaluated. Among them, 46 cases (52.9%) were males and 41 (47.1%) were females. The most affected age group was patients in their third decade with a mean age of 29.61 ± 14.27 (minimum 7 years and maximum 70 years). Clinical symptoms of the patients are shown in <xref ref-type="table" rid="T1">Table 1</xref>.</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Symptoms of a pulmonary hydatid cyst in the studied groups</p></caption><graphic xlink:href="LI-31-361-g001"/></table-wrap><p>The incidence of hepatic involvement was 19.5% (17 cases) and the hepatic localization was not complicated in the chest in16 patients. We operated a lung hydatid cyst at first and then a liver cyst operated in the second stage (6 weeks later). One patient was referred due to biloptysis and he was treated for both lung and liver cysts in first stage. The surgical technique approach for the patient was right posterolateral thoracotomy incision. We found a cyst in the middle lobe and fistula between diaphragm and lower lobe. We evacuated a lung cyst and capitonized. Then diaphragm was opened and the hepatic cyst was drained and was capitonized. Sub-diaphragmatic drain and chest tube were inserted.</p><p>The rate of bilateral hydatid cyst was 22 cases (25.3%) and unilateral hydatid cyst was 65 cases (74.7%).</p><p>Among 87 patients, 61 cases (70.1%) had a cyst with size of <10 cm and 26 (29.9%) had cyst with size of ≥10 cm. In terms of the number of cysts, 5 patients (5.7%) had more than three cysts and 67 cases (77%) one cyst, 9 (10.3%) two cysts, and 6 (6.9%) three cysts.</p><p>About the location of the cysts, in 19 cases (21.8%), the location of cyst was right lower lobe, in 12 (13.8%) right upper lobe, in 26 (29.9%) left lower lobe, in 11 (12.6%) left upper lobe, in 3 (3.4%) middle lobe, in 5 (5.7%) we observed pleural involvement and multi-lobe involvement in 11 (12.6%).</p><p>The surgical technique approach according to the location of the cysts in the patients with a pulmonary hydatid cyst is shown in <xref ref-type="table" rid="T2">Table 2</xref>. There was significant relation between the existence of the cyst in the middle lobe and performing lobectomy (<italic>P</italic> = 0.016).</p><table-wrap id="T2" position="float"><label>Table 2</label><caption><p>Type of operation according to the location of cysts</p></caption><graphic xlink:href="LI-31-361-g002"/></table-wrap><p>The choice of operation technique according to the size of the cysts in the patients with a pulmonary hydatid cyst is shown in <xref ref-type="table" rid="T3">Table 3</xref>. No significant relation was found between the size of cyst and lobectomy (<italic>P</italic> = 0.682).</p><table-wrap id="T3" position="float"><label>Table 3</label><caption><p>Type of operation according to the size of cysts</p></caption><graphic xlink:href="LI-31-361-g003"/></table-wrap><p>Selection of operation technique according to the number of the cysts in the patients with pulmonary hydatid cyst is shown in <xref ref-type="table" rid="T4">Table 4</xref>. No significant relation was observed between the number of cysts and lobectomy (<italic>P</italic> = 0.344). The results show that the choice of surgical technique is based on the location, not the size and number of cysts.</p><table-wrap id="T4" position="float"><label>Table 4</label><caption><p>Type of operation according to the number of cysts</p></caption><graphic xlink:href="LI-31-361-g004"/></table-wrap><p>In terms of hospitalization, nine cases had hospital stay for more than 9 days and five of them had undergone lobectomy.</p><p>The most common postoperative complication in the studied patients was prolonged air leak, which occurred in 12 cases. Other postoperative complications were empyema in 1 case, hemoptysis in 5 patients and bile secretion from drain in 1 case. In the last patient we performed conservative treatment because the patient was severely ill. After 2 months bile leakage subsided. We observed no complication in 68 patients. The rate of mortality was 1.15% (one case). He had lung and mediastinal cysts. The mediastinal cyst was contagious to great vessels. After opening the cyst, the patient had severe bleeding. We managed bleeding process during operation but the patient expired 2 days later in ICU due to multiorgan failure.</p><p>The mean follow-up time was 1 month. Among the referred patients, 96.8% had no severe problem and 3.2% had complication and 25 patients didn’t refer for follow-up.</p></sec><sec sec-type="discussion" id="sec1-4"><title>DISCUSSION</title><p>A hydatid cyst is a parasitic infestation caused mostly by echinococcal granulosus. It is an important health problem in regions where people earn the living by agriculture and livestock raising, while veterinary services, public health and preventive policies are poorly offered.[<xref rid="ref1" ref-type="bibr">1</xref>] Common complaints of patients with lung hydatid cysts are dry cough, hemoptysis and feeling pressure in the thorax. Symptoms are related to size, location and eventual rupture of the cysts. Hydatid disease when is located in lungs can reach a certain size without causing any symptom.[<xref rid="ref3" ref-type="bibr">3</xref>] A cyst is identifiable in chest x-ray and computed tomography (CT) scanning as a round or oval homogenous opacity that can be differentiated from pulmonary parenchyma.[<xref rid="ref4" ref-type="bibr">4</xref>] Serological investigations have a limited value in diagnosis of lung hydatid cysts.[<xref rid="ref5" ref-type="bibr">5</xref>] Some studies reported that in patients with pulmonary hydatid disease, the lung parenchyma should be preserved as much as possible and radical procedures must be avoided.[<xref rid="ref2" ref-type="bibr">2</xref>] Karavdic <italic>et al</italic>. reported that surgical treatment is related to size, localization, clinical manifestation and eventual complication of the cysts.[<xref rid="ref6" ref-type="bibr">6</xref>]</p><p>The most common localization of a hydatid cyst is the liver (50-60%) and secondly the lungs (10-30%).[<xref rid="ref7" ref-type="bibr">7</xref>] A hydatid cyst should be treated as soon as diagnosis is made, since it may cause serious complications by means of rupture into bronchi and pleural cavity or vital organ compression.[<xref rid="ref8" ref-type="bibr">8</xref>] Surgical treatment is preferred in pulmonary hydatid cysts.[<xref rid="ref9" ref-type="bibr">9</xref>] Medical treatment is considered for small cysts without complication, in patients who are high risk for surgery and those who reject the surgery.[<xref rid="ref10" ref-type="bibr">10</xref>] The choice of surgical technique is based on the location, size and the number of cysts. Since, hydatid disease is endemic in some parts of the world including Iran, we aimed to perform this study to evaluate whether the location of cyst affects the choice of surgical technique or not.</p><p>In this study, the most affected age group was 21-30 years with a mean age of 30 years and when compared with other studies, the same age group was involved.[<xref rid="ref11" ref-type="bibr">11</xref>] Although it may occur in any age group, it is more common in the third and fourth decades and is more prevalent in men.[<xref rid="ref12" ref-type="bibr">12</xref>] In this study, the rate of incidence of a pulmonary hydatid cyst was 52.9% in male and 48.1% in female. In a study by Bulent <italic>et al</italic>. no significant differences were found between the incidence rates by the gender.[<xref rid="ref13" ref-type="bibr">13</xref>] However, our result about the gender-related incidence rate was similar to those reported in the study performed by Bilgin <italic>et al</italic>.[<xref rid="ref14" ref-type="bibr">14</xref>] But, the study of Ghaffarifar reported that the incidence rate is more common in females.[<xref rid="ref15" ref-type="bibr">15</xref>]</p><p>In the referred patients of this study, 16.1% were asymptomatic and 83.9% were symptomatic. Common complaints were dry cough and chest pain. This was similar to the study of Bagheri <italic>et al</italic>. which reported that in the patients with a pulmonary hydatid cyst, cough and chest pain were the most common complaints (62%, 56%, respectively).[<xref rid="ref2" ref-type="bibr">2</xref>] Sehitogullari <italic>et al</italic>. in the study with 102 patients, 62 males and 40 females, reported that there is a relation between symptoms of patients and localization and size of the hydatid lung cysts.[<xref rid="ref16" ref-type="bibr">16</xref>]</p><p>In this study, the incidence of hepatic involvement was 19.5% and the rate of bilateral hydatid cyst was 25.3%. Various studies have reported multifocal involvement rates of 8% to 30%. Dogan <italic>et al</italic>. in the study involving a series of 1055 patients reported that the incidence rate of concomitant hepatic and pulmonary involvement was less than 10%.[<xref rid="ref17" ref-type="bibr">17</xref>] In the study of Karavdic <italic>et al</italic>. cyst localization was unilateral in 87.5% and bilateral in 12.5%.[<xref rid="ref6" ref-type="bibr">6</xref>]</p><p>In the present study, 70.1% of the patients had a cyst with size of <10 cm and 29.9% had a cyst with size of ≥10 cm. No significant relation was found between the size of cyst and lobectomy (<italic>P</italic> = 0.682). Usluer <italic>et al</italic>. performed a study on two groups of the patients with a hydatid cyst: Those smaller than 10 cm (group A) and those 10 cm or greater (group B). There were 462 patients (86%) in group A and 75 patients (14%) in group B. Lobectomy rates were significantly higher in group B compared with group A (<italic>P</italic> = 0.038).[<xref rid="ref18" ref-type="bibr">18</xref>] Also, Isitmangil <italic>et al</italic>. reported that one of the indications for lobectomy is large cysts involving more than 50% of the lobe.[<xref rid="ref19" ref-type="bibr">19</xref>] Moreover, in this study, we didn’t find any significant relation between the number of cysts and performing lobectomy (<italic>P</italic> = 0.344). However, the literature review demonstrated one of the major indications of lobectomy, the number and not the size of the cysts.[<xref rid="ref11" ref-type="bibr">11</xref>]</p><p>In our study, the left lower lobe was the most frequently affected area of the lung (29.9%) and the right lower lobe was affected as 21.8%. Ulku <italic>et al</italic>. concluded that the right lower lobe was the most frequently attacked area of the lung.[<xref rid="ref10" ref-type="bibr">10</xref>] The study of Sehitogullari showed that the right lower lobe was the most affected area of the lung.[<xref rid="ref17" ref-type="bibr">17</xref>] Their finding is not similar to our study. Also, in the present study, there was a significant relation between the existence of a cyst in the middle lobe and lobectomy (<italic>P</italic> = 0.016). Rochan <italic>et al</italic>. stated that lobectomy should be performed in cases where more than half of the lobe is involved.[<xref rid="ref7" ref-type="bibr">7</xref>] Of course, no study has been performed so far to evaluate the relation between the location of cysts and lobectomy.</p><p>Postoperative morbidity rates have varied between 0% to 17%, with air leak as the most common complication.[<xref rid="ref17" ref-type="bibr">17</xref>] In our study also, the most common postoperative complication was air leak in 12 patients of which 25% of cases of air leak was observed in the patients who underwent lobectomy. There was a significant relation between postoperative air leak and lobectomy (<italic>P</italic> = 0.052).</p><p>In the medical literature, mortality rates ranged from 0% to 2%.[<xref rid="ref20" ref-type="bibr">20</xref>] In our study, the rate of mortality was 1.15%.</p></sec><sec sec-type="conclusion" id="sec1-5"><title>CONCLUSION</title><p>In spite of having not enough cases (89 patients) and cases with a middle lobe cyst were few (7 patients), our data indicate if a hydatid cyst is located in the middle lobe, it may need lobectomy. Although size and number of cyst are determining factors for the selection of operation, but the location and the ratio of destruction of lung are important factors too.</p><p>Also, in patients with underlying pulmonary diseases if the lesion is completely removable with lobectomy, it is better not to insist on conservative surgical therapy, because it leads to incomplete tissue removal and therefore more complications would be expected. Moreover, since, pulmonary hydatid disease is endemic in the area of Khorasan, registry of the cases for better organizing the health, medical and statistical burden and prevalence of this disease is recommended. This will help to reduce the incidence of this disease. However, more studies with larger sample size are required to obtain more accurate results.</p></sec> |
Ease-of-use, preference, confidence, and satisfaction with Revolizer<sup>®</sup>, a novel dry powder inhaler, in an Indian population | <sec id="st1"><title>Context:</title><p>While prescribing an inhaler device, it is important to take into account the usability, preference, confidence, and satisfaction of the patients.</p></sec><sec id="st2"><title>Aims:</title><p>The present study assessed these parameters with Revolizer<sup>®</sup>, a novel dry powder inhaler (DPI), in patients with obstructive airway diseases and in device-naïve healthy participants.</p></sec><sec id="st3"><title>Settings and Design:</title><p>In this open-label, prospective, multicentre study with 100 participants [<italic>n</italic> = 50 healthy participants, <italic>n</italic> = 45 mild asthma patients, and <italic>n</italic> = 5 mild chronic obstructive pulmonary disease (COPD) patients], all participants were instructed and trained on the use of Revolizer and then the participants subsequently demonstrated the inhalation technique at two visits.</p></sec><sec id="st4"><title>Materials and Methods:</title><p>The average time required to execute three correct consecutive attempts and the number of errors (including critical errors) were recorded. Participants were asked about the ease of use, preference, confidence, and satisfaction by means of a questionnaire at each visit.</p></sec><sec id="st5"><title>Results:</title><p>The average time required by the participants to achieve three correct consecutive attempts at visit 1 was 3.75 ± 2.10 min, which significantly reduced at visit 2 (3.07 ± 1.32 min, <italic>P</italic> < 0.01). The number of errors decreased from visit 1 to visit 2. More than 85% participants found the Revolizer easy to use, and it was preferred by more than 75% participants. Revolizer scored high on the confidence and satisfaction of all participants at both visits.</p></sec><sec id="st6"><title>Conclusions:</title><p>Revolizer is an easy-to-use and a preferred device in patients with mild asthma and COPD, as well as in healthy participants with no previous experience of using inhalation devices. The participants felt confident and satisfied using the Revolizer.</p></sec> | <contrib contrib-type="author"><name><surname>Rajan</surname><given-names>Sujeet K.</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Gogtay</surname><given-names>Jaideep A.</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib> | Lung India : Official Organ of Indian Chest Society | <sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Administering drugs through the inhalation route is the most efficacious form of drug delivery in obstructive airway diseases (OADs) such as asthma and chronic obstructive pulmonary disease (COPD).[<xref rid="ref1" ref-type="bibr">1</xref>] In addition to being environmental-friendly and requiring minimum maintenance, the key aspects of an ideal inhalation drug delivery system include ease of use, reproducible dosing with optimal drug deposition, and a feedback mechanism to ensure correct dosing.[<xref rid="ref1" ref-type="bibr">1</xref>]</p><p>Dry powder inhalers (DPIs) are one of the most preferred inhalation drug delivery systems. Their advantages of being breath-actuated, easy to use, and compact make the DPIs reasonably well accepted by patients and their prescribers.[<xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref3" ref-type="bibr">3</xref>]</p><p>The two important factors that should be considered when prescribing an inhalation device are ease of use and patient preference, as these adversely impact a patient's adherence to prescribed therapy, possibly resulting in poor disease control and deteriorating quality of life.[<xref rid="ref1" ref-type="bibr">1</xref>] The confidence and satisfaction with the inhaler device will improve patient acceptance towards the device and the therapy and may improve the treatment outcomes, potentially reducing the healthcare costs associated with uncontrolled disease.[<xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref5" ref-type="bibr">5</xref>]</p><p>There are various DPIs now available, and several studies document the safety and efficacy of these DPIs in OADs.[<xref rid="ref3" ref-type="bibr">3</xref>] However, few studies have evaluated the ease of use, satisfaction, confidence, and patient preference with DPIs,[<xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref8" ref-type="bibr">8</xref><xref rid="ref9" ref-type="bibr">9</xref>] though these factors are associated with compliance and, therefore, efficacy.</p><p>Revolizer<sup>®</sup> is a novel unit dose DPI device having adequate drug deposition[<xref rid="ref10" ref-type="bibr">10</xref>] and a simple inhalation technique, allowing patients to confirm dose delivery as they can see, hear, and taste the drug. This is the first study to assess the usability, confidence, satisfaction, and patient preference with Revolizer in healthy volunteers and in patients with asthma and COPD in India.</p></sec><sec sec-type="materials|methods" id="sec1-2"><title>MATERIALS AND METHODS</title><sec id="sec2-1"><title>Study design and methods</title><p>This was an open-label, prospective, multicentre study assessing the usability, preference, confidence, and satisfaction with Revolizer in healthy volunteers and in patients with asthma and COPD. Each participant enrolled in one of the two study centers had to complete two study visits. During visit 1 (Day 1), the investigator explained and demonstrated the inhalation technique of Revolizer and noted the time taken by the participants to achieve three consecutive correct attempts. At visit 2 (Day 4), the participants demonstrated the inhalation technique without retraining and the number and types of errors were noted. Irrespective of whether the technique was performed correctly or incorrectly, the participants were trained twice and the time taken to perform three consecutive correct attempts and the number and types of errors noted. At both visits, the participants were asked about the ease of use, preference, confidence, and satisfaction with Revolizer using a questionnaire adapted from Schulte <italic>et al</italic>.[<xref rid="ref9" ref-type="bibr">9</xref>] The questionnaire was self-administered with 20 questions divided into four domains of usability, preference, confidence, and satisfaction [<xref ref-type="app" rid="APP1">Appendix 1</xref>]. The questionnaire was based on a Likert scale where the responses for usability, preference, and confidence ranged from 1 to 6 and for satisfaction ranged from 1 to 5 (a score of 6 or 5 implied a positive response and a score of 1 implied a negative response). Revolizer is a novel unit dose DPI developed, manufactured, and marketed by Cipla Limited in India. It is a simple-to-use device and consists of a mouthpiece, capsule chamber, and base [<xref ref-type="fig" rid="F1">Figure 1</xref>].</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Study device – the Revolizer<sup>®</sup></p></caption><graphic xlink:href="LI-31-366-g001"/></fig></sec><sec id="sec2-2"><title>Participants</title><p>The study included healthy volunteers and patients of asthma and COPD above 18 years of age [<xref ref-type="fig" rid="F2">Figure 2</xref>]. As the questionnaire had been developed in English, participants were included in the study only if they were able to speak and write English. Participants with either mental or physical impairment (such as Parkinson's disease, tremors) were excluded.</p><fig id="F2" position="float"><label>Figure 2</label><caption><p>Patient flow and follow-up diagram</p></caption><graphic xlink:href="LI-31-366-g002"/></fig></sec><sec id="sec2-3"><title>Endpoints</title><p>The primary endpoint was the average (±SD) time in minutes taken by the participants at visit 1 and visit 2 for three consecutive correct attempts in using the Revolizer. Correct attempt was defined as demonstration of all the steps involved in the proper use of the Revolizer [<xref ref-type="app" rid="APP2">Appendix 2</xref>].</p><p>The secondary endpoints included the average (±SD) number of attempts required for the first correct attempt and the number and type of errors while using the inhaler device during both the visits. The errors were classified as critical errors – those that can have a significant effect on the drug delivery to the lungs (they include slow inhalation and not holding breath after inhalation) or non-critical errors – those that do not affect dose delivery to the lungs. These errors were classified as being critical or non-critical prior to the study by an expert committee which included the principal investigators, and were not revealed to the participant [<xref ref-type="app" rid="APP3">Appendix 3</xref>].</p><p>The other secondary endpoints included the average (±SD) number and type of errors without retraining at visit 2, assessment of usability, preference assessment in patients using other devices prior to study enrollment, and an assessment of overall participant response to qualitative attributes, confidence, and satisfaction with the Revolizer.</p><p>The study was performed in accordance with the Good Clinical Practices and Declaration of Helsinki and was registered with the Clinical Trials Registry of India (CTRI number: CTRI/2009/091/000215). Ethics committee approval was obtained prior to the initiation of the study. The participants were explained the purpose of the study and the study procedures and a written informed consent was obtained. The device and study sponsorship was provided by Cipla Limited, India. Data management and statistical analysis were performed by an independent agency.</p></sec><sec id="sec2-4"><title>Statistical analysis</title><p>The demographic and baseline characteristics were analyzed and presented descriptively. All categorical variables were presented as counts and percentages. The primary and secondary endpoint of the number of attempts required to achieve first correct attempt was analyzed using paired <italic>t</italic>-test at 5% level of significance. The other secondary endpoints were described in terms of frequency and percentage of each event, and the mean difference was compared between the two visits.</p></sec></sec><sec sec-type="results" id="sec1-3"><title>RESULTS</title><sec id="sec2-5"><title>Baseline and demographic characteristics</title><p>One hundred participants (46 males and 54 females; mean age ± SD = 42 ± 14 years) were screened and enrolled. They consisted of 50 healthy volunteers, 45 patients with mild asthma, and 5 patients with mild COPD (based on the clinical diagnosis), and all completed the study. All patients with asthma and COPD (except two) were experienced in using an inhalation device other than the Revolizer (DPIs, <italic>n</italic> = 43; pressurized metered-dose inhaler (pMDI) alone, <italic>n</italic> = 3; and pMDI + spacer, <italic>n</italic> = 2), whereas all healthy volunteers (and two patients) did not have experience of using any inhalation device [<xref ref-type="table" rid="T1">Table 1</xref>].</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Baseline and demographic characteristics</p></caption><graphic xlink:href="LI-31-366-g003"/></table-wrap></sec><sec id="sec2-6"><title>Primary endpoint</title><p>The average (±SD) time taken for three consecutive correct attempts was 3.75 (±2.10) min at visit 1 and 3.07 (±1.32) min at visit 2 (<italic>P</italic> < 0.01) [<xref ref-type="fig" rid="F3">Figure 3</xref>]. The time taken for three consecutive correct attempts by the healthy participants was 4.07 (±2.34) min at visit 1 and 3.20 (±1.32) min (<italic>P</italic> = 0.004) at visit 2 [<xref ref-type="fig" rid="F3">Figure 3</xref>]. In patients with asthma or COPD, the average time taken for three consecutive correct attempts was 3.44 (±1.81) min at visit 1 and 2.95 (±1.33) min at visit 2 (<italic>P</italic> = 0.018) [<xref ref-type="fig" rid="F3">Figure 3</xref>].</p><fig id="F3" position="float"><label>Figure 3</label><caption><p>Time taken by participants for three consecutive correct attempts</p></caption><graphic xlink:href="LI-31-366-g004"/></fig></sec><sec id="sec2-7"><title>Secondary endpoints</title><sec id="sec3-1"><title>Number of attempts required for correct attempt</title><p>The mean (±SD) number of attempts required for the first correct attempt was significantly reduced at visit 2 [2.17 (±1.53) at visit 1 and 1.82 (±1.11) at visit 2, <italic>P</italic> = 0.005]. The average number of attempts required for the first correct attempt at visits 1 and 2 for healthy participants was 2.48 (±1.72) and 1.96 (±1.14) (<italic>P</italic> = 0.017), respectively, and for patients was 1.86 (±1.26) and 1.68 (±1.08) (<italic>P</italic> = 0.151), respectively [<xref ref-type="fig" rid="F4">Figure 4</xref>].</p><fig id="F4" position="float"><label>Figure 4</label><caption><p>Number of attempts required by the participants for the first correct attempt</p></caption><graphic xlink:href="LI-31-366-g005"/></fig></sec><sec id="sec3-2"><title>Number and type of errors while using the study device during both the visits</title><p>The most frequent errors while using the device included improper or inadequate exhalation prior to inhalation and slow inhalation. The overall number and type of errors (including critical errors) reduced at visit 2 compared to visit 1, with patients already experienced in using inhaler devices making fewer errors than the healthy participants [<xref ref-type="fig" rid="F5">Figure 5</xref>].</p><fig id="F5" position="float"><label>Figure 5</label><caption><p>Overall number and type of errors</p></caption><graphic xlink:href="LI-31-366-g006"/></fig></sec><sec id="sec3-3"><title>Number and type of errors without retraining at visit 2</title><p>Without retraining at visit 2, 44% of the participants completed single attempt with no error (critical or non-critical) and 74% of participants completed single attempt with no critical error. Fewer errors were made by patients compared to healthy participants at visit 2 (without retraining), of which the most frequently performed errors were slow inhalation and not holding breath after inhalation.</p></sec><sec id="sec3-4"><title>Assessment of usability of the study device</title><p>An average of 93.84% participants found the Revolizer easy to operate, understand, and remember when usability was assessed at both the visits [<xref ref-type="fig" rid="F6">Figure 6</xref>].</p><fig id="F6" position="float"><label>Figure 6</label><caption><p>Ease-of-use with Revolizer<sup>®</sup>-in participants</p></caption><graphic xlink:href="LI-31-366-g007"/></fig></sec><sec id="sec3-5"><title>Preference and overall participant response to qualitative attributes of the study device</title><p>Of the total number of asthma and COPD patients using an inhalation device other than the study device, 78% preferred the Revolizer over their current device.</p><p>Additionally, at both the visits, an average of 89.67% participants responded positively in terms of various qualitative attributes such as shape, color, portability, and likeability, and the comfort while operating and handling the device [<xref ref-type="table" rid="T2">Table 2</xref>].</p><table-wrap id="T2" position="float"><label>Table 2</label><caption><p>Participant responses towards qualitative attributes of the Revolizer<sup>®</sup></p></caption><graphic xlink:href="LI-31-366-g008"/></table-wrap></sec><sec id="sec3-6"><title>Confidence and satisfaction of the participants with the study device</title><p>At both the visits, the confidence and satisfaction was assessed with Revolizer [Figures <xref ref-type="fig" rid="F7">7</xref> and <xref ref-type="fig" rid="F8">8</xref>]. Overall, the participants felt confident and satisfied using the device. They found it easy to use and reported preference for the device.</p><fig id="F7" position="float"><label>Figure 7</label><caption><p>Participants’ confidence with Revolizer<sup>®</sup></p></caption><graphic xlink:href="LI-31-366-g009"/></fig><fig id="F8" position="float"><label>Figure 8</label><caption><p>Participants’ satisfaction with Revolizer<sup>®</sup> at both the visits</p></caption><graphic xlink:href="LI-31-366-g010"/></fig><p>The Revolizer scored high on usability, response toward qualitative attributes, confidence, and satisfaction of patients at both the visits [<xref ref-type="table" rid="T3">Table 3</xref>].</p><table-wrap id="T3" position="float"><label>Table 3</label><caption><p>Overall assessment scores of the participants</p></caption><graphic xlink:href="LI-31-366-g011"/></table-wrap><p>During the study period, no adverse events were reported in any patient.</p></sec></sec></sec><sec sec-type="discussion" id="sec1-4"><title>DISCUSSION</title><p>Chronic respiratory diseases such as asthma and COPD have an increasing prevalence worldwide.[<xref rid="ref11" ref-type="bibr">11</xref>] Inhalation therapy is the most recommended and accepted form of treating these chronic respiratory diseases.[<xref rid="ref12" ref-type="bibr">12</xref>] Recent years have seen an exponential increase in the types of inhaler devices both in India and the world over. However, not all inhalers are patient-friendly and easy to use, with each having their own advantages and disadvantages.</p><p>DPIs are the widely prescribed inhalation devices globally.[<xref rid="ref3" ref-type="bibr">3</xref>] Revolizer, a novel DPI, is widely marketed in India and our study demonstrates that it scores high on the ease of use, confidence, satisfaction, and preference in patients with asthma and COPD, as well as in healthy participants with no previous experience of using inhalation devices. Similar studies have assessed the handling, preference, and satisfaction with different DPIs in asthma and COPD.[<xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref9" ref-type="bibr">9</xref><xref rid="ref13" ref-type="bibr">13</xref>] This was the first study to assess all the parameters which need to be taken into account while selecting an inhaler device for the patient, such as ease of use, preference, confidence, and satisfaction in patients as well as in healthy participants.</p><p>It is further important to analyze whether the device prescribed is easy for the patient to remember and use.[<xref rid="ref12" ref-type="bibr">12</xref>] The average time taken by the participants in our study to execute three consecutive correct attempts in using the Revolizer was less than 4 min. The participants in our study were able to correctly use the Revolizer for the first time in less than three attempts at visit 1 and required a maximum of two attempts at visit 2, suggesting that the Revolizer has an inhalation technique which is easy to remember. Remembering the inhaler technique is an important aspect as it can significantly impact the adherence to therapy and can possibly affect disease control.[<xref rid="ref14" ref-type="bibr">14</xref><xref rid="ref15" ref-type="bibr">15</xref>] More than 90% of the participants in our study found the Revolizer technique easy to understand and operate at both the visits, which could probably mean that the Revolizer has a potential to become a device of choice for patients having cognitive or physical impairment, though this would need confirmation in clinical studies.</p><p>The inhalation technique varies with each type of DPI, and like any other inhaler device, optimum drug delivery depends on the correct inhalation technique. Errors made by the patients while using an inhaler device can lead to reduced drug delivery adversely impacting the disease control.[<xref rid="ref16" ref-type="bibr">16</xref>] The most common errors observed in our study while using Revolizer were inadequate exhalation prior to inhalation and slow inhalation, which are not device dependent but patient dependent and can be overcome by proper instruction and training.[<xref rid="ref17" ref-type="bibr">17</xref>] Before retraining at visit 2, the most common errors observed were slow inhalation and not holding the breath after taking the inhalation dose, which were identified as critical errors, and around 74% participants were able to complete a single attempt without any critical error. The patients with asthma and COPD made fewer errors while using the Revolizer compared to the healthy participants throughout the study. The reason for this difference could be that the patients were already using some device prior to the study. Healthy volunteers, on the other hand, probably had no experience with the inhalation therapy at all. The number of errors and critical errors decreased from visit 1 to visit 2 in both patients and healthy volunteers. Our results indicate that with proper instruction and training, correct inhalation technique can be achieved.</p><p>In addition to ensuring correct inhalation technique, it is equally important to take the patient preference into account for an inhalation device and this approach is also recommended by the Global Initiative of Asthma Management (GINA).[<xref rid="ref6" ref-type="bibr">6</xref>] Patients’ attitudes and preference vary from one device to another, even within the DPI category, as seen in previous studies.[<xref rid="ref18" ref-type="bibr">18</xref>] In our study, 78% patients preferred the Revolizer over their previous inhaler device that included the pMDI with or without a spacer. The major reasons for such a preference were that the device was easy to remember, use, operate, carry, and clean. More than 75% participants found the Revolizer favorable in terms of shape, color, handling, comfort, portability, and inhalation technique. Patient preference for an inhalation device has the potential to improve satisfaction with the device as well as therapy.[<xref rid="ref19" ref-type="bibr">19</xref>]</p><p>We not only assessed satisfaction but also confidence with the Revolizer because if participants including the device-naïve were confident of using the prescribed device, they would also be confident of the prescribed therapy, eventually leading to better adherence. To our knowledge, the present study is the only one to have assessed the confidence of the participants about using an inhaler device. There is definitely a need to develop more validated instruments that can assess the patient confidence along with the ease of use, preference, and satisfaction with an inhaler device.</p><p>Generally, such device handling studies are of short duration as single- or two-visit studies to ascertain the usability, preference, confidence, and satisfaction with a particular device.[<xref rid="ref8" ref-type="bibr">8</xref><xref rid="ref9" ref-type="bibr">9</xref>] The parameters such as usability, preference, confidence, and satisfaction for an inhaler device should ideally be assessed in a larger and heterogeneous patient population including non-English speaking patients, preferably with a comparator device. These have not been done in this study, which is a study limitation. We are in the process of developing questionnaires on the currently assessed parameters to suit the linguistic diversity for capturing more data from a larger population.</p><p>We did not measure the breathing pattern such as rapid and deep inhalation objectively, which could be one of the limitations of our study. The lesser number of COPD patients and short study duration were the other limitations of our study. As errors generally develop over time, it would be worthwhile to perform a study analyzing the number and type of errors with a significant time gap between training and retraining visits. It would also be interesting to repeat a similar study in this population which consists mainly of elderly individuals.</p><p>In conclusion, we confirm that the Revolizer is an easy-to-use and a preferred device. It scores high on the satisfaction and confidence in patients and individuals who have never used an inhaler device. Revolizer is a patient-friendly, portable, and comfortable device which is easy to operate and handle. It has the potential to become the device of choice for both patients and physicians alike.</p></sec> |
Potential impact of fireworks on respiratory health | <p>The world-wide use of fireworks with their consequent detrimental effect on the air quality is widely recognized with elevated ambient air levels of particulate matter and its several metallic components and gases identified in several studies carried out during such events. Exposed individuals may be at risk following inhalation of such produced pollutants. This review focuses on the impact of fireworks on air quality and the potential effect of fireworks on the respiratory system of healthy individuals as well as those suffering from underlying respiratory diseases, particularly asthma and chronic obstructive pulmonary disease (COPD). This applies not only to spectators including children but also to pyrotechnicians themselves. An extensive Medline search revealed that a strong evidence of the impact of fireworks on respiratory health is lacking in susceptible as well as healthy individuals with no formal studies on COPD or asthma, other than a few case reports in the latter. The implementation of global strategies to control the use of fireworks and hence improve air quality could possibly reduce their likely detrimental effect on human respiratory health in exposed individuals, but clearly a more targeted research is needed.</p> | <contrib contrib-type="author"><name><surname>Gouder</surname><given-names>Caroline</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Montefort</surname><given-names>Stephen</given-names></name><xref ref-type="aff" rid="aff1"/></contrib> | Lung India : Official Organ of Indian Chest Society | <sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>The tradition of celebrating national and cultural events with fireworks is recognized the world over. For anyone who has enjoyed a spectacular fireworks display, a part of the scene was most definitely obscured by the cloud of smoke from the burning fireworks themselves. Over recent years, short-term air quality degradation events have been studied intensively.[<xref rid="ref1" ref-type="bibr">1</xref><xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref3" ref-type="bibr">3</xref>] Several studies carried out in various countries across the world, with the majority in Europe, India, Canada, and China have identified a sharp increase in the concentrations of several firework constituents in the atmosphere following firework displays with resultant variations in air quality. Most of these studies were carried out during world-renowned celebrations such as the Diwali Festival in India, Yanshui Festival in Taiwan, Montreal International Fireworks competition, Lantern Festival in Beijing, Guy Fawkes Night in the United Kingdom, amongst others. According to an article published by the European Respiratory Society,[<xref rid="ref4" ref-type="bibr">4</xref>] all fireworks contain carbon and sulfur, which are necessary for burning. In addition, during fireworks manufacture, a range of substances are added such as arsenic, manganese, sodium oxalate, aluminum, iron dust powder, potassium perchlorate, strontium nitrate and barium nitrate, which act as stabilizers, oxidizers and added colors. The burning of fireworks releases a large amount of air pollutants, particularly sulfur dioxide (SO<sub>2</sub>), carbon dioxide (CO<sub>2</sub>), carbon monoxide (CO), and particulate matter (PM) along with several metal salts, for example aluminum, manganese, and cadmium.</p><p>There is increasing recognition of the detrimental effect of urban air pollution on human health both in the long- and in the short term.[<xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref7" ref-type="bibr">7</xref>] Adults exposed to high levels of ambient air pollution have shown increased prevalence of chronic cough, phlegm, and breathlessness[<xref rid="ref8" ref-type="bibr">8</xref>] and are therefore at an increased risk of developing respiratory symptoms, asthma, chronic obstructive pulmonary disease (COPD), allergic rhinitis, lower respiratory tract infections, and lung cancers.[<xref rid="ref4" ref-type="bibr">4</xref>]</p><p>The objective of this paper is to review current literature to attempt to identify the potential detrimental effect of these fireworks on the respiratory health of healthy as well as susceptible individuals.</p></sec><sec sec-type="materials|methods" id="sec1-2"><title>MATERIALS AND METHODS</title><p>Medline was consulted for this literature search. Search key words included fireworks, asthma, COPD, lung, health, air quality, respiratory system as well as particular firework constituents. Only articles and studies published in English were included. All articles on firework-related injuries were excluded.</p><p>Several articles identified the effect of air pollution from a number of sources and its effect on respiratory health and individual lung conditions. When the literature search was narrowed down to the impact of fireworks on air quality and the respiratory system, 43 articles were identified and reviewed. Twenty five addressed the effect of fireworks on air quality, two identified the effect of fireworks on asthma, none identified the effect of fireworks on COPD, and four assessed the association between fireworks and respiratory health in general. So it is evident that a direct correlation between fireworks and the respiratory system has not been extensively studied.</p></sec><sec sec-type="discussion" id="sec1-3"><title>DISCUSSION</title><sec id="sec2-1"><title>Fireworks and air quality</title><p>The harmful effects of ambient air pollutants are caused by the formation of reactive oxygen species, which in turn induce oxidative stress in the lungs resulting in a powerful cellular and mediator inflammatory response.[<xref rid="ref9" ref-type="bibr">9</xref>] When fireworks are set off, chemicals used in their composition react to ignite and propel the explosives with a resultant noticeable and odorous cloud of PM in the atmosphere. Inhalation of such particles is one of the most important routes of exposure to elevated concentrations of these emissions. PM deposition in the respiratory system mainly depends on the particle size.[<xref rid="ref10" ref-type="bibr">10</xref>]</p><p>Several metallic elements of PM were found to be at substantially elevated levels in a number of studies during firework events: Aluminum,[<xref rid="ref11" ref-type="bibr">11</xref><xref rid="ref12" ref-type="bibr">12</xref><xref rid="ref13" ref-type="bibr">13</xref><xref rid="ref14" ref-type="bibr">14</xref><xref rid="ref15" ref-type="bibr">15</xref><xref rid="ref16" ref-type="bibr">16</xref>] barium,[<xref rid="ref11" ref-type="bibr">11</xref><xref rid="ref13" ref-type="bibr">13</xref><xref rid="ref15" ref-type="bibr">15</xref><xref rid="ref16" ref-type="bibr">16</xref><xref rid="ref17" ref-type="bibr">17</xref><xref rid="ref18" ref-type="bibr">18</xref><xref rid="ref19" ref-type="bibr">19</xref>] copper,[<xref rid="ref11" ref-type="bibr">11</xref><xref rid="ref13" ref-type="bibr">13</xref><xref rid="ref18" ref-type="bibr">18</xref>] strontium,[<xref rid="ref11" ref-type="bibr">11</xref><xref rid="ref13" ref-type="bibr">13</xref><xref rid="ref15" ref-type="bibr">15</xref><xref rid="ref18" ref-type="bibr">18</xref><xref rid="ref19" ref-type="bibr">19</xref><xref rid="ref20" ref-type="bibr">20</xref>] antimony,[<xref rid="ref11" ref-type="bibr">11</xref><xref rid="ref13" ref-type="bibr">13</xref>] lead,[<xref rid="ref13" ref-type="bibr">13</xref><xref rid="ref19" ref-type="bibr">19</xref><xref rid="ref20" ref-type="bibr">20</xref>] magnesium,[<xref rid="ref12" ref-type="bibr">12</xref><xref rid="ref13" ref-type="bibr">13</xref><xref rid="ref14" ref-type="bibr">14</xref><xref rid="ref16" ref-type="bibr">16</xref><xref rid="ref18" ref-type="bibr">18</xref><xref rid="ref19" ref-type="bibr">19</xref><xref rid="ref20" ref-type="bibr">20</xref>] and potassium.[<xref rid="ref13" ref-type="bibr">13</xref><xref rid="ref14" ref-type="bibr">14</xref><xref rid="ref15" ref-type="bibr">15</xref><xref rid="ref16" ref-type="bibr">16</xref><xref rid="ref18" ref-type="bibr">18</xref><xref rid="ref19" ref-type="bibr">19</xref><xref rid="ref20" ref-type="bibr">20</xref>] SO<sub>2</sub>,[<xref rid="ref9" ref-type="bibr">9</xref><xref rid="ref19" ref-type="bibr">19</xref><xref rid="ref21" ref-type="bibr">21</xref>] nitrogen dioxide (NO<sub>2</sub>),[<xref rid="ref9" ref-type="bibr">9</xref><xref rid="ref21" ref-type="bibr">21</xref>] nitric oxide,[<xref rid="ref20" ref-type="bibr">20</xref>] PM10,[<xref rid="ref9" ref-type="bibr">9</xref><xref rid="ref16" ref-type="bibr">16</xref><xref rid="ref17" ref-type="bibr">17</xref><xref rid="ref19" ref-type="bibr">19</xref><xref rid="ref21" ref-type="bibr">21</xref>] total suspended particulate (TSP) matter,[<xref rid="ref9" ref-type="bibr">9</xref><xref rid="ref18" ref-type="bibr">18</xref>] PM1,[<xref rid="ref13" ref-type="bibr">13</xref>] PM2,[<xref rid="ref13" ref-type="bibr">13</xref>] PM2.5;[<xref rid="ref19" ref-type="bibr">19</xref><xref rid="ref20" ref-type="bibr">20</xref><xref rid="ref21" ref-type="bibr">21</xref>] benzene, toluene, ethylbenzene, and xylene-volatile aromatic compounds (BTEX),[<xref rid="ref13" ref-type="bibr">13</xref>] perchlorate,[<xref rid="ref21" ref-type="bibr">21</xref>] and chloride[<xref rid="ref14" ref-type="bibr">14</xref><xref rid="ref19" ref-type="bibr">19</xref>] also found to be elevated. Concentrations varied to different degrees in the various studies since different fireworks vary in the combination of metals they contain.[<xref rid="ref22" ref-type="bibr">22</xref>] There was no standardization as to the method utilized to analyze the pollutants and measurements were taken from varying distances from where the fireworks were let off. Ambient noise level was 1.2 to 1.3 times higher than a normal day,[<xref rid="ref23" ref-type="bibr">23</xref>] further contributing to another type of environmental pollution.</p></sec><sec id="sec2-2"><title>Fireworks and respiratory health</title><p>A number of health warnings have been issued on the worldwide web for susceptible individuals to avoid firework displays and avoid the inhalation of resultant fumes and airborne PM. A good number of papers have reported associations between airborne particles and a range of respiratory outcomes from symptoms to mortality,[<xref rid="ref24" ref-type="bibr">24</xref>] while others have reported associations with pulmonary function deficits, emergency room visits, hospital admissions, and daily deaths.[<xref rid="ref25" ref-type="bibr">25</xref>] Despite PM components having widely different physicochemical properties, they may induce similar cellular responses[<xref rid="ref24" ref-type="bibr">24</xref>] with a range of sensitivities to pollutants across different “at risk” groups,[<xref rid="ref26" ref-type="bibr">26</xref>] particularly patients suffering from lung and heart diseases, namely asthma and COPD in the former group. The individuals’ sensitivity to pollution is said to be related to their pulmonary antioxidant defences.[<xref rid="ref27" ref-type="bibr">27</xref>] Effects of fine particles on human health depend on the size, shape, number, and mass concentration together with chemical composition[<xref rid="ref28" ref-type="bibr">28</xref>] and may implicate that their detrimental effects on human health can affect more individuals for a prolonged period of time.</p><p>The impact of fireworks on human health has been reported in the literature and has been attributed to the large amount of chemical components that have been used during the manufacture of these fireworks. In 1975, Bach <italic>et al</italic>. reported a 113% statistically significant increase in treated respiratory illness during a fireworks event but a non-significant 8% decline in pulmonary function.[<xref rid="ref29" ref-type="bibr">29</xref>] According to the Swiss study on air pollution and lung diseases in adults,[<xref rid="ref4" ref-type="bibr">4</xref>] increments in NO<sub>2</sub>, ozone (O<sub>3</sub>), and TSPs were associated with decrements in forced expiratory volume in 1 second (FEV<sub>1</sub>) and increments in NO<sub>2</sub> and TSPs were associated with decrements in FVC.[<xref rid="ref4" ref-type="bibr">4</xref>] Six panel studies performed on patients suffering from chronic pulmonary diseases identified a decrease of peak expiratory flow and an increase in daily symptom and medication use in association with elevated daily particle concentrations.[<xref rid="ref30" ref-type="bibr">30</xref>] No studies were identified on the effect of firework emission exposure and the effect on lung function. Joly <italic>et al</italic>. in their study reported on the emissions of fireworks, which was carried out at the level of where people usually stand, and conducted during nine separate firework shows at an amusement park. They concluded that the particles emitted include extremely high levels of PM2.5 and are much more likely to enter the respiratory system.[<xref rid="ref14" ref-type="bibr">14</xref>] Hirai <italic>et al</italic>. (2000) also found that the inhalation of smoke from fireworks causes cough, fever, and dyspnea and lead to some cases of acute eosinophilic pneumonia.[<xref rid="ref31" ref-type="bibr">31</xref>]</p><p>Children seem to be particularly susceptible to the harmful effects of ambient air pollution. Compared with adults, children have poor defences against PM and gaseous air pollutants, have a differential ability to metabolize and detoxify environmental agents, and have an airway epithelium that is more permeable to inhaled air pollutants. Also, children have a greater level of physical activity than adults; hence, their intake of air into the lungs is much greater than that of adults per day.[<xref rid="ref5" ref-type="bibr">5</xref>] No studies involving paediatric patients were identified in this field.</p></sec><sec id="sec2-3"><title>Fireworks and asthma</title><p>Few investigators have identified the association between asthma and exposure to fireworks. According to researchers from the Institute of Environmental Assessment and Water Research (IDAEA-CSIC), metallic particles in the smoke emitted from the fireworks pose a health risk, particularly to asthmatics.[<xref rid="ref22" ref-type="bibr">22</xref>] In India, a 30% to 40% increase in the cases of wheezing, respiratory diseases, exacerbation of bronchial asthma, and bronchitis patients of all ages and gender were reported during the Diwali festival.[<xref rid="ref32" ref-type="bibr">32</xref>] A published case report of one fatal and another near-fatal asthma exacerbation within hours of exposure to fireworks were identified.[<xref rid="ref33" ref-type="bibr">33</xref>] Numerous articles offering advice to asthmatics to avoid exposure as well as how to manage an exacerbation following exposure were found.[<xref rid="ref34" ref-type="bibr">34</xref><xref rid="ref35" ref-type="bibr">35</xref>]</p></sec><sec id="sec2-4"><title>Fireworks and COPD</title><p>We could not find any publications identifying an association between COPD and firework exposure. However, several epidemiological studies have implicated exposure to a variety of ambient air particles and gases to exacerbate symptoms of COPD, resulting in increased morbidity and mortality, despite the variances in PM measures used and the health endpoints studied.[<xref rid="ref7" ref-type="bibr">7</xref>] Dominici <italic>et al</italic>. reported a near doubling in admissions for COPD exacerbations for every 10 μg<sup>3</sup> of increase in PM2.5. Since a number of studies have identified deterioration in COPD patients following exposure to air pollutants, one could speculate that fireworks are most likely to have a detrimental effect on these patients.[<xref rid="ref25" ref-type="bibr">25</xref>]</p></sec><sec id="sec2-5"><title>Individual firework constituents/emissions and respiratory health</title><p>Prolonged inhalation of fine dusts and chemicals during the manufacturing process may pose a risk to pyrotechnicians themselves following prolonged hours of exposure, especially if no protective equipment is worn. No published data were identified on the relation between inhalation of firework constituents during their manufacture and its effect on the respiratory health of those involved. Underlying respiratory co-morbidities as well as a smoking history may be additional risk factors for such individuals. However, the effects of the certain components used in their manufacture have been studied with respect to their effect on the lungs when used in different fields.</p><p>Studies as early as in 1961 on the effect of aluminum from alternative sources have shown that inhalation of finely powdered aluminum has a causative fibrotic effect on the lungs.[<xref rid="ref36" ref-type="bibr">36</xref>] Aluminum smelting fumes can also cause bronchoconstriction in susceptible individuals.[<xref rid="ref37" ref-type="bibr">37</xref>] Cast-house workers in the aluminum industry reported significantly more respiratory symptoms than controls, namely continuous trouble with breathing, repeated trouble with breathing, wheezing, asthma attacks (ever), and doctor-diagnosed asthma.[<xref rid="ref38" ref-type="bibr">38</xref>] The existence of occupational asthma in aluminum pot room workers has been confirmed by characteristic patterns of repeated peak flow measurements, supported by changes in methacholine responsiveness in workers with suspected work-related asthma.[<xref rid="ref39" ref-type="bibr">39</xref>]</p><p>Barium chromate was identified as being cytotoxic and genotoxic to human lung cells[<xref rid="ref40" ref-type="bibr">40</xref>] but no acute, intermediate, or chronic-duration inhalation estimates of exposure levels posing risk to humans have been carried out. Benign pneumoconiosis in several workers exposed to barium sulfate was reported in one particular study.[<xref rid="ref41" ref-type="bibr">41</xref>]</p><p>Most respiratory physicians recognize that chronic exposure to respirable cadmium in the workplace may lead to emphysema.[<xref rid="ref42" ref-type="bibr">42</xref>] It has been observed that there was an increased number of lung cancers in a United States cohort of cadmium-exposed workers, which has further been strengthened by further data.[<xref rid="ref43" ref-type="bibr">43</xref>]</p><p>Exposure to copper dust is associated with an excess of deaths from lung cancer in copper miners[<xref rid="ref44" ref-type="bibr">44</xref>] and a higher serum copper has been associated with lower FEV1[<xref rid="ref45" ref-type="bibr">45</xref>] while a long-term inhalation of antimony can potentiate pneumoconiosis.[<xref rid="ref46" ref-type="bibr">46</xref><xref rid="ref47" ref-type="bibr">47</xref>]</p><p>Consistent evidence has shown that asthmatic patients are more sensitive to the effects of inhaled SO<sub>2</sub> than are healthy subjects, with varying responses among asthmatic individuals in the degree of airway resistance and resultant changes in lung function.[<xref rid="ref26" ref-type="bibr">26</xref>] A study carried out in Birmingham identified a significant increase in hospital admissions for asthma and respiratory disease in association with daily variations in smoke and SO<sub>2</sub>.[<xref rid="ref48" ref-type="bibr">48</xref>]</p><p>Exposure to ozone, a highly reactive gas, has been noted to result in small changes in airway resistance and repeated exposure has been associated with increased asthma-related symptoms in asthmatic individuals.[<xref rid="ref26" ref-type="bibr">26</xref>] It has also been noted to cause decreased lung function, increased airway hyperreactivity, and pulmonary inflammation in healthy individuals.[<xref rid="ref27" ref-type="bibr">27</xref>] Ganguly identified a higher concentration of ozone during the fireworks season during Diwali, which has been associated with pneumonia, influenza, asthma and a persistent decrease in lung function.[<xref rid="ref49" ref-type="bibr">49</xref>]</p><p>An association between repeated exposure to firework emissions and respiratory symptoms has not been definitely identified. However, the association between long-term exposure to fine particulate air pollution has been associated with lung cancer mortality.[<xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref3" ref-type="bibr">3</xref>] Thus one might extrapolate this to the heavy exposure to such matter during and after fireworks displays, but formal correlative studies are lacking.</p><p>One can argue that typical exposure to such pollutants is limited, as assessed by Singh <italic>et al</italic>.,[<xref rid="ref50" ref-type="bibr">50</xref>] ranging from a few hours to a few days, depending on the duration of the firework festival, thus probably only causing minimal exposure. Also, fireworks are often let off following sunset when most people would go indoors to sleep after seeing the show, hence limiting exposure time to the ambient air pollutants. However, Barman <italic>et al</italic>. reported that particles remain suspended for up to 20 hours[<xref rid="ref28" ref-type="bibr">28</xref>] and a study carried out in Spain identified a “reservoir of metalliferous particles” which persisted for days after the festival, which was affected by wind, traffic, and other activity.[<xref rid="ref22" ref-type="bibr">22</xref>] Short-term exposure to such pollutants for an occasional day may not have such a detrimental effect but in countries like Malta where given the small geographical size of the archipelago of 316 km<sup>2</sup>, 86 religious feasts are held between June and September, and each celebrated with fireworks over approximately three nights of the week. Camilleri and Vella reported significantly higher PM10 and heavy metal concentration levels in the Maltese air between July and August as opposed to September and October when 59 and 11 feasts are celebrated respectively.[<xref rid="ref11" ref-type="bibr">11</xref>]</p><p>There is limited literature describing the physical characteristics of firework particles themselves including size distribution, number concentrations, modal characteristics as well as particle density.[<xref rid="ref51" ref-type="bibr">51</xref>] The latter has been identified as a direct result of particle deposition in the lungs.[<xref rid="ref52" ref-type="bibr">52</xref>] In a study performed during the Chinese New Year's firework events, emissions from fireworks were associated with substantial changes in particle size distributions as well as increasing overall particle number concentrations,[<xref rid="ref51" ref-type="bibr">51</xref>] which could influence lung deposition and subsequently trigger a pathological process.</p><p>Chemical components and combustion products of fireworks displays and their effect on air quality and health utilized different methods of analysis of the emissions. These alone depended on the distances of the letting off the fireworks from the scene and the timing and duration of collection of data. Additional sources of PM readily available for inhalation must also be taken into consideration, mainly cigarette smoke as well as ambient air pollutants from other sources such as vehicles and industrial pollutants, as these are confounding factors which may further contribute to developing or exacerbating lung disease.</p><p>All but one of the studies identified during this literature review were carried out outdoors. The only one performed indoors was carried out in the New Orleans Superdome during which high levels of particulate mass concentrations were initially noted but which decreased rapidly due to the ventilation system. The authors argued that indoor pyrotechnic displays are potentially more hazardous to health since particles are dispersed in a small volume of air and are detonated closer to the spectators than are outdoor pyrotechnic displays.[<xref rid="ref53" ref-type="bibr">53</xref>]</p><p>During the Spring Festival in Beijing, meteorological conditions seemed to be the main controlling factors of air quality, with high-concentration pollution mainly being related to wind speed, temperature inversion, and humidity.[<xref rid="ref54" ref-type="bibr">54</xref>] A two-year data review of ambient air quality in Delhi, India, showed a temporal variation in PM10, PM2.5, and PM1 with a 1.5 time decrease in the second year which was attributed to the higher mixing height, as well as temperature and wind speed variations.[<xref rid="ref55" ref-type="bibr">55</xref>] Weather conditions could therefore influence individuals depending on the weather conditions during the event and may be utilized by the patients to limit their exposure by, for instance, noting the wind direction.</p></sec></sec><sec sec-type="conclusion" id="sec1-4"><title>CONCLUSION</title><p>Following a thorough review of the literature available, further studies are necessary to consolidate current evidence and speculation. Implementation of strategies and enforcement of regulations to limit the harmful contaminants being used in the fireworks industry as well as legal control on the quantity of fireworks let off, together with additional surveillance of air quality during periods of increased risk should aid to avoid unnecessary morbidity. We recommend that susceptible individuals, mainly patients suffering from respiratory conditions, should avoid heavy exposure so as to avoid the possibility of exacerbations of their illness. Increasing awareness amongst the high-risk groups should decrease morbidity and the number of unscheduled health care visits and hospitalizations.</p><p>Further knowledge on the physical properties of firework aerosol particles could improve our understanding of the influence of fireworks on respiratory health. Opting for more environmental friendly techniques using newer technology other than the conventional methods is a further possible solution.</p><p>Assessing the respiratory health of firework manufacturers and identifying any potential negative impact are necessary to promote health and safety among these individuals with the possibility of avoiding possible morbidity and mortality.</p></sec> |
Pleuro-peritoneal lymphomatosis with concurrent tonsillar involvement in T-cell nonHodgkin's lymphoma: Clinical presentation mimicking disseminated tuberculosis | <p>A young male patient had been evaluated for pleural effusion at another center wherein on the basis of exudative, lymphocyte predominant pleural effusion with high pleural fluid adenosine deaminase (ADA) levels and tuberculin skin test (TST) reactivity, antituberculous medications had been administered. Reevaluation in view of worsening symptoms led to confirmation of the diagnosis of T-cell lymphoblastic lymphoma with pleuro-peritoneal lymphomatosis and tonsillar involvement. This case highlights the fact that elevated ADA levels should not be taken as surrogate for a diagnosis of TB in the absence of histopathological/microbiological confirmation even in countries with high tuberculosis (TB) prevalence. Tonsil in an uncommon site of involvement in patients with T-cell lymphoma. As the diagnosis in our patient was confirmed from biopsy of a tonsillar mass, a thorough physical examination should be performed in all patients with a suspected diagnosis of lymphoma.</p> | <contrib contrib-type="author"><name><surname>Tiwari</surname><given-names>Pavan</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Madan</surname><given-names>Karan</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Jain</surname><given-names>Deepali</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib><contrib contrib-type="author"><name><surname>Kumar</surname><given-names>Rakesh</given-names></name><xref ref-type="aff" rid="aff3">2</xref></contrib><contrib contrib-type="author"><name><surname>Mohan</surname><given-names>Anant</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Guleria</surname><given-names>Randeep</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib> | Lung India : Official Organ of Indian Chest Society | <sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>In high prevalence countries, it is usual to consider a diagnosis of tuberculosis (TB) when the pleural/peritoneal fluid levels of adenosine deaminase (ADA) are elevated in the setting of an exudative pleural effusion/ascites with predominance of lymphocytes on cytological examination. However, there are limitations in the sensitivity and specificity of elevated ADA levels in the diagnosis of TB. Also, tuberculin reactivity is very often inappropriately misused as an indicator for active TB.</p><p>Lymphomas are the third most important cause of malignant pleural effusion next to lung and breast cancer. Disseminated involvement with lymphoma can have a presentation that can closely mimic disseminated TB. We herein describe the case of a young male patient wherein a clinical picture resembling disseminated TB was subsequently diagnosed as disseminated involvement with T-cell lymphoblastic lymphoma (T-LL).</p></sec><sec id="sec1-2"><title>CASE REPORT</title><p>A young male patient presented with history of fever, shortness of breath and right sided chest pain for 4 weeks duration. Fever used to be more during the evening hours. Chest pain was pleuritic in character. There was no history of hemoptysis, wheezing, leg swelling, dysphagia, odynophagia, orthopnea, stridor, hoarseness of voice, or generalized swellings. Patient had lost 6 kg weight over the past 2 months and there was loss of appetite. Past and family history was unremarkable. The patient was a lifetime nonsmoker and nonalcoholic. There was history of contact with a patient of TB in the recent past. HIV-ELISA test was negative.</p><p>For the above symptoms, patient had been evaluated at another center where diagnosis of right pleural effusion was made and thoracentesis had been performed. Straw colored clear fluid had been aspirated. Fluid was an exudate with predominance of lymphocytes on cytological examination and pleural fluid ADA levels were elevated (70 IU/L). Tuberculin skin test demonstrated induration of 18 mm at the end of 48 h. With this profile, a possibility of tubercular pleural effusion was considered and patient was initiated on four drug antitubercular treatment (ATT) with four first line drugs namely Isoniazid, Rifampicin, Ethambutol, and Pyrazinamide. There was no improvement in symptoms and patient developed worsening shortness of breath, abdominal distension, and high grade fever within 2 weeks of initiation of treatment. He was referred to our center. A pleural pigtail catheter had been inserted for drainage of the reaccumulating right side pleural effusion.</p><p>On general physical examination, pulse rate was 92/min, blood pressure (BP) was 122/80 mmHg and there was no peripheral lymph node enlargement or venous prominences. Examination of the oral cavity showed unilateral enlargement of the left palatine tonsil. The surface of the tonsil had whitish plaques and there was no erythema or bleeding from its surface. Examination of the respiratory system was suggestive of bilateral pleural effusion and on abdominal examination shifting dullness was present suggestive of ascites. Chest radiograph demonstrated blunting of bilateral costophrenic angles suggesting bilateral pleural effusion [<xref ref-type="fig" rid="F1">Figure 1</xref>, Left panel]. Review of computed tomography (CT) scan of the thorax demonstrated bilateral pleural effusion (right > left) and small mediastinal lymph node enlargement with normal appearing lungs [<xref ref-type="fig" rid="F1">Figure 1</xref>, Right panel].</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Chest radiograph showing blunting of both costophrenic angles suggestive of bilateral pleural effusion. Pleural pigtail catheter is seen <italic>in situ</italic> on the right side (Left panel). CECT scan of the thorax demonstrating bilateral pleural effusion, Right > Left side. (Right panel)</p></caption><graphic xlink:href="LI-31-380-g001"/></fig><p>Pleural fluid and ascitic fluid examination were performed. Both fluids showed predominance of small lymphocytes with low glucose (5 mg/dl), elevated proteins (5-6 g/dl) and very high ADA (500 U/L) levels. Gram stain and cultures of both the fluids were negative. Whole body Fluorodeoxyglucose positron emission tomography computed tomography (FDG-PET-CT) examination [<xref ref-type="fig" rid="F2">Figure 2</xref>] demonstrated intense uptake in left tonsillar region, along with FDG-avid mediastinal and mesenteric lymph nodes. Abdominal PET-CT images showed omental caking along with diffuse peritoneal thickening and ascites, suggestive of diffuse peritoneal infiltration.</p><fig id="F2" position="float"><label>Figure 2</label><caption><p>PET-CT scan showing pattern of whole body FDG uptake (Panel A). Enlargement of and intense FDG-avidity is noted in the left tonsil (Panel B). Bilateral pleural effusion and intensely FDG-avid mediastinal lymph nodes are noted (Panel C). Omental caking, with diffuse peritoneal thickening and ascites with diffuse FDG uptake is also seen. (Panel D)</p></caption><graphic xlink:href="LI-31-380-g002"/></fig><p>In view of extremely high levels of ADA in the body fluids and radiological findings, a diagnostic possibility of lymphoma was considered and punch biopsy from left tonsillar area and repeat examination of pleural and ascitic fluid for immunocytochemical analysis were performed. Pleural and ascitic fluid examinations and examination of tonsillar biopsy [<xref ref-type="fig" rid="F3">Figure 3</xref>] subsequently confirmed a diagnosis of T-LL. On immunohistopathological examination of the tonsillar biopsy, tumor cells were immunopositive for CD3 (diffusely), CD5 (diffusely), and Tdt (focally), while tumor cells were immunonegative for CD20 and CD10. A final diagnosis of T-LL with pleuro-peritoneal lymphomatosis was established. Bone marrow examination showed all normal hemopoietic cells with adequate megakaryocytes. No lymphoma deposit or granulomas was seen. The patient subsequently received chemotherapy for lymphoma but had a progressively worsening course and expired a few days later.</p><fig id="F3" position="float"><label>Figure 3</label><caption><p>Microphotograph of tonsil biopsy shows expansion of the subepithelium by atypical lymphoid cells. H and E, ×200 (Left panel). The cells express nuclear TdT (Middle panel) and diffuse cytoplasmic CD3 (T-cell) antigen (Right panel)</p></caption><graphic xlink:href="LI-31-380-g003"/></fig></sec><sec sec-type="discussion" id="sec1-3"><title>DISCUSSION</title><p>Lymphomas are one of the leading causes of malignant pleural effusion. Pleural effusions and body cavity involvement with lymphoma are more commonly observed with nonHodgkin's lymphoma (NHL) especially the large cell variety. Cytological fluid examination is a sensitive modality for establishing the correct diagnosis.</p><p>T-LL is an infrequent type of NHL, representing about 2% of NHL cases. There is bimodal incidence, and patients younger than 20 years or those older than 50 years are usually affected. T-LL comprised 6% of all NHLs according to one lymphoma registry from India.[<xref rid="ref1" ref-type="bibr">1</xref>] Patients usually present with cervical, supraclavicular or axillary lymphadenopathy (50%), or anterior mediastinal mass (50-75%). Presentation as anterior mediastinal mass is usually associated with pleural/pericardial effusions or superior vena cava syndrome. Stage IV disease (80%) and B symptoms (50%) are common. Extranodal disease is less common and abdominal dissemination is unusual. Bone marrow involvement is infrequent at the time of presentation; however, up to two-thirds of patients eventually develop marrow infiltration. Involvement of the central nervous system is uncommon and usually occurs in advanced disease with concurrent bone marrow involvement.[<xref rid="ref2" ref-type="bibr">2</xref>] Involvement of Waldeyer's ring as was seen in our patient has been only rarely reported.[<xref rid="ref3" ref-type="bibr">3</xref>] In treated patients, Stage II/IV disease, late achievement of remission or elevated lactate dehydrogenase (LDH) (>500) have been found to be associated with poor prognosis.[<xref rid="ref4" ref-type="bibr">4</xref>] The unusual features in our patient were absence of peripheral lymphadenopathy and predominant involvement of the serous cavities and Waldeyer's ring (palatine tonsil) at the time of presentation. Although tonsil is not an uncommon site of involvement in lymphoma, tonsillar involvement in T-cell lymphoma is unusual.</p><p>Peritoneal lymphomatosis, as seen in our patient, is a rare condition in malignant lymphoma, which mimics metastatic carcinoma. Other radiologic differential diagnoses include tuberculous peritonitis, mesothelioma, and peritoneal desmoids. In young patients, CT findings like abdominal lymphadenopathy, aneurysmal dilatation of a gut segment with wall thickening and poor delineation at the mesenteric border or enlargement of liver/spleen should lead to the consideration of lymphoma as the leading differential diagnosis. Cytological/histopathological sampling is imperative for establishing a definitive diagnosis.[<xref rid="ref5" ref-type="bibr">5</xref>]</p><p>Apart from TB, high levels of ADA have also been reported in various noninfectious conditions associated with pleural fluid lymphocytosis, including malignant conditions (like adenocarcinomas, leukemias, and lymphomas) and collagen vascular diseases (like rheumatoid pleuritis and Systemic lupus erythematosus (SLE)). Others include fungal infections like coccidioidomycosis and histoplasmosis and bacterial infections like brucellosis. It has been highlighted repeatedly in literature that none of the biomarkers in pleural fluid are specific for tubercular pleuritis therefore interpretation of pleural fluid ADA assay results should always be done in the light of clinical findings and ideally along with results of histopathological findings.[<xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref7" ref-type="bibr">7</xref>] The maximum joint sensitivity and specificity of ADA for the diagnosis of tuberculous pleuritis was observed to be 93%.[<xref rid="ref8" ref-type="bibr">8</xref>] Therefore, ADA assay should not be considered as an alternative to methods of definitive diagnosis like biopsy and mycobacterial culture. With very high levels of ADA, possibility of lymphoma should always be considered as a diagnostic possibility.</p></sec> |
Superior vena caval syndrome and ipsilateral pleural effusion: A rare presentation of anterior mediastinal thymoma | <p>Incidence of thymic malignancies is very low. Thymoma, a tumor of thymus gland, is of epithelial origin and is most common anterior mediastinal tumor. In most cases, thymomas are localized and locally advanced thymomas may rarely present with superior vena caval obstruction (SVCO) and malignant pleural deposits. Microscopically, capsular invasion is noted in case of locally advanced thymomas, which behave like a malignant neoplasm. Complete surgical removal of the tumor along with intact capsule is the treatment modality of choice in case of localized tumors. Neoadjuvant radiotherapy (RT) and chemotherapy followed by surgical resection of residual tumor is useful in case of locally advanced tumors. RT is especially useful in case of SVCO to relieve the distressing respiratory symptoms. Here, we report a rare case of locally advanced thymoma, complicated by SVCO and ipsilateral pleural effusion in a 53-year-old male patient.</p> | <contrib contrib-type="author"><name><surname>Das</surname><given-names>Anirban</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Pandit</surname><given-names>Sudipta</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Choudhury</surname><given-names>Sabyasachi</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Das</surname><given-names>Sibes K.</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Basuthakur</surname><given-names>Sumitra</given-names></name><xref ref-type="aff" rid="aff1"/></contrib> | Lung India : Official Organ of Indian Chest Society | <sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Thymoma is of epithelial origin arising from the epithelium of thymus gland. Incidence of thymic malignancies is 2.5-3.2/10,00,000 population.[<xref rid="ref1" ref-type="bibr">1</xref>] Thymoma comprises 20-25% of all mediastinal tumors and is most common anterior mediastinal tumor.[<xref rid="ref1" ref-type="bibr">1</xref>] The tumour is rare in children, peak age of occurrence is between 40 and 60 years, with equal gender predilection.[<xref rid="ref2" ref-type="bibr">2</xref>] Two-third to three-quarter cases are benign in nature and do not show gross or microscopic invasion of surrounding structures. Capsular invasion is surrogate marker of malignant nature of the thymomas, which is a rarity. Invasive thymomas show local invasion, local recurrence following removal and rarely lymphatic and hematogenous spread. Pleural seeding and obstruction of superior vena cava are two rare manifestations of local invasion of invasive thymoma. Here, we report a case of superior vena caval obstruction (SVCO) and ipsilateral pleural effusion due to malignant thymoma in 53-year-old male teacher.</p></sec><sec id="sec1-2"><title>CASE REPORT</title><p>This was a case report of a non-smoker, non-alcoholic 53-year-old male patient presented with gradually progressive breathlessness at rest and dry cough for last 8 months. He also complained low grade, intermittent fever in initial 2 months, which was resolved spontaneously. Recently, he noticed a swelling of face, neck and arm for last 1 month which was gradually progressing and aggravated on lying down position. Cough and shortness of breath were increased in severity in lying down position. He also complained dysphagia, but no hoarseness of voice, hemoptysis and chest pain. The patient was normotensive and non-diabetic and had no past history of pulmonary tuberculosis. There was no history of altered sensorium, convulsion, nausea, visual disturbance and dizziness. History of significant weight loss and anorexia were present. Aspiration of red colored fluid from right pleural space was done and category I regimen of anti-tuberculous drugs (ATD) (rifampicin, isoniazid, pyrazinamide and ethambutol) was advised by local DOTS Clinic, before he was presenting to us. At that time, pleural fluid analysis revealed lymphocyte predominant, exudative fluid with high adenosine deaminase (ADA) value. Ziehl Neelen stain and Papanicolaou stain were negative for acid fast bacilli and malignant cells respectively.</p><p>On general survey, clubbing was present, but no pallor, icterus, peripheral lymphadenopathy, cyanosis and edema were present. Swelling of face, neck and upper extremities of both sides were noticed along with facial plethora, bilateral conjunctival suffusion and edema and engorged, non-pulsatile neck veins on both sides without positive abdomino-jugular reflux. All findings were more pronounced on raising the hands over the head. His axillary temperature was 97°F, respiratory rate 24 breaths/min, pulse rate 110 beats/min, blood pressure 110/80 mmHg and SpO<sub>2</sub> 82% at room air. The patient preferred upright posture without forward bending. Purpuric spot or other manifestations of bleeding disorders or coagulopathy were absent.</p><p>On examination of respiratory system, there was restricted movement and fullness of right side of the chest with engorged, tortuous veins over the anterior and lateral chest wall and also over anterior abdominal wall. Accessory muscles of respiration were grossly working and intercostal suction was present. Trachea was shifted to left and apex beat was at the 1 cm lateral to left mid-clavicular line in 5<sup>th</sup> intercostals space. Measurement of chest expansion was 3 cm. Direction of venous blood flow was from above downwards. On percussion, there was stony dullness from 3<sup>rd</sup> intercostal space downwards along the right mid-clavicular line, from 4<sup>th</sup> intercostal space downwards along mid-axillary line and from 6<sup>th</sup> intercostals space downwards on the back. Sternal percussion was dull (anterior D’ Espine sign was positive). On left side, resonance note was noted; Traube's space was tympanitic which was present in 6<sup>th</sup> and 7<sup>th</sup> intercostals spaces. On auscultation, diminished vesicular breath sounds were audible over right infraclavicular, axillary, lower interscapular areas, breath sounds were absent over right mammary, infra-axillary and infra-scapular areas. On left side, normal vesicular breath sound was audible in all areas. Bronchial breath sound was noted in upper interscapular area, below the fourth thoracic spine (posterior D’ Espine sign was positive). No added sounds were audible in any areas. Vocal resonance was diminished over all areas of right side, except suprascapular and upper interscapular areas. Examination of abdomen revealed no organomegaly, ascites, tenderness and lymphadenopathy. Examination of other systems revealed no abnormality.</p><p>Complete hemogram and blood biochemistry were normal. Chest X-ray (postero-anterior view) showed right sided pleural effusion with right para-tracheal mass lesion. Sputum for acid fast bacilli and malignant cells were negative. Spirometry showed restrictive defect. Arterial blood gas analysis showed partial pressure of oxygen 56 mm Hg and that of carbon dioxide 42 mm Hg with bicarbonate value 23 mEq/L. Contrast enhanced computed tomography (CECT) scan of thorax showed anterior mediastinal mass with ipsilateral pleural effusion [<xref ref-type="fig" rid="F1">Figure 1</xref>]. Pleural fluid analysis revealed lymphocyte predominant (95%), exudative red colored fluid with raised values of lactate dehydogenase (LDH - 1654 IU/L) and ADA (271.9 IU/L). Total cell count was 2,000/cmm. Papanicolaou stain showed predominance of atypical lymphoid cells mixed with few lymphocytes and reactive mesothelial cells. Gram stain and Ziehl Neelsen stain were negative. Pyogenic and mycobacterial culture showed no growth. Closed pleural biopsy was inconclusive. Serum LDH, uric acid and alpha-fetoprotein values were normal. CT-guided fine needle aspiration cytology taken from the mediastinal mass showed high cellularity comprised of monotonous population of small cells having round to slightly irregular nuclei and very scanty cytoplasm arranged mostly in discrete pattern and occasion aggregates in background of blood, favoring the possibilities of lymphoma or small cell carcinoma of lung. CT - guided tru-cut biopsy taken from the mediastinal mass revealed a growth composed of plump epithelial cells having pale eosinophilic cytoplasm and oval nuclei, which show vague rosette formations accompanied by small lymphoid infiltrates. Scanty hyalinized stroma was also found [<xref ref-type="fig" rid="F2">Figure 2</xref>]. This picture was suggestive of thymoma. On immunohistochemistry, it was found that scattered small lymphoid cells were positive for CD3 and 80% tumor cells were positive for Ki-67 and pan-cytokeratin, but negative for CD20 [<xref ref-type="fig" rid="F3">Figure 3</xref>]. Ultrasound of whole abdomen revealed no abnormality. CECT brain showed no metastatic deposits. Hence, Final diagnosis was anterior mediastinal thymoma (World Health Organization type B1) complicated with SVCO and ipsilateral pleural effusion, hence as per thymoma staging system of Masaoka and Koga, our case was stage IVa.[<xref rid="ref3" ref-type="bibr">3</xref><xref rid="ref4" ref-type="bibr">4</xref>] After the diagnosis, he had a rapidly downhill clinical course due to progressively increasing dyspnea as a result of rapidly increasing pleural effusion and increase in the severity of superior vena caval compression. Ultimately, the patient succumbed to his illness before starting chemo-radiotherapy (RT).</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Contrast enhanced computed tomography scan of thorax showing anterior mediastinal mass with ipsilateral pleural effusion</p></caption><graphic xlink:href="LI-31-383-g001"/></fig><fig id="F2" position="float"><label>Figure 2</label><caption><p>Microphotograph of tru-cut biopsy specimen showing plump epithelial cells having pale eosinophilic cytoplasm and oval nuclei, with vague rosette formations accompanied by small lymphoid infiltrates, suggestive of thymoma (H and E, ×10)</p></caption><graphic xlink:href="LI-31-383-g002"/></fig><fig id="F3" position="float"><label>Figure 3</label><caption><p>Microphotograph of immunohistochemistry of biopsy specimen showing scanty CD3 positivity, (a) Pan-cytokeratin (b) and Ki-67 (c) Positivity and CD20 negativity (d) (×10)</p></caption><graphic xlink:href="LI-31-383-g003"/></fig></sec><sec sec-type="discussion" id="sec1-3"><title>DISCUSSION</title><p>Most common cause of superior vena caval syndrome and malignant pleural effusion is lung cancer.[<xref rid="ref5" ref-type="bibr">5</xref>] In this respect, these two presentations are rarely reported in association with thymoma. Histologically, thymomas are of three types: Epithelial type, where 80% of neoplastic cells are epithelially derived, Lymphocytic type, where 80% of neoplastic cells are lymphocytically derived and third one, mixed or lymphoepithelial type. Prognosis usually depends upon the clinical stage of the tumor.[<xref rid="ref6" ref-type="bibr">6</xref>] However, aggressiveness of the thymoma also depends upon the appearance of the neoplastic epithelial cells and their numerical predominance over lymphocytes.[<xref rid="ref7" ref-type="bibr">7</xref>] Hence, capsular invasion and adjacent structure involvement are more common in epithelial type and therefore malignant behavior. SVCO and malignant pleural effusion are more common in this type for the same reason. Nearly 40% of thymomas are asymptomatic, 40% patients have local compressive symptoms like SVCO and rest 20% has systemic manifestations.[<xref rid="ref1" ref-type="bibr">1</xref>] Parathymic syndromes such as myasthenia gravis, pure red cell aplasia, hypogammaglobulinemia etc., may rarely be associated with thymomas,[<xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref8" ref-type="bibr">8</xref>] which were absent in our case.</p><p>On the other hand, in majority, metastatic pleural seeding is characterized by exudative, lymphocyte predominant pleural effusion with low ADA level. Lymphocyte predominant, exudative pleural effusion with high ADA value (>60 IU/L) is seen in tuberculosis, complicated para-pneumonic effusion, empyema thoracis, rheumatoid arthritis, chronic lymphatic leukemia, mesothelioma and lymphoma.[<xref rid="ref9" ref-type="bibr">9</xref><xref rid="ref10" ref-type="bibr">10</xref>] After extensive search of the literature, we found that very few cases of malignant pleural deposits in thymomas are reported,[<xref rid="ref11" ref-type="bibr">11</xref><xref rid="ref12" ref-type="bibr">12</xref>] the pleural fluid was exudative and lymphocytic in all these cases, but they have remained silent regarding pleural ADA values. This may be due to the fact that unlike in a tuberculosis-prevalent country like India where ADA value is routinely advised in the evaluation of exudative pleural effusion to confirm or exclude tuberculosis, this may not be the routine practice in the developed countries. Hence, high ADA value is a unique feature of this case. Besides that huge anterior mediastinal mass (clinico-radiologically, in most of the time mistakenly diagnosed as mediastinal lymphadenopathy and variegated appearance indicating necrotizing lymph nodes of tuberculous origin) with lymphocyte predominant exudative pleural effusion with high ADA value frequently provoke the clinicians to start ATD (in fact, Category - I ATD was completed in this case before presenting to us), especially in tuberculosis endemic zones, like our country, but think twice, as misdiagnosis and wrong treatment may lead to adverse outcome. Hence, tissue diagnosis is essential in case of mediastinal mass before starting ATD.</p><p>Another interesting point is that combination of mediastinal mass simulating lymphadenopathy and lymphocyte predominant exudative pleural effusion with high ADA and lactate dehydrogenase values in most cases is diagnosed as lymphoma on histo-pathological examination. However, this combination without peripheral lymphadenopathy is unusual in lymphoma. Hence, in our case, absence of peripheral lymphadenopathy helps us to think about the diagnosis other than lymphoma.</p><p>Surgical removal of both the tumor and the entire surrounding gland is the primary treatment modality for localized disease.[<xref rid="ref13" ref-type="bibr">13</xref>] Combined modality approach using RT, cytotoxic chemotherapy and surgical resection is very much effective in case of locally advanced tumor presenting with SVCO.[<xref rid="ref14" ref-type="bibr">14</xref>] Neoadjuvant RT and chemotherapy lead to significant tumor regression and finally surgical resection of residual tumor results in complete recovery in significant number of the cases.[<xref rid="ref15" ref-type="bibr">15</xref>] Local palliative RT (30 Gy in 10 fractions) is useful for relieving SVCO with distressing respiratory symptoms.[<xref rid="ref3" ref-type="bibr">3</xref>] On the other hand, combination chemotherapy, comprising of cisplatin, doxorubicin, vincristine and cyclophosphamide, is helpful in treatment of pleural deposits.[<xref rid="ref16" ref-type="bibr">16</xref>] In our case, unfortunately the patient succumbed to his illness before staring chemo-RT.</p></sec> |
Surgical lung biopsy to diagnose Behcet's vasculitis with adult respiratory distress syndrome | <p>A 34-year-old female presented with fever and abdominal pain. Past medical history includes Crohn's and Behcet's disease. Examination revealed multiple skin ulcerations, oral aphthae, and bilateral coarse rales. She developed respiratory distress with diffuse bilateral alveolar infiltrates on chest radiograph requiring intubation. PaO<sub>2</sub>/FiO<sub>2</sub> ratio was 132. The chest computed tomography revealed extensive nodular and patchy ground-glass opacities. Bronchoalveolar lavage demonstrated a predominance of neutrophils. Methylprednisolone 60 mg every 6 h and broad-spectrum antimicrobials were initiated. No infectious etiologies were identified. Surgical lung biopsy demonstrated diffuse alveolar damage (DAD) mixed with lymphocytic and necrotizing vasculitis with multiple small infarcts and thrombi consistent with Behcet's vasculitis. As she improved, steroids were tapered and discharged home on oral cyclophosphamide. Pulmonary involvement in Behcet's is unusual and commonly manifests as pulmonary artery aneurysms, thrombosis, infarction, and hemorrhage. Lung biopsy findings demonstrating DAD are consistent with the clinical diagnosis of adult respiratory distress syndrome. The additional findings of necrotizing vasculitis and infarcts may have led to DAD.</p> | <contrib contrib-type="author"><name><surname>Vydyula</surname><given-names>Ravikanth</given-names></name><degrees>MD</degrees><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Allred</surname><given-names>Charles</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Huartado</surname><given-names>Mariana</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Mina</surname><given-names>Bushra</given-names></name><xref ref-type="aff" rid="aff1"/></contrib> | Lung India : Official Organ of Indian Chest Society | <sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Behcet's disease is an idiopathic vasculitis characterized by recurrent mucocutaneous ulcerations and uveitis. The diagnosis is made on the basis of clinical findings.[<xref rid="ref1" ref-type="bibr">1</xref><xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref3" ref-type="bibr">3</xref>] It is very prevalent in Turkey, but also seen worldwide.[<xref rid="ref4" ref-type="bibr">4</xref>] Although Behcet's disease can affect any organ system, pulmonary involvement is uncommon and typically manifests as pulmonary vascular lesions. Pulmonary artery aneurysms involving the large proximal branches of the pulmonary arteries are the most common finding on chest computed tomography[<xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref8" ref-type="bibr">8</xref>] and hemoptysis is a common presenting symptom. Pulmonary small vessel vasculitis and lung parenchymal involvement is rarely reported, although the true incidence is uncertain given that histopathologic correlation is seldom pursued.[<xref rid="ref9" ref-type="bibr">9</xref>] This is an unusual case of Behcet's disease complicated by hypoxemic respiratory failure and acute respiratory distress syndrome. To our knowledge, this is the first case of adult respiratory distress syndrome associated with Behcet's disease with diffuse alveolar damage (DAD) and vasculitis on lung biopsy.</p></sec><sec id="sec1-2"><title>CASE REPORT</title><p>A 34 year-old, non-Turkish, Caucasian female with a history of Behcet's disease refractory to multiple immunosuppressive regimens presents with severe diffuse abdominal pain. She also reported a recent exacerbation of her Behcet's disease with skin ulcerations and orogenital aphthae. Past medical history included chronic sinusitis, migraine headaches, and Crohn's disease. She smokes one pack of cigarettes a day. Since the diagnosis of Behcet's, she had failed single-agent therapies, and therefore had required treatment with various combinations of immunosuppressants. Her current regimen included concomitant use of mycophenolate mofetil, methotrexate, azathioprine, infliximab, and prednisone at a dose of 50 mg daily. She self-discontinued methotrexate 2 weeks prior to her presentation due to gastrointestinal upset and the prednisone dose was increased to 80 mg given her current symptoms. The prednisone dose was then reduced to 50 mg several days later to avoid side effects from high-dose steroids. On day 2 of admission, she experienced rapidly progressive shortness of breath and hypoxemia requiring intubation and mechanical ventilation. She was febrile with a temperature of 102.7°F. Physical examination revealed a cushingoid-appearing female with a left mandibular healing ulcer, multiple punched out mucocutaneous ulcerations, and bilateral diffuse wheeze on auscultation. Her PaO<sub>2</sub>/FiO<sub>2</sub> ratio was 132. An echocardiogram was normal. Chest radiography showed extensive bilateral alveolar infiltrates [<xref ref-type="fig" rid="F1">Figure 1a</xref>]. Computed tomography of the chest revealed numerous small nodules and patchy areas of ground glass opacity [<xref ref-type="fig" rid="F1">Figure 1b</xref>]. Bronchoscopy reveals numerous upper airway aphthous ulcers. Bronchoalveolar lavage showed a predominance of neutrophils. Intravenous methylprednisolone 60 mg every 6 h and empiric broad-spectrum antimicrobials were initiated. Bacterial, fungal, and viral cultures, including studies for Aspergillus and <italic>Pneumocystis jirovecii</italic> were unremarkable. Serology and immunology including polymerase chain reaction (PCR) for cytomegalovirus, flu antigens A and B, rapid human immunodeficiency virus (HIV) antibody, <italic>Mycoplasma pneumoniae</italic> antibody immunoglobulin (Ig) G and IgM, Legionella, and Cryptococcal antigens were negative. CD4 count was 96. Vasculitis panel, which includes serum myeloperoxidase antibodies (cytoplasmic antineutrophil cytoplasmic antibodies (c-ANCA)), serum proteinase 3 antibody (perinuclear-ANCA (p-ANCA)), rheumatoid factor, serum antinuclear antibodies (ANA) titers, creatinine kinase, and aldolase levels were all unremarkable. Urine analysis was normal and renal function remained stable. Antibiotics were subsequently discontinued several days later. On day 5, a surgical lung biopsy was performed that revealed areas of necrosis and DAD mixed with lymphocytic and necrotizing vasculitis with multiple small infarcts and thrombi [Figures <xref ref-type="fig" rid="F1">1c</xref> and <xref ref-type="fig" rid="F1">d</xref>]. She recovered over the next 10 days and was successfully extubated. Steroids were slowly tapered and she was discharged home on combination therapy with cyclophosphamide and prednisone.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>(a) Extensive bilateral infiltrates. (b) Numerous patchy areas of ground glass opacities. (c) Diffuse alveolar damage characterized by intra-alveolar fibrin and hyaline membranes (hematoxylin and eosin stain (H and E)). (d) Multiple thrombi and infracts (H and E)</p></caption><graphic xlink:href="LI-31-387-g001"/></fig></sec><sec sec-type="discussion" id="sec1-3"><title>DISCUSSION</title><p>This is a case of Behcet's disease complicated by an acute fulminant pulmonary syndrome. The findings of necrotizing vasculitis with multiple infarcts and thrombi are consistent with previously described pulmonary manifestations of Behcet's disease.[<xref rid="ref10" ref-type="bibr">10</xref>] The literature pertaining to pulmonary involvement in Behcet's disease is mostly limited to case reports and case series describing large vessel vasculitis such as pulmonary artery aneurysms and their sequelae. Data on lung parenchymal involvement in Behcet's disease is scarce. The various pathologic findings reported include pulmonary infarction, hemorrhage, interstitial lung disease, and small vessel vasculitis. DAD in association with Behcet's disease has rarely been described.[<xref rid="ref11" ref-type="bibr">11</xref>] As surgical lung biopsy in Behcet's disease is seldom pursued, true incidence of pulmonary Behcet's disease is likely underdiagnosed. To our knowledge, this is the first case of Behcet's disease with pulmonary involvement manifesting as DAD mixed with necrotizing vasculitis on lung biopsy.</p><p>DAD is a well-described lung injury pattern which can occur in many settings including infection, collagen vascular disease, drug toxicity, inhalational injury, and uremia. It may be idiopathic. In the absence of any identifiable cause, the clinical diagnosis of acute interstitial pneumonia is appropriate. In this case, the coexistent vasculitis with multiple small infarcts consistent with lung involvement by Behcet's disease may have triggered the onset of DAD, and it provides an explanation for the clinical presentation. It is possible the abrupt tapering of steroids prior to her presentation led to an exacerbation of Behcet's disease.</p><p>Diffuse alveolar hemorrhage (DAH), another diagnosis to consider in the setting of vasculitis, is a syndrome characterized by injury or inflammation of the pulmonary arterioles, venules, or alveolar septal capillaries, and is associated with three different histologic patterns, including pulmonary capillaritis, bland alveolar hemorrhage, and DAD. DAH can result from a variety of conditions, such as coagulation disorders, drugs, toxins, collagen vascular diseases, and mitral stenosis. DAH is also commonly associated with ANCA positive vasculitis presenting as a pulmonary renal syndrome. Alveolar hemorrhage in Behcet's vasculitis is rare but may occur in the setting of DAD.[<xref rid="ref12" ref-type="bibr">12</xref><xref rid="ref13" ref-type="bibr">13</xref>]</p><p>Drug-induced lung injury is another possible explanation for her presentation. Immunosuppressive therapy is the mainstay of treatment for Behcet's disease, although evidence supporting specific agents is limited. Several immunomodulators and biologic agents, some of which our patient was using, have been reported to cause lung injury. Methotrexate, azathioprine, mycophenolate mofetil, as well as the tumor necrosis factor alpha inhibitor infliximab, have all been associated with varying degrees of pulmonary toxicity, including DAD.[<xref rid="ref14" ref-type="bibr">14</xref>] A cohort study of 514 patients with acute lung injury reported an incidence of drug-associated lung injury of 9.5%.[<xref rid="ref15" ref-type="bibr">15</xref>]</p><p>Pulmonary involvement in Behcet's disease, although uncommon, is a real entity and can be life-threatening. Physicians who treat patients with Behcet's disease, especially those with unexplained respiratory complaints, must keep this in their differential. Patients with Behcet's disease, especially those on immunosuppressant therapy should be frequently evaluated for any such evolving respiratory complications as well as drug-related pulmonary toxicity. For those on chronic steroid therapy, abrupt withdrawal or rapid tapering of steroids should be avoided to prevent an exacerbation of underlying vasculitis. High-dose corticosteroids appeared to have benefited our patient who had a complete recovery. Future lung biopsy-confirmed cases of DAD in association with Behcet's disease may help elucidate pathophysiologic mechanisms and optimize treatment.</p><p>In conclusion, this is a case of pulmonary Behcet's disease presenting as adult respiratory distress syndrome with DAD and vasculitis on lung biopsy. Physicians must maintain a high index of suspicion and early lung biopsy may help in the diagnosis and management of patients with Behcet's disease who present with unexplained respiratory failure.</p></sec> |
Bilateral pleural effusion with APLA positivity in a case of rhupus syndrome | <p>Rhupus syndrome is a rare syndrome characterized by overlap of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Our patient was a diagnosed case of RA and developed SLE 2 years after. She was a middle-aged woman, presented with bilateral pleural effusion with exacerbation of skin and joint symptoms of SLE. We diagnosed the case as tubercular pleural effusion by positive <italic>Mycobacterium tuberculosis</italic> in bactec 460 culture. She had also anti-phospholipid antibody positivity without any symptoms and signs of thrombosis.</p> | <contrib contrib-type="author"><name><surname>Saha</surname><given-names>Kaushik</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Saha</surname><given-names>Arnab</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Mitra</surname><given-names>Mrinmoy</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib><contrib contrib-type="author"><name><surname>Panchadhyayee</surname><given-names>Prabodh</given-names></name><xref ref-type="aff" rid="aff3">2</xref></contrib> | Lung India : Official Organ of Indian Chest Society | <sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Rhupus syndrome is the combination of clinical and immunological features of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). The patients of this syndrome have features of RA at beginning and few years later the manifestations of SLE develops. It is a very rare clinical entity and still now a very few number of well-documented cases are available in literature. We are reporting a twenty-eight year old young lady of rhupus syndrome with bilateral tubercular pleural effusion and associated positive anti-phospholipid antibody (APLA).</p></sec><sec id="sec1-2"><title>CASE REPORT</title><p>A twenty-eight year old married female was diagnosed as rheumatoid arthritis 2 years back based on stiffness, polyarthritis of small joints of both hand, raised rheumatoid arthritis (RA) factor (1:498), positive anti-cyclic citrullinated peptide (anti-CCP) 55 IU; (normal range 0-17 IU). She was on treatment with methotrexate, hydroxychloroquine and episodic analgesics. She had attended our pulmonary medicine outdoor with complaints of dry cough and bilateral pleuritic chest pain with evening rise of temperature for last one month. On enquiry she gave history suggestive of photosensitivity; genital ulcerations; oral mucosal ulcerations; loss of appetite; joint pain with swelling of bilateral small joints of hand and wrist joints and elbows for last three months. She denied any history suggestive of Raynaud's phenomenon, hemoptysis, menstrual irregularities, dysphagia, dryness of mouth and muscle weakness or seizures.</p><p>On examination of face, there was presence of mucosal ulcerations in the lower lips with butterfly like rash over malar region [<xref ref-type="fig" rid="F1">Figure 1</xref>]. Joints examination revealed tenderness with swelling of bilateral proximal and distal interphalangeal joints; metacarpophalangeal joints, wrist and elbow joints without any deformity and restriction of movement. There was absence of skin thickening, lymphadenopathy and hepato-splenomegaly but pallor present. Examination of chest revealed bilateral stony dull percussion note over infra-axillary and infra-scapular area with diminished vesicular breath sound suggestive of bilateral pleural effusion. He denied any history of anti-tubercular drug intake, treatment with corticosteroids and contact history of tuberculosis.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Face examination showing presence of mucosal ulcerations in the lower lips with butterfly like rash over malar region</p></caption><graphic xlink:href="LI-31-390-g001"/></fig><p>Complete blood count was normal except normocytic normochromic anemia with hemoglobin 9.0 gm/dl and raised erythrocyte sedimentation rate 65 mm at 1<sup>st</sup> hour. Urine examination showed albuminuria 2+, occasional red blood cells and 24-hour urinary protein as 900 mg/day. Liver function and renal function tests were normal. The X-ray of both hands showed lucency within bilateral triquetral bones and erosion at base of right sided 5<sup>th</sup> metacarpal bone suggestive of erosive arthritis [<xref ref-type="fig" rid="F2">Figure 2</xref>]. Her chest X-ray postero-anterior view showed bilateral mild pleural effusion [<xref ref-type="fig" rid="F3">Figure 3</xref>]. Her electrocardiography, echocardiography and ultrasonography of whole abdomen were normal. The pleural fluid was straw in colour and its analysis revealed total cell count 750 cells/mm<sup>3</sup> (lymphocytes 95%, and neutrophil 05%), protein 5.1 mg/dl, sugar 64 mg/dl, lactate dehydrogenase 450 U/L and adenosine deaminase 72.9 U/L (normal < 30 U/L). Gram stain, pyogenic culture and Ziehl-Neelsen (Z-N) stain of pleural fluid was negative but bactec 460 culture by <sup>14</sup>C-label substrate medium confirmed the presence of <italic>Mycobacterium tuberculosis</italic>. The consecutive 3 days sample of sputum for acid-fast bacilli was negative. On Special investigations, the index value of serum anti-nuclear antibody (ANA) was 6.92 (positive: More than 1.0) with anti- double-stranded DNA (anti-dsDNA) by enzyme immune assay (EIA) 3.73 (cutoff value - 0.52). Serum anti-phospholipid IgG antibody (APLA) by EIA was 2.56 (the cutoff value 0.52) without any symptoms and signs of arterial and venous thrombosis and remains positive on repeat examination after 12 weeks. Serum international normalised ratio (INR) was 0.88. Pleural fluid for lupus erythematosus (LE) cells, ANA and RA factor was negative.</p><fig id="F2" position="float"><label>Figure 2</label><caption><p>X-ray of both hands showing lucency within bilateral triquetral bones and erosion at base of right sided 5<sup>th</sup> metacarpal bone suggestive of erosive arthritis (black arrow)</p></caption><graphic xlink:href="LI-31-390-g002"/></fig><fig id="F3" position="float"><label>Figure 3</label><caption><p>Chest X-ray (postero-anterior view) showing bilateral mild pleural effusion</p></caption><graphic xlink:href="LI-31-390-g003"/></fig><p>Patient fulfilled 6 out of 11 American College of Rheumatology (ACR) revised criteria for classification of SLE and had a score of 8/10 based on the 2010 American College of Rheumatology/European League against rheumatism classification criteria for RA.[<xref rid="ref1" ref-type="bibr">1</xref><xref rid="ref2" ref-type="bibr">2</xref>] We had diagnosed the case as bilateral tubercular pleural effusion with APLA positivity in a case of rhupus syndrome and started treatment with daily anti-tubercular therapy consisting of isoniazid 300 mg, rifampicin 450 mg, pyrazinamide 1250 mg and ethambutol 1000 mg and hydroxy-chloroquine (400 mg once daily). Topical hydrocortisone ointment was advised to apply on the oral mucosal and genital ulceration sites thrice daily. Sunscreen lotion containing octinoxate 7.5% w/w, avobenzone 2% w/w, oxybenzone 3% w/w, zinc oxide 2% w/w, with 26 sun-protection factor (SPF) was also advised to apply on the face mainly on the hyper-pigmented spots thrice daily.</p></sec><sec sec-type="discussion" id="sec1-3"><title>DISCUSSION</title><p>In the year 1974, Schur coined the terminology ‘rhupus syndrome’ for the first time and he showed the presence of features of both RA as well as SLE.[<xref rid="ref3" ref-type="bibr">3</xref>] The rhupus syndrome is usually diagnosed by inflammatory symmetrical polyarthritis, raised RA factor, clinical manifestations suggestive of SLE (cutaneous manifestations such as butterfly skin rashes, alopecia, photosensitivity; hematological manifestations such as leucopenia and thrombocytopenia; serositis such as pleural and pericardial effusion and mucosal involvement), positive anti-dsDNA or anti-smith auto antibodies.[<xref rid="ref4" ref-type="bibr">4</xref>] Amezcua-Guerra LM <italic>et al</italic>., had used anti-CCP to differentiate RA and rhupus from SLE.[<xref rid="ref5" ref-type="bibr">5</xref>] The early manifestations of rhupus consist of features of RA and among them the most common presentation is erosive symmetrical polyarthritis followed by rheumatoid nodules (40% cases).[<xref rid="ref6" ref-type="bibr">6</xref>] Newer imaging modalities like ultrasound (US) with doppler and magnetic resonance imaging (MRI) of joints helps in differentiation of rhupus arthritis from SLE or RA arthritis and detects the severity of joints early with prognostic implications.[<xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref8" ref-type="bibr">8</xref>] Tani C <italic>et al</italic>., described differences in the clinical features and joint imaging scores between rhupus patients and SLE patients.[<xref rid="ref8" ref-type="bibr">8</xref>] In this study at least one pathological finding (synovitis or bone erosion) was observed by US examination of hand or wrist joints in all rhupus patients and statistically significant higher scores on US was detected in respect to SLE patients but RA patients had similar scores. MRI of joints also showed the similar findings like US.[<xref rid="ref8" ref-type="bibr">8</xref>] Rhupus patients had significantly lesser involvement of kidney compared with SLE patients, but no differences were observed regarding the involvement of hematological, serositis, neuropsychiatric and cutaneous by Tani C <italic>et al</italic>.[<xref rid="ref8" ref-type="bibr">8</xref>] The features of SLE develop several years after the onset of the features of RA probably due to precipitation of some hormonal factors.[<xref rid="ref9" ref-type="bibr">9</xref>] We diagnosed our case as rhupus syndrome by the clinical features of RA and SLE with erosive arthritis, positive RA, Anti-CCP, ANA and Anti-dsDNA. In our patient manifestations of SLE appeared two years after the diagnosis of RA made. In rhupus syndrome extra-articular major organ involvement is extremely unusual though lupus encephalitis and lupus pnemonitis were reported.[<xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref10" ref-type="bibr">10</xref>] These patients have APLA positivity in high titers but symptomatic thrombosis is very rare.[<xref rid="ref11" ref-type="bibr">11</xref>]</p><p>Pleural effusion in a patient of rhupus syndrome can occur due to different reasons like rheumatoid pleuritis, lupus pleuritis, parapneumonic, tuberculosis etc., Predominant cell type in parapneumonic effusion is neutrophilic but in other causes (rheumatoid, lupus and tubercular) of effusions is lymphocytic mostly. Patients with lupus pleuritis have higher pleural fluid pH (>7.35), higher pleural fluid glucose levels (>60 mg/dl), and lower pleural fluid LDH levels (<500 IU/L or < 2 times the upper limit of normal for serum) than patients with rheumatoid pleuritis and tubercular effusion.[<xref rid="ref12" ref-type="bibr">12</xref>] There are no available reports still yet of association of tubercular pleural effusion in a case of rhupus syndrome. The confirmatory diagnosis of tubercular pleural effusion is very difficult because of presence of very low number of bacilli in pleural fluid. The overall diagnostic sensitivity of acid-fast bacilli smear by Z-N staining is only 17.5% and Bactec culture is 45% in a recent study.[<xref rid="ref13" ref-type="bibr">13</xref>] The Bactec 460 TB system is very much superior to Lowenstein-Jensen (LJ) medium for detection of Mycobacteria especially for extrapulmonary tuberculosis.[<xref rid="ref14" ref-type="bibr">14</xref>] In our case tubercular pleural effusion was diagnosed by Bactec 460 culture. However, we would like to highlight the necessity of Bactec 460 culture in a case of pleural effusion in rhupus.</p><p>Treatment of arthritis in rhupus patients mainly involves symptomatic treatment with non-steroidal anti-inflammatory drugs in case of occasional arthralgia and disease modifying rheumatic drugs like hydroxyl chloroquine or methotrexate in case of persistent arthritis. In refractory rhupus patients, rituximab is a safe and effective therapeutic option.[<xref rid="ref15" ref-type="bibr">15</xref>]</p></sec> |
Common variable immunodeficiency disorder - An uncommon cause for bronchiectasis | <p>Bronchiectasis continues to be a common respiratory problem of varied etiology. Common variable immunodeficiency disorder (CVID) is an uncommon cause for bronchiectasis. However, the prevalence of bronchiectasis remains very high in patients with CVID. This remains largely an underdiagnosed entity as primary immunodeficiency is not suspected in adults as a cause of bronchiectasis and hence, serum immunoglobulin (Ig) levels are not measured routinely. In addition to bronchiectasis, patients with CVID usually present with various extrapulmonary symptoms. I report here a case of young man who presented with bronchiectasis and multisystem complains who was diagnosed as CVID.</p> | <contrib contrib-type="author"><name><surname>Panigrahi</surname><given-names>Manoj Kumar</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib> | Lung India : Official Organ of Indian Chest Society | <sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Bronchiectasis is a common respiratory disorder of diverse etiology. Usually, extensive investigation to identify the cause of bronchiectasis is not done in routine clinical practice as it is perceived to have little influence on its management. Conventionally, in absence of an obvious cause, bronchiectasis is often considered as post-infective or idiopathic. However, establishing a specific etiological diagnosis of bronchiectasis may be significant for guiding appropriately tailored therapy in some patients with a favorable outcome.[<xref rid="ref1" ref-type="bibr">1</xref><xref rid="ref2" ref-type="bibr">2</xref>] Primary humoral immunodeficiency (PHI), an underdiagnosed entity, comprises of heterogeneous group of disorders. Common variable immunodeficiency disorder (CVID) represents the most commonly diagnosed form of PHI. CVID usually manifests as recurrent respiratory infections that often lead to bronchiectasis later on. CVID remains underevaluated as an etiology of bronchiectasis.[<xref rid="ref3" ref-type="bibr">3</xref>] I present here a case of 34-year-old male diagnosed as CVID who presented with bronchiectasis and multisystem involvement.</p></sec><sec id="sec1-2"><title>CASE REPORT</title><p>A 34-year-old male presented with fever, cough with purulent expectoration, and increased breathlessness for a week. He had mild right chest pain and occasional abdominal pain. He gave history of recurrent respiratory infections since childhood. He had recurrent diarrhea with intermittent steatorrhea and right otorrhea for last 7 years. The stool consistency was semisolid mixed with mucus but no blood. He was a farmer, married, and had two children. He had never smoked. He received empirical antitubercular therapy twice in the past in the year 1994 and 2007 for suspected smear negative pulmonary tuberculosis and empyema thoracis, respectively. He was emaciated, had pallor and clubbing. On admission his vitals were as follows: Pulse rate = 96 beats/min, blood pressure = 90/60 mmHg, respiratory rate = 32/min, and SpO<sub>2</sub> = 95% in room air. Auscultation revealed normal breath sound with bilateral diffuse coarse crackles. Inflammatory arthritis was noted in small joints, ankle, and knee. Blood metabolic panel, electrolytes, and hemogram were normal except for mild anemia. Repeated sputum examination for acid fast bacilli was negative. Blood, urine, and stool cultures were sterile. Stool for occult blood was negative. Stool microscopy showed hookworm ova. Chest radiograph showed patchy opacities in right lower and left upper zones [<xref ref-type="fig" rid="F1">Figure 1</xref>]. High resolution computed tomography (HRCT) chest confirmed bilateral diffuse bronchiectasis [Figure <xref ref-type="fig" rid="F2">2a</xref> and <xref ref-type="fig" rid="F2">b</xref>]. In view of multisystem involvement and recurrent respiratory infections, serum immunoglobulin (Ig) estimation was done to rule out primary immunodeficiency. All Ig levels were significantly reduced (IgG < 1.37 g/l, IgA < 0.231 g/l, IgM < 0.17 g/l) (nephelometry) suggesting CVID. Antibody tests for human immunodeficiency virus, hepatitis B and C virus were negative. Sputum culture grew <italic>Pseudomonas aeruginosa</italic>. Spirometry demonstrated reversible airway obstruction. Evaluation of right otorrhea showed a small central perforation. Gastrodudonescopy was normal. Fibreoptic bronchoscopy revealed minimally inflamed airway and bronchoalveolar lavage microbiology was noncontributory. Considering the constellation of symptoms like recurrent respiratory infections/bronchiectasis, previous empyema, chronic diarrhea, suppurative otitis media, inflammatory arthritis, and low Ig levels; the diagnosis of CVID was made. He received intravenous ceftriaxone and ceftazidime for 2 weeks and recovered from the present infective exacerbation. Intravenous Ig (IVIG) therapy could not be administered as the cost was prohibitive for the patient.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Chest X-ray showing nonhomogenous opacity in right lower and left upper zones</p></caption><graphic xlink:href="LI-31-394-g001"/></fig><fig id="F2" position="float"><label>Figure 2</label><caption><p>(a) High resolution computed tomography (HRCT) chest showing bilateral diffuse bronchiectasis with lower lobes predominance. (b) HRCT chest showing upper lobes bronchiectasis</p></caption><graphic xlink:href="LI-31-394-g002"/></fig></sec><sec sec-type="discussion" id="sec1-3"><title>DISCUSSION</title><p>PHI is a broad terminology comprising of several disorders that share defective antibody production as common abnormality. Broadly PHI is divided into two groups: One having a defined genetic linkage (e.g. X-linked agammaglobulinemia and autosomal recessive agammaglobulinemia) and one of unknown genetic base. The latter group constitutes the largest one and includes CVID, IgG subclass deficiencies, IgA deficiency, and selective antibody deficiency. The first group of diseases appear mainly during childhood and the second group appears later on.[<xref rid="ref3" ref-type="bibr">3</xref>] Secondary immunodeficiency usually results from neoplasms like lymphoma, chronic lymphatic leukemia and myeloma, protein energy malnutrition, protein loosing enteropathy, severe trauma, corticosteroid, and cytotoxic drugs therapy and infections like human immunodeficiency virus and cytomegalovirus.[<xref rid="ref4" ref-type="bibr">4</xref>] PHI usually manifests as recurrent respiratory infections like sinusitis, bronchitis, pneumonia, and bronchiectasis. CVID remains the most common symptomatic form of PHI diagnosed in adulthood. This is characterized by hypogammaglobulinemia, recurrent bacterial infections and autoimmune dysfunction. The reported incidence of CVID varies from 1 in 10,000 to 1 in 50,000 and equal in both sexes.[<xref rid="ref3" ref-type="bibr">3</xref>] Familial occurrence is reported in about 20% cases. Majority present in their 2<sup>nd</sup> or 3<sup>rd</sup> decade. The average delay from the onset of symptoms to diagnosis ranges from 6 to 8 years.[<xref rid="ref5" ref-type="bibr">5</xref>] This delay is mainly due to two reasons: (i) Primary immunodeficiency is not suspected or considered in adults and (ii) the initial symptoms are usually considered to be nonspecific. For most patients, the early symptoms of CVID are recurrent sinusitis or bronchitis. Unfortunately, serum Ig levels are not measured routinely in such cases. The fundamental immunological defect in CVID is reduced number of switch memory B cells with a failure to produce significant antibody response to specific antigens. The diagnosis of CVID is made mostly by exclusion of secondary causes of immunodeficiency and other primary immunodeficiency disorders. Though there is no consensus definition of CVID, the most agreed one as proposed by European Society for Immunodeficiencies is reduced (below 2 standard deviations of the mean) levels of IgG with reduced IgA and/or IgM, together with failure to mount a significant antibody response to vaccination, in the absence of a known cause. Functional Ig deficiency should be considered in patients who have recurrent respiratory infections and normal Ig and complement levels. These patients fail to produce measurable serological response to pneumococcal vaccine and tetanus toxoid.[<xref rid="ref3" ref-type="bibr">3</xref>]</p><p>Bronchiectasis is a late manifestation in CVID and signatory to repeated infective insults in the past. Bronchiectasis remains the most common pulmonary pathology detected in CVID patients with a reported prevalence varying from 17 to 76%. HRCT scan is the single best imaging tool for the diagnosis and monitoring of bronchiectasis in CVID. Bronchiectasis is generally cylindrical, bilateral, and diffuse. It affects mostly the middle or lower lobes, and less commonly the upper lobes. The other pulmonary manifestations are emphysema, fibrosis, granulomatous disease mimicking sarcoidosis, and interstitial lung disease. In addition to lung, virtually any organ system can be involved in CVID. Extrapulmonary manifestations of CVID include recurrent diarrhea and malabsorption, autoimmune disorders like idiopathic thrombocytopenic purpura and autoimmune hemolytic anemia, and malignancy like lymphoma and gastric cancer. Presence of bronchiectasis and liver disease at diagnosis carries a poor prognosis in CVID patients. Bronchiectasis is not a feature in isolated IgA or IgM deficiencies but more likely if these occur in association with selective IgG subclass deficiencies.[<xref rid="ref3" ref-type="bibr">3</xref><xref rid="ref6" ref-type="bibr">6</xref>]</p><p>The differential diagnoses those closely mimic CVID include primary ciliary dyskinesia (PCD), adult-onset cystic fibrosis (CF), alpha-1 antitrypsin deficiency, and non-tuberculous mycobacterial (NTM) infection. Male infertility is a common feature in both PCD and adult-onset CF. However, presence of rhinitis since neonatal period and a classic triad of sinusitis, situs inversus, and bronchiectasis if present (in 50% cases) will differentiate PCD from adult-onset CF, the later may present with recurrent pancreatitis. The diagnosis of PCD is established by ultrastructural study of cilia, whereas sweat chloride estimation and/or genetic analysis is required in adult-onset CF. Alpha-1 antitrypsin deficiency is usually associated with concomitant emphysema and affects the lower lobes, whereas NTM infection has a predilection for middle lobe and lingula with nodular bronchiectasis and tree-in-bud appearance.</p><p>Subjecting every patient of bronchiectasis to an array of exhaustive investigations may not be justified. However, such an approach may be rewarding in well-selected group of patients.[<xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref8" ref-type="bibr">8</xref><xref rid="ref9" ref-type="bibr">9</xref>] In a recent study, a cause for bronchiectasis was identified in 74% (122/165) of patients that influenced the management in 61 (37%). Seven had primary immune deficiency.[<xref rid="ref7" ref-type="bibr">7</xref>] Another study involving 150 patients of bronchiectasis showed etiological diagnosis in 47% cases and this altered the management in 15%.[<xref rid="ref8" ref-type="bibr">8</xref>] Patients with immune deficiency are younger than those with idiopathic or post-infective bronchiectasis.</p><p>IVIG remains the mainstay of therapy in CVID. The standard recommendation for IVIG is 400-600 mg/kg body weight every 3-4 weeks. An IgG trough level (the IgG level before the next infusion) of at least 5 g/l should be attained. Patients with bronchiectasis or diarrhea may require higher IVIG doses (500-600 mg/kg) to reach the mandatory trough level. The dose, frequency and route of administration of IVIG should be individualized to achieve maximum success. Antimicrobial therapy is the other main component of CVID therapy, because Ig replacement alone may not adequately prevent or treat local and/or persistent infections. Intravenous antibiotic for a longer duration (10-14 days) is recommended for control of acute infective exacerbation of bronchiectasis in CVID patients to prevent relapse. Those with recurrent infections despite IVIG therapy may benefit from suppressive antibiotic therapy especially azithromycin given three times a week. Other usual therapy for bronchiectasis like bronchodilators, inhaled corticosteroids, and airway clearance techniques should be optimized for maximum benefit. In addition, the accompanying diseases and sequel of CVID require adequate treatment. Corticosteroids and cyclosporine A are effective for granulomatous manifestations and autoimmune diseases, although long-term treatment efficacy may be limited due to side effects. Newer agents like infliximab or etanercept have been successfully used in few cases.[<xref rid="ref3" ref-type="bibr">3</xref><xref rid="ref6" ref-type="bibr">6</xref>]</p><p>In conclusion, primary immunodeficiency should be considered as a possibility for bronchiectasis in young patients who have recurrent respiratory infections and multisystem involvement. Serum Ig estimation should be done in such patients.</p></sec> |
Granulomatosis with polyangitis with mononeuritis multiplex-immunosuppressives playing a double-edged sword | <p>A 52-year-old female was diagnosed with rheumatoid arthritis and was on methotrexate and prednisolone. She developed fever, cough, hemoptysis, and cavitary lesion on chest skiagram. She was put on antitubercular therapy without any improvement, meanwhile she developed painful right foot drop. Clinicoradiology and C-ANCA study confirmed the diagnosis of granulomatosis with polyangitis (GPA). She was started on cyclophosphamide, corticosteroid, and co-trimoxazole. While her treatment was being continued she showed significant improvement of pulmonary manifestations. About 1 year later, there was reappearance of fever, cough, and radiological opacity with oropharyngeal candidiasis. She became very ill with disseminated intravascular coagulation (DIC)-like features. Immunological markers were negative but bronchoalveolar lavage fluid study showed growth of <italic>Aspergillus spp</italic>. The patient was promptly put on intravenous voriconazole but unfortunately she succumbed to her illness.</p> | <contrib contrib-type="author"><name><surname>Mukherjee</surname><given-names>Subhasis</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Biswas</surname><given-names>Debabani</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Begum</surname><given-names>Shabana</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib><contrib contrib-type="author"><name><surname>Bhanja</surname><given-names>Pulakesh</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Misra</surname><given-names>Amartya Kumar</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Chatterjee</surname><given-names>Partha</given-names></name><xref ref-type="aff" rid="aff3">2</xref></contrib> | Lung India : Official Organ of Indian Chest Society | <sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Wegener's granulomatosis (WG), which has been recently renamed as Granulomatosis with polyangitis (GPA)[<xref rid="ref1" ref-type="bibr">1</xref><xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref3" ref-type="bibr">3</xref>] is a systemic vasculitis involving small and medium vessels. Although classical GPA presents with a triad of upper respiratory tract, pulmonary and kidney involvement, uncommonly patients with atypical presentation puts the clinicians in diagnostic as well as therapeutic dilemma. Here we report a case of GPA with mononeuritis multiplex with more than 1-year follow up showing the therapeutic challenges that clinicians may come across during the course of treatment.</p></sec><sec id="sec1-2"><title>CASE REPORT</title><p>A 52-year-female was on methotrexate and prednisolone since September 2011 with a diagnosis of rheumatoid arthritis. She developed fever and cough with scanty hemoptysis in March 2012; she was thought as a case of smear negative pulmonary tuberculosis in a patient on immunosuppressives and was put on antitubercular drugs. She took the antitubercular drugs for 2 months but her clinical condition worsened and she presented to our department. On further enquiry, she gave a history of symmetrical arthralgia of small and medium joints of hands for last 2 years without a definite history of joint swelling or morning stiffness and history of recurrent attacks of ear infections. She denied the history of oliguria, hematuria, skin lesions, oral or genital ulcer, or ocular symptoms. On examination, there was pallor, tachypnoea, tachycardia, but cyanosis and clubbing were absent, her blood pressure was 160/90 mm-Hg. Examination of the respiratory system was unremarkable apart from the presence of crepitations in both lower zones, examination of other systems were within normal limits. On the third day of admission, she developed tingling sensation in the right leg in the morning with development of painful sudden foot drop in the same day evening [<xref ref-type="fig" rid="F1">Figure 1</xref>]. Neurological examination revealed absent ankle jerk, very weak (1/5) power of both dorsiflexors (tibialis anterior, extensor hallucis longus and extensor digitorum longus and extensor digitorum brevis), and plantar flexors (gastrocnemius, soleus, flexor hallucis longus and flexor digitorum longus); she was also unable to evert or invert the right foot. Sensory testing revealed mixed sensory loss over anterolateral aspect of lower leg and right foot not conforming to any specific nerve area. Neurological examination was normal in all other limbs. Her ear examination revealed the presence of bilateral chronic suppurative otitis media.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Photograph showing right foot drop</p></caption><graphic xlink:href="LI-31-397-g001"/></fig><p>Complete hemogram revealed - hemoglobin - 8 gram/dl, total leucocyte count (TLC) - 14600/cmm with a neutrophilic predominance, blood sugar fasting was 156 mg/dl and postprandial sugar was 212 mg/dl. Serum urea and creatinine were within the normal limits and urine for routine and microscopic examination did not show any RBC or cast. Sputum for AFB was negative on two occasions. Chest X-ray PA view revealed consolidation and nodular shadows in both lower zones [<xref ref-type="fig" rid="F2">Figure 2</xref>]. Contrast-enhanced computed tomography (CECT) scan of thorax also corroborated with the chest x-ray findings [<xref ref-type="fig" rid="F3">Figure 3</xref>]. Immunological markers showed rheumatoid factor positive in high titer (100.8 IU), but anti-CCP antibody was negative; antinuclear factor (ANF) and anti-ds-DNA antibody were negative; cytoplasmic antineutrophilic cytoplasmic antibody (C-ANCA) was positive in high titer (96.7 IU by the ELISA method) but P-ANCA was negative. Nerve conduction velocity (NCV) revealed nonrecordable compound muscle action potential (CMAP) in right peroneal and grossly reduced CMAP in right tibial nerve with absent sensory nerve action potential (SNAP) in right sural nerve. Right tibial and peroneal “F” responses were absent and so was right “H” reflex. NCV study of all four limbs were consistent with sensory-motor axonal involvement in right tibial and peroneal nerves and was consistent with mononeuritis multiplex of the right leg. Echocardiography and ultrasound of abdomen were normal. Patient was diagnosed as GPA with pulmonary involvement and mononeuritis multiplex.</p><fig id="F2" position="float"><label>Figure 2</label><caption><p>Chest x-ray (March 2012) showing consolidation with multiple nodules in both lower zones</p></caption><graphic xlink:href="LI-31-397-g002"/></fig><fig id="F3" position="float"><label>Figure 3</label><caption><p>CT scan of thorax (March 2012) showing multiple nodules in both lower lobes</p></caption><graphic xlink:href="LI-31-397-g003"/></fig><p>She was immediately started on intravenous pulse cyclophosphamide (750 mg) on day 1 with intravenous pulse methylprednisolone 1 g for three consecutive days (day 1-3). Patient showed significant clinical improvement regarding pulmonary symptoms. She was then maintained on prednisolone 40 mg/day, oral cyclophosphamide 100 mg/day, and cotrimoxazole tablet. Follow-up chest X-ray showed significant improvement at the end of 3 months and near complete resolution of radiological lesions after 6 months [<xref ref-type="fig" rid="F4">Figure 4</xref>] with negative C-ANCA; but her foot drop persisted. Steroid was tapered gradually and was stopped at the end of 9 months while cyclophosphamide and co-trimoxazole were being continued. She was euglycaemic with oral hypoglycemic agents. In June 2013, she again developed fever with cough, anorexia, and dysphagia; on examination pallor was present and there were multiple ecchymoses over both hands. Examination of oropharynx revealed oropharyngeal candidiasis. Chest x-ray showed reappearance of consolidation with a cavity in right lower zone; sputum for AFB was negative on two occasions, blood counts showed anemia, TLC- 3100/cmm with neutropenia, thrombocytopenia (45000/cmm); serum urea, creatinine, blood sugar, electrolytes were within normal limit; serum bilirubin was 2.1 mg/dl with increased liver enzymes (SGPT- 274, SGOT- 233), though hepatitis profile was negative. C- ANCA was negative as well. Septicaemia with disseminated intravascular coagulation (DIC) was suspected; blood culture (aerobic and anaerobic) did not yield any growth and her coagulation profile showed prolongation of prothrombin time and activated partial thromboplastin time, with increased level of fibrin degradation products-compatible with the diagnosis of DIC. Cyclophosphamide was stopped and patient was put on broad spectrum antibiotics with intravenous meropenem and moxifloxacin with intravenous fluconazole, but she did not show any clinicoradiological response in next 48 h; current CECT Thorax showed cavity in the lower-right lobe with surrounding satellite nodules [<xref ref-type="fig" rid="F5">Figure 5</xref>]; fiberoptic bronchoscopy was performed and bronchoalveolar lavage fluid culture showed growth of <italic>Aspergillus spp</italic>. Fluconazole was replaced with intravenous voriconazole (6 mg/kg twice a day). Unfortunately, the patient succumbed from the invasive fungal infection on fifth day after starting voriconazole.</p><fig id="F4" position="float"><label>Figure 4</label><caption><p>Follow-up chest x-ray after 6 months showing near complete resolution of radiological opacities</p></caption><graphic xlink:href="LI-31-397-g004"/></fig><fig id="F5" position="float"><label>Figure 5</label><caption><p>CT scan of thorax (June 2013) showing single large cavitary lesion in lower-right lobe with satellite nodules</p></caption><graphic xlink:href="LI-31-397-g005"/></fig></sec><sec sec-type="discussion" id="sec1-3"><title>DISCUSSION</title><p>GPA is a systemic pauci-immune vasculitis associated with ANCA. The clinical entity was first described by Friedrich Wegener in 1936 and since then it is widely known as WG in the medical literature; the disease nosology was changed of late to GPA.[<xref rid="ref1" ref-type="bibr">1</xref><xref rid="ref3" ref-type="bibr">3</xref>] Pulmonary involvement is common, being present in 90% cases; manifestations are protean but multiple nodules with or without cavitation, fleeting pneumonia, unilateral or bilateral pleural effusion, and diffuse alveolar hemorrhage are common.[<xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref6" ref-type="bibr">6</xref>] Recurrent otitis media and epistaxis are common upper airway symptoms. Although neurological involvement is more prevalent in churg-strauss syndrome, it is seen in GPA as well. Rarely neurological manifestations may be the initial presentation.[<xref rid="ref7" ref-type="bibr">7</xref>] Peripheral neuropathy in the form of mononeuritis multiplex is the commonest neurological manifestation.[<xref rid="ref8" ref-type="bibr">8</xref>] Demonstration of necrotizing vasculitis and granuloma on biopsy is confirmatory for diagnosis of GPA, but a high index of clinical suspicion combined with a positive C-ANCA in high titer by the ELISA method is also sufficient for the diagnosis of GPA in the absence of biopsy.[<xref rid="ref9" ref-type="bibr">9</xref>] Prognosis of GPA has dramatically improved with the introduction of combined cyclophosphamide and prednisolone, with high remission and 5-year survival rate.[<xref rid="ref10" ref-type="bibr">10</xref>] Worsening of a patient during therapy or remission phase with reappearance of pulmonary symptoms and new opacity on chest x-ray are often due to flaring up or relapse of the primary pathology, but infections with organisms such as <italic>Mycobacterium tuberculosis</italic>, <italic>Pneumocystis jiroveci</italic>, or opportunistic fungal infections have to be seriously considered, too, specially because of ongoing immunosuppressive therapy for such a long duration.[<xref rid="ref11" ref-type="bibr">11</xref><xref rid="ref12" ref-type="bibr">12</xref>] Older age, renal involvement, and infection are found to be the adverse prognostic factors and infection is reported to be the one of the commonest cause of mortality.[<xref rid="ref11" ref-type="bibr">11</xref><xref rid="ref13" ref-type="bibr">13</xref>] Abtahi <italic>et al</italic>.[<xref rid="ref14" ref-type="bibr">14</xref>] reported a case of sinusal mucormycosis in a case of WG, 2.5 years after the initial diagnosis. Berenguer <italic>et al</italic>.[<xref rid="ref15" ref-type="bibr">15</xref>] and Noguiera[<xref rid="ref16" ref-type="bibr">16</xref>] <italic>et al</italic>. have also reported pulmonary mucormycosis mimicking disease relapse in patients with WG on immunosuppressive therapy. Invasive pulmonary aspergillosis in patients with GPA have also been reported in the literature.[<xref rid="ref17" ref-type="bibr">17</xref><xref rid="ref18" ref-type="bibr">18</xref>] Incidence of disseminated fungal infection leading to DIC is very rare but is associated with a fatal outcome with high mortality.[<xref rid="ref19" ref-type="bibr">19</xref>] In our case also the patient showed very good response to cyclophosphamide plus prednisolone therapy, but unfortunately developed oropharyngeal candidiasis and pulmonary aspergillosis with DIC and ultimately succumbed from the infection.</p><p>To conclude, in the management of systemic vasculitis, immunosuppressive agents can act as a double-edged sword; on one hand these are life saving and have improved the prognosis of vasculitides to a great extent and on the other hand they make patients more vulnerable to infections with opportunistic pathogens like fungi, which may disseminate and can be fatal. Clinicians should keep a very close vigil on the vasculitis patients on immunosuppresants for earliest detection and appropriate aggressive therapy to combat these opportunistic fungal infections.</p></sec> |
Endobronchial ultrasound guided needle aspiration of a paraspinal mass with prior failed multiple diagnostic interventions: A case report and literature review | <p>The increasing frequency of tuberculosis (TB) in both developed and developing countries has continued to make spinal TB an important health problem. The present case report is about a patient who presented to us with progressive back pain and paraspinal mass. We performed endobronchial ultrasound guided needle aspiration from the paraspinal mass. The cytology showed granulomatous inflammation suggestive of TB.</p> | <contrib contrib-type="author"><name><surname>Sinha</surname><given-names>Nishant</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Padegal</surname><given-names>Vivek</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Jermely</surname><given-names>Deepthi</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib><contrib contrib-type="author"><name><surname>Satyanarayana</surname><given-names>Satish</given-names></name><xref ref-type="aff" rid="aff3">2</xref></contrib><contrib contrib-type="author"><name><surname>Santosh</surname><given-names>H. K.</given-names></name><xref ref-type="aff" rid="aff4">3</xref></contrib> | Lung India : Official Organ of Indian Chest Society | <sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Spinal involvement occurs in less than 1% of patients with tuberculosis (TB).[<xref rid="ref1" ref-type="bibr">1</xref><xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref3" ref-type="bibr">3</xref>] Linear probe endobronchial ultrasound (EBUS), with the ability to perform real-time transbronchial needle aspiration (EBUS-TBNA), has increased the diagnostic possibilities for mediastinal and hilar lymphadenopathy, including lymph nodes metastasis of lung cancer, sarcoidosis, tuberculous lymphadenopathy, and lymphoma. However, there is limited experience on usefulness of EBUS-TBNA in the diagnosis of non-lymph node lesions like tubercular involvement of thoracic spine.</p></sec><sec id="sec1-2"><title>CASE REPORT</title><p>This was a case report of a 65-year-old gentleman who was admitted in the neurosurgery ward with gradually progressive back ache for 3 month. He had no history of cough, fever, loss of weight or appetite. Routine blood examinations were normal. Neurological examination, bowel and bladder functions were normal. Systemic examination was normal. Serum protein electrophoresis was normal. Skin test for <italic>Mycobacterium tuberculosis</italic> (Purified Protein Derivative with 5 TU) was negative. Serum Brucella titer was negative and he was seronegative for human immunodeficiency virus. There was no other significant medical illness. He was earlier treated empirically with anti-tuberculosis (TB) medications for 2 weeks, which the patient has stopped prior to coming to us.</p><p>Contrast enhanced magnetic resonance imaging (MRI) of dorsal spine showed predominantly enhancing soft-tissue swelling in the pre/para-vertebral and anterior epidural aspect of the spinal canal at T6-T7 level causing thecal sac compression. It extended superiorly up to T4 vertebral level and inferiorly until T8 vertebral level. There was no spinal instability or cord compression requiring any urgent neurosurgical intervention.</p><p>Computed tomography (CT) guided fine needle aspiration cytology (FNAC) was done from the posterior approach and cytology showed only degenerated cells and no definite comment could be made by the pathologist. CT guided tru-cut biopsy through the posterior approach to the right side of T7-T8 spine showed fibro adipose tissue, focal lymphoid aggregates, which were partially crushed. There were no definite granulomas or malignant cells. Bone marrow aspirate and bone biopsy of T7-T8 spine did not show granulomas, acid fast bacilli (AFB) or malignant cells. Second CT guided tru-cut biopsy also could not yield a diagnosis.</p><p>Subsequently rigid pleuroscopy guided biopsy was done from paravertebral tissue and mediastinal pleura under general anesthesia. The histopathological examination showed sclerainflammatory pathology and mediastinal pleural biopsy showed no definite pathology.</p><p>We were consulted when patient complained of acute onset breathlessness in the ward. CT pulmonary angiogram showed filling defects in the subsegmental branches of right and left pulmonary arteries suggestive of acute pulmonary embolism. The proximal extent of the mass was seen up to posterior carinal and subcarinal level [<xref ref-type="fig" rid="F1">Figure 1</xref>]. There was no pulmonary parenchymal abnormality, no adenopathy or pleural effusion. Subsequently, bronchoscopic guided endobronchial ultrasound transbronchial needle aspiration (EBUS-TBNA) was planned.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Computed tomography pulmonary angiogram image at level of main carina, showing vertebral body destruction and soft-tissue lesion at D4-D5 level</p></caption><graphic xlink:href="LI-31-401-g001"/></fig><p>Bronchoscopy was done using the linear EBUS scope (BF-UC 180F; Olympus Medical Systems, Japan) with a compatible endoscopic ultrasound unit (EU-M E1; Olympus Medical Systems, Japan). The patient received nebulized lignocaine (4% solution) immediately before the procedure. Conscious sedation with injection midazolam and fentanyl were used – 2 mg/25 mcg respectively titrated up to 6 mg/150 mcg to achieve a good level of sedation. Topical 10% lignocaine spray was applied in the oropharynx. The procedure was done in the supine position through the oral route. The paraspinal mass was visualized with the EBUS scope placed in the medial wall of right and left main bronchi, seen best with the scope placed in the medial wall of left main bronchus [<xref ref-type="fig" rid="F2">Figure 2</xref>]. TBNA specimens were obtained using a dedicated, disposable, 22-gauge, EBUS needle (NA-201SX-4022 Olympus Medical Systems, Japan), using the jabbing method under real-time ultrasound control. Continuous suction was applied with a dedicated 20 ml syringe (VacLok) while the catheter was moved back and forth for up to a maximum of 10 times. Four passes were made from each side of the carina with the scope placed along the medial wall of right and left main bronchi.</p><fig id="F2" position="float"><label>Figure 2</label><caption><p>Endobronchial ultrasound image of subcarinal area shows the mass with transbronchial needle aspiration needle in it. The mass appears as homogeneous well-defined round structure, in the upper part of image, with specs of calcification (whitish dots) in it</p></caption><graphic xlink:href="LI-31-401-g002"/></fig><p>Bedside cytology showed epitheliod cell granulomas suggestive of TB [<xref ref-type="fig" rid="F3">Figure 3</xref>]. AFB stain was negative in the TBNA smear. Cytology of the aspirate showed no malignant cells. Polymerase chain reaction (PCR) of the TBNA aspirate (multiplex PCR, <italic>Mycobacterium</italic>) was positive for <italic>M. tuberculosis</italic> species. Gram stain and culture of the aspirate ruled out pyogenic or fungal infection. Patient was started on isoniazid, rifampicin, ethambutol and pyrazinamide according to World Health Organization (WHO) recommended weight regimen. He was discharged with anti-tubercular medications and vitamin K antagonists for pulmonary embolism. After 1 month of follow-up, he had significant relief of back pain and repeat erythrocyte sedimentation rate was 30 mm/h when compared to earlier value of 105 mm/h, 1 month back. TBNA aspirate culture by <italic>Mycobacterium</italic> growth indicator tube (MGIT, colorimetric based method) was negative for <italic>M. tuberculosis</italic> species. At 3 months follow-up, patient had remarkable clinical improvement with complete subsidence of back pain.</p><fig id="F3" position="float"><label>Figure 3</label><caption><p>Photomicrograph of transbronchial needle aspiration aspirate showing Epitheliod cell Granulomas (H and E, ×400)</p></caption><graphic xlink:href="LI-31-401-g003"/></fig></sec><sec sec-type="discussion" id="sec1-3"><title>DISCUSSION</title><p>The first modern case of spinal TB was described in 1779 by Percival Pott.[<xref rid="ref4" ref-type="bibr">4</xref>] There are two distinct types of spinal TB, the classic form or spondylodiscitis and an increasingly common atypical form which is spondylitis without disc involvement.[<xref rid="ref5" ref-type="bibr">5</xref>] The anterior aspect of the verte-bral body adjacent to the subchondral plate is commonly involved.[<xref rid="ref6" ref-type="bibr">6</xref>]</p><p>Differential diagnosis of spinal TB includes pyogenic and fungal infections as well as metaststic and primary spinal tumors and it may be difficult when only clinical and radiographic findings are considered.[<xref rid="ref7" ref-type="bibr">7</xref>] CT provides bony detail while MRI evaluates the involvement of soft-tissue and abscess formation. In comparison to pyogenic discitis, the most distinguishing feature of spinal TB is bony destruction with relative preservation of the intervertebral disc and heterogeneous enhancement. In pyogenic discitis, bone destruction and homogenous enhancement is more frequently observed.[<xref rid="ref8" ref-type="bibr">8</xref>] The presence of an abscess and bone fragments differentiate spinal TB from neoplasia and if there is any doubt an image-guided biopsy is indicated.[<xref rid="ref9" ref-type="bibr">9</xref>] In 88.5-96.4% of the cases, a CT guided FNAC biopsy is helpful and yields a diagnosis.[<xref rid="ref10" ref-type="bibr">10</xref><xref rid="ref11" ref-type="bibr">11</xref>]</p><p>Spinal TB is a medical disease and anti-TB drugs have a main role in the recovery and response of patients.[<xref rid="ref12" ref-type="bibr">12</xref>] Combination of rifampicin, isoniazid, ethambutol and pyrazinamide for 2 months followed by combination of rifampicin and isoniazid for a total period of 6, 9, 12 or 18 months is the most frequent protocol used for treatment of spinal TB.[<xref rid="ref13" ref-type="bibr">13</xref>] The proposed regimen of WHO with total duration of 6 months consists of primary treatment with isoniazid, rifampicin, pyrazinamide and ethambutol for 2 months followed by 4 months of therapy with isoniazid and rifampicin. American Thoracic Society recommends 9 months of treatment with the same 4 drugs for initial 2 months followed by 7 months of therapy with isoniazid and rifampicin in the continuation phase, whereas the Canadian Thoracic Society recommends a total time of treatment as long as 9-12 months.</p><p>CT guided biopsy was attempted twice in this patient, but futile. Possible reasons could be: (1) The bulk of the lesion was pre-vertebral and located anteriorly in the spinal column. (2) The biopsy and FNAC were taken from the posterior spinal column along with the bone biopsy, which was taken from posterior spinal pedicle, and the bulk of lesion was located anteriorly in the spinal column. (3) As the patient was partially treated with anti-TB medication there might have been peripheral healing and aspirates taken from the periphery of the lesion repeatedly showed only degenerated cellular pathology.</p><p>Our patient presented with Pott's spine, however no definitive diagnosis could be made with the best available tests, hence we tried aspiration through endobronchial route via bronchoscopy (EBUS). To the best of our knowledge, this is the first reported case from India of EBUS TBNA from Pott's spine in a patient with acute pulmonary embolism.</p></sec><sec sec-type="conclusion" id="sec1-4"><title>CONCLUSION</title><p>Base on the above report it can be concluded that endobronchial ultrasound guide needle aspiration is a safe procedure for difficult to diagnose pre/para-vertebral soft-tissue lesions, provided the lesion involves D4 vertebral level (at the level of main carina). It can be tried as second choice if CT guided biopsy is negative and there is diagnostic dilemma.</p></sec> |
Primary amyloidoma of lung treated with radiation: A rare case report | <p>Amyloidosis is a collection of diseases in which different proteins are deposited as insoluble beta-pleated sheets, disrupting organ function. Distribution of these deposits may be diffuse or localized throughout the body, depending on the pathophysiology of the underlying amyloid type. Isolated deposition of amyloid proteins in lung is a very rare entity. They are frequently misdiagnosed as bronchogenic carcinoma, metastatic disease, or focal fungal infections. The treatment of solitary pulmonary amyloidosis is not well-defined. We have treated a 65-year-old female patient with external beam radiation and corticosteroids in palliative intent and she is leading a good quality of life after six months of follow up.</p> | <contrib contrib-type="author"><name><surname>Kapoor</surname><given-names>Rakesh</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Bhattacharyya</surname><given-names>Tapesh</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Bahl</surname><given-names>Amit</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Agarwal</surname><given-names>Ritesh</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib><contrib contrib-type="author"><name><surname>Bal</surname><given-names>Amanjit</given-names></name><xref ref-type="aff" rid="aff3">2</xref></contrib><contrib contrib-type="author"><name><surname>Gulati</surname><given-names>Ajay</given-names></name><xref ref-type="aff" rid="aff4">3</xref></contrib> | Lung India : Official Organ of Indian Chest Society | <sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Amyloidosis is a heterogenous group of disease characterized by deposition of misfolded insoluble proteins in extracellular space. Amyloidosis can be either primary or secondary, and deposition of those amyloid proteins can be systemic or localized. Localized amyloid deposition in respiratory tract or lung parenchyma is a rare entity.</p></sec><sec id="sec1-2"><title>CASE REPORT</title><p>A 65-year-old female presented with fever for eight months, which was insidious in onset, high-grade intermittent in nature. She also had a long-standing history of intermittent cough and shortness of breath. She smoked 4-6 bidis per day for last 40 years. The physical examination was unremarkable. Her routine blood investigations were within normal limits. CECT (Contrast-enhanced computed tomography) chest revealed an ill-defined 6 × 4 × 7 cm heterogeneously enhancing mass causing abrupt cut off the left lower lobe bronchus with the consequent distal collapse/consolidation of the left lower lobe. The left pulmonary artery is variably encased and attenuated by the lesion. The left inferior pulmonary vein is displaced anterioly by the lesion. Multiple enlarged conglomerate mediastinal lymph nodes with left-sided pleural effusion were also seen [<xref ref-type="fig" rid="F1">Figure 1</xref>]. Bronchoscopy revealed submucosal infiltration in left and right bronchial tree with nodules at secondary carina on left side and anterior segment of right upper lobe. Left lower lobe segment could not be visualized due to stenosis. Endobronchial biopsy revealed multiple fragments lined by respiratory epithelium with focal mucinous metaplasia. The subepithelium showed nodular deposits of eosinophilic, homogenous material with intervening scanty lymphomononuclear infiltrate. These nodular deposits were congophilic and showed apple green birefringence on polarized light. No granuloma or any evidence of malignancy was seen. Features were of amyloidoma [Figures <xref ref-type="fig" rid="F2">2</xref> and <xref ref-type="fig" rid="F3">3</xref>]. FNAC (Fine Needle Aspiration Cytology) from abdominal fat pad came out to be negative. Rest of the systemic amyloidosis work up was negative. Patient was not taken up for resection because of the vascular encasement of the lesion. She was treated with corticosteroids simultaneously along with palliative external beam radiation. Thirty Gy was delivered in ten fractions over two weeks period with photon energy of 6 MV with two fields (AP-PA portals). The steroids were tapered and stopped after one week. After six months of follow up, patient is leading a good quality of life with clinical and radiological complete response.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Contrast-enhanced computed tomography chest revealed an ill-defined 6 × 4 × 7 cm heterogeneously enhancing mass causing abrupt cut off the left lower lobe bronchus with the consequent distal collapse/consolidation of the left lower lobe</p></caption><graphic xlink:href="LI-31-404-g001"/></fig><fig id="F2" position="float"><label>Figure 2</label><caption><p>Endobronchial biopsy revealed multiple fragments lined by respiratory epithelium with focal mucinous metaplasia. The subepithelium showed nodular deposits of eosinophilic, homogenous material with intervening scanty lymphomononuclear infiltrate. These nodular deposits were congophilic</p></caption><graphic xlink:href="LI-31-404-g002"/></fig><fig id="F3" position="float"><label>Figure 3</label><caption><p>Nodular deposits of eosinophilic, homogenous material with intervening scanty lymphomononuclear infiltrates showing apple green birefringence</p></caption><graphic xlink:href="LI-31-404-g003"/></fig></sec><sec sec-type="discussion" id="sec1-3"><title>DISCUSSION</title><p>Amyloidosis is a disease characterized by deposition of abnormal proteins in extracellular tissue. The term amyloid was first used by Rudolf Virchow in 1854.[<xref rid="ref1" ref-type="bibr">1</xref>] Amyloidosis localized to respiratory tract was first recognized by Lesser in 1877.[<xref rid="ref2" ref-type="bibr">2</xref>] The locations of the isolated forms of pulmonary amyloidosis are divided into three main anatomical areas; large airway amyloidosis of the larynx and bronchus, parenchymal pulmonary amyloidosis, and mediastinal amyloid.[<xref rid="ref3" ref-type="bibr">3</xref>] Tracheobronchial amyloidosis is the most common form of localized primary pulmonary amyloidosis.[<xref rid="ref4" ref-type="bibr">4</xref>] Patients with tracheobronchial amyloidosis present with obstructive manifestations such as cough, dyspnea, and hemoptysis. The second pattern of pulmonary involvement is nodular parenchymal amyloidosis, which is characterized by circumscribed nodule with occasional interstitial and perivascular extension. Clinically, those lesions resemble a neoplasm.[<xref rid="ref5" ref-type="bibr">5</xref>] The amyloidoma of the lung, when solitary, may be confused with primary bronchogenic carcinoma. When the lesions are multiple, it may be difficult to differentiate these from metastatic cancer of the lung. The slow progression of the lesions, the presence of calcification, and the absence of a primary tumor would suggest the non-malignant nature of the lesion; however, there are no reliable clinical or roentgenographic criteria that can help in making diagnosis of amyloidoma. Diffuse amyloid deposition in the lung parenchyma is usually associated with systemic AL amyloidosis. It is characterized by widespread amyloid deposition involving small vessels and the interstitium.[<xref rid="ref6" ref-type="bibr">6</xref>]</p><p>Solitary pulmonary nodular amyloidosis is an uncommon diagnosis. More common conditions that present similarly should be excluded first, such as neoplastic, infectious, or inflammatory conditions. In an event when clinical findings, radiographic appearances, and pathological conclusions are incongruent with these common diagnoses, amyloid of the lung should be considered.[<xref rid="ref7" ref-type="bibr">7</xref>]</p><p>Due to the variability of amyloid involvement in the pulmonary system, the treatment for respiratory amyloidosis ranges from observation to bronchoscopic or surgical resection based on severity and symptomatology. Management decisions are mostly based on an individual basis. Systemic amyloidosis can be treated with chemotherapy while localized forms are typically treated with local interventions. Infiltrative and systemic disease has been amenable to chemotherapy with oral melphalan and prednisolone.[<xref rid="ref8" ref-type="bibr">8</xref>] A prospective study done at Mayo Clinic in patients with systemic amyloidosis showed that combination therapy with melphalan, prednisone, and colchicine or melphalan and prednisone resulted in prolonged survival compared to colchicine alone. The median duration of survival after randomization was 8.5 months in the colchicine group, 18 months in the group assigned to melphalan and prednisone, and 17 months in the group assigned to melphalan, prednisolone, and colchicine.[<xref rid="ref9" ref-type="bibr">9</xref>] Excisional therapy is the standard approach to managing TBA (Tracheobronchial Amyloidosis). In some nodular forms of TBA, neodymium: Yttrium-aluminum-garnet (Nd: YAG) laser treatment removes tissue and eliminates further amyloid deposition in the field, perhaps by laser-induced thermal injury to underlying plasma cells.[<xref rid="ref10" ref-type="bibr">10</xref><xref rid="ref11" ref-type="bibr">11</xref>]</p><p>External beam radiotherapy has been successfully utilized in tracheobronchial amyloidosis in a patient believed not to be a candidate for endoluminal therapy due to the diffuse nature of the airways disease.[<xref rid="ref12" ref-type="bibr">12</xref>] A total of 24 Gy was delivered in 12 fractions, and colchicine was given as an adjunctive therapy. Improvements were measured by sequential pulmonary function testing, radiographic imaging, bronchoscopic evaluation, and performance status. Kurrus <italic>et al</italic>. published a case report demonstrating a benefit of external beam radiation (20 Gy in 10 fractions over two weeks) in causing local response in a patient with localized tracheobronchial disease. They treated the patient based on the hypothesis that plasma cells that secrete amyloidogenic protein are radiosensitive.[<xref rid="ref13" ref-type="bibr">13</xref>] Other hypotheses proffered include radiation injures cells other than plasma cells that may secrete amyloidogenic proteins, and free radicals generated by radiation may modify and enhance the degradation of amyloid protein deposits. Combined endobronchial and radiation therapy have demonstrated a similar beneficial effect.[<xref rid="ref14" ref-type="bibr">14</xref>]</p><p>The index patient has also responded very well both clinically and radiologically with radiotherapy. Hence, radiotherapy can be a viable option for the treatment of solitary pulmonary amyloidoma.</p></sec> |
Shrinking lung syndrome in systemic lupus erythematosus-scleroderma overlap | <p>Shrinking lung syndrome (SLS) is a infrequently reported manifestation of systemic lupus erythematosus (SLE). Reported prevalence of SLS is about 0.5% in SLE patients. Pathogenesis is not fully understood and different therapeutic modalities have been employed with variable results, as only 77 cases of SLS have been documented in literature. SLS in SLE-Scleroderma overlap has not been reported yet. We report a patient of SLE - scleroderma overlap presenting with dyspnea, intermittent orthopnea and pleuritic chest pain. Evaluation revealed elevated hemidiaphragms and severe restrictive defect. She was eventually diagnosed as a case of SLS. This case report is a reminder to the medical fraternity that SLS although a rare complication must be thought of in the special subset of patients of SLE having respiratory symptoms.</p> | <contrib contrib-type="author"><name><surname>Guleria</surname><given-names>Vivek S.</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Singh</surname><given-names>Pradeep K.</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Saxena</surname><given-names>Puneet</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Subramanian</surname><given-names>Shankar</given-names></name><xref ref-type="aff" rid="aff1"/></contrib> | Lung India : Official Organ of Indian Chest Society | <sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Pleuropulmonary manifestations occur in 60-80% of patients with systemic lupus erythematosus (SLE), frequently in the form of pleuritis with or without pleural effusion, pneumonia, interstitial fibrosis, acute lupus pneumonitis and pulmonary hypertension.[<xref rid="ref1" ref-type="bibr">1</xref>] Shrinking lung syndrome (SLS) is an uncommon pulmonary manifestation of SLE, although the exact prevalence is not known, as only 77 cases of SLS have been documented in the literature.<sup>[</sup><sup>2]</sup> SLS in SLE-scleroderma overlap has not been reported so far. This condition might be unrecognized and the estimates of the prevalence may be inaccurate. The pathogenesis of SLS is not yet clear and different therapeutic approaches have been employed with variable success. We describe here a patient with SLE-scleroderma overlap presenting with pulmonary complaints and eventually being diagnosed as SLS.</p></sec><sec id="sec1-2"><title>CASE REPORT</title><p>A 28-year-old woman, a resident of Maharashtra, India, presented in January 2013 with 2 years history of Raynaud's phenomenon, polyarthralgia, recurrent oral ulcers and diffuse hair loss from scalp. She continued to have these symptoms off and on since 2011; however, since last 2 months she had developed progressive darkening and tightening of skin (left more than right), restricted mouth opening and dysphagia especially to solids. On clinical examination she was having pallor, induration and hyperpigmentation of limbs, sclerodactyly with fixed flexion deformities in fingers of left hand, ulcers in buccal mucosa, diffuse alopecia, restricted mouth opening (3 cm) [<xref ref-type="fig" rid="F1">Figure 1</xref>] and chest expansion of 2 cm. Her systemic examination was essentially normal.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Reduced mouth opening (03 cm)</p></caption><graphic xlink:href="LI-31-407-g001"/></fig><p>Laboratory investigations [<xref ref-type="table" rid="T1">Table 1</xref>] showed anemia. Liver and kidney function tests were within normal range except for 24 hours urinary protein of 400 mg/day. Immunological work up revealed antinuclear antibody (ANA) and anti-ds DNA positive, anti-centromere antibody: 1.56 μ/ml (<3.0), SCL-70: 2.53 U/ml (<3.00), anti-Sm antibody: 97.94 IU/L (>15 positive), anticardiolipin antibodies (ACLA) IgG positive and anti-neutrophil cytoplasmic antibodies (ANCA) negative. A renal biopsy performed on her showed ANCA-negative pauci immune necrotizing glomerulonephritis.</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Investigations of the patient</p></caption><graphic xlink:href="LI-31-407-g002"/></table-wrap><p>She was thus diagnosed as a case of SLE-scleroderma overlap and started on oral prednisolone 40 mg OD, mycophenolate mofetil (MMF) 500 mg BD, hydroxychloroquine (HCQ) 200 mg BD. She was stabilized on this treatment and discharged.</p><p>In March 2013, while on treatment, she developed dyspnea on exertion with inability to walk for even 100 m on even ground, associated with intermittent episodes of orthopnea. She also complained of right sided chest pain which was pricking type exacerbated on deep inspiration with no history of cough or hemoptysis. Clinically she had tachycardia (pulse 110/min), tachypnea (respiratory rate 28/min, rapid shallow breathing) with normal saturation. Jugular venous pressure was not raised and there was no pedal edema. Chest examination showed bilaterally reduced lung volumes and chest expansion. Breath sounds were normal with no adventitious sounds. Second heart sound was not accentuated.</p><p>Chest radiograph showed elevation of both hemidiaphragms (right more than left) and bilaterally reduced lung volumes. Lung fields were normal [<xref ref-type="fig" rid="F2">Figure 2</xref>] with high-resolution computed tomography (HRCT) of the chest being normal [<xref ref-type="fig" rid="F3">Figure 3</xref>]. Ultrasonography of abdomen with sniff test revealed no infradiaphragmatic pathology or diaphragmatic palsy. Her 2D echocardiography was normal. Pulmonary function tests showed severe restrictive defect [<xref ref-type="table" rid="T2">Table 2</xref>]. She could not hold her breath for DLCO maneuver which, thus, could not be done.</p><fig id="F2" position="float"><label>Figure 2</label><caption><p>Chest radiograph showing elevation of both hemidiaphragms (right more than left) and bilaterally reduced lung volumes right hemidiaphragm</p></caption><graphic xlink:href="LI-31-407-g003"/></fig><fig id="F3" position="float"><label>Figure 3</label><caption><p>Normal HRCT scan</p></caption><graphic xlink:href="LI-31-407-g004"/></fig><table-wrap id="T2" position="float"><label>Table 2</label><caption><p>Pulmonary function tests</p></caption><graphic xlink:href="LI-31-407-g005"/></table-wrap><p>Arterial blood gas analysis showed pH 7.53, pO<sub>2</sub> 74.9, HCO<sub>3</sub> 19.8, pCO<sub>2</sub> 29.3. Brain natriuretic peptide (BNP) was normal. Based on these findings she was diagnosed as SLS and metered dose inhalers (with spacer) Formetrol + Budesonide (200 μg) 02 puffs BD were started along with tab Deriphylline (Theophyllin) 150 mg BD and oral prednisolone was continued. She responded to the treatment and her orthopnea improved after 2 days of therapy. Breathlessness gradually improved over next 5 days and the patient was eventually discharged.</p></sec><sec sec-type="discussion" id="sec1-3"><title>DISCUSSION</title><p>Pleuropulmonary involvement in SLE occurs in 60 to 80% of patients.[<xref rid="ref1" ref-type="bibr">1</xref><xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref3" ref-type="bibr">3</xref>]</p><p>SLS is a rare complication of SLE, with a reported prevalence of 0.5% of this overall population. First named in 1965 by Hoffbrand and Beck[<xref rid="ref4" ref-type="bibr">4</xref>] SLS was used to describe a SLE patient who presented with dyspnea, radiological evidence of raised diaphragm, and a restrictive pattern of pulmonary function test. The precise pathogenetic mechanism underlying the SLS remains to be elucidated. Studies found no evidence of major parenchymal lung or pleural disease on thoracic CT scanning.[<xref rid="ref2" ref-type="bibr">2</xref>] Pulmonary surfactant deficiency was initially thought to be the cause of alveolar microatelectasis and hyaline membrane formation. Later investigators found abnormalities in transdiaphragmatic pressures consistent with diaphragm dysfunction.[<xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref6" ref-type="bibr">6</xref>]</p><p>Phrenic nerve involvement and myopathy were thought to be the cause of diaphragm dysfunction,[<xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref7" ref-type="bibr">7</xref>] but this has not been established till date. The majority of neurophysiological studies yielded normal nerve conduction velocity that excluded the presence of a demyelinating neuropathy as the cause of diaphragmatic weakness. Pérez <italic>et al</italic>.[<xref rid="ref8" ref-type="bibr">8</xref>] reported a case of SLS caused by lupus myopathy, who went into respiratory failure. The authors proposed that this may be due to infiltration of the chest wall muscles and the diaphragm by T lymphocytes (as they had demonstrated T cells in the deltoids sample). However, elevation of CPK, which is an indicator of myositis, has not been reported in patients with SLS.</p><p>Our patient presented with classical triad of SLS namely, dyspnea, raised diaphragm and a restrictive pulmonary defect. Parenchymal lung disease was unlikely with clinical examination and normal chest X-ray (other than small lung volumes and raised hemidiaphragms) and HRCT scan. Cardiovascular cause was ruled out by an essentially normal echocardioy and BNP. Reduced total lung capacity with preserved residual volume and normal FEV1/FVC and absence of air trapping on radiology points towards an extrapulmonary/neuro-muscular cause of restriction as is expected in SLS. This probably is the first reported case of SLS in SLE-scleroderma overlap.</p><p>She was already on steroids; theophylline and MDI of β-agonist + inhaled steroids were added to her prescription to which she responded subjectively with resolution of orthopnea. These drugs are thought to improve contractility by acting on beta receptors on the diaphragm. Since the pathogenesis is not fully understood, different therapeutic approaches were reported to treat SLS. However, no RCTs have been carried out or consensus reached regarding optimal therapy.</p></sec><sec sec-type="conclusion" id="sec1-4"><title>CONCLUSION</title><p>We have reported a case of SLS in a patient of SLE-Scleroderma overlap. Although this is a rare manifestation of the disease process but must be kept in hindsight in a patient of SLE who has dyspnea and orthopnea and is found to have normal chest examination and imaging with a restrictive pattern of PFT. The pathogenesis and optimal therapy for this condition has not yet been fully elucidated and continued research and possibly a RCT would be required to layout them out clearly.</p></sec> |
Pulmonary tumor thrombotic microangiopathy in an unknown primary cancer | <p>Pulmonary tumor thrombotic microangiopathy (PTTM) is a highly fatal complication of cancer leading to acute cor pulmonale and pulmonary hypertension. We present a case of 47-year-old male patient who developed acute breathlessness and died suddenly. The pulmonary vessels at autopsy on histopathologic examination showed the presence of fibrocellular intimal proliferation, fibrin thrombi and few tumor emboli consisting of malignant adenocarcinoma cells. There was associated lymphangiosis carcinomatosis. No primary visceral tumor was found despite extensive search. The patient had died following acute cor pulmonale with sudden pulmonary hypertension due to PTTM. This entity (PTTM) must be kept as a differential diagnosis in patients presenting with acute breathlessness especially in cases of cancers.</p> | <contrib contrib-type="author"><name><surname>Amonkar</surname><given-names>Gayathri P.</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Jashnani</surname><given-names>Kusum D.</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Pallewad</surname><given-names>Sandhya</given-names></name><xref ref-type="aff" rid="aff1"/></contrib> | Lung India : Official Organ of Indian Chest Society | <sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Pulmonary tumor thrombotic microangiopathy (PTTM) also known as carcinomatosis endarteritis is a highly fatal respiratory complication seen in cases of malignancies especially adenocarcinomas of the stomach.[<xref rid="ref1" ref-type="bibr">1</xref><xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref3" ref-type="bibr">3</xref>] It was first described by von Herbay <italic>et al</italic>.[<xref rid="ref1" ref-type="bibr">1</xref>] There are only around 80 cases reported in literature so far including less than 10 cases where the primary malignancy has not been detected even after extensive search.[<xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref5" ref-type="bibr">5</xref>] The clinical diagnosis is extremely difficult and is most often detected at autopsy. The patients develop sudden pulmonary hypertension and acute cor pulmonale.[<xref rid="ref6" ref-type="bibr">6</xref>]</p></sec><sec id="sec1-2"><title>CASE REPORT</title><p>We present an autopsy case of a 47-year-old male patient who developed acute breathlessness and died suddenly. At autopsy there was mild right ventricular dilatation and lungs showed few firm areas and prominent bronchovasculature. No mass lesion was noted anywhere. On microscopy, the lungs showed the presence of florid fibrocellular intimal proliferation of the arterioles and small arteries [<xref ref-type="fig" rid="F1">Figure 1</xref>]. Vessels showed fibrin thrombi and occasional vessel showed tumor emboli composed of neoplastic adenocarcinoma cells showing glandular and occasional signet ring morphology. There was evidence of lymphangiosis carcinomatosis [<xref ref-type="fig" rid="F2">Figure 2</xref>]. There were similar tumor emboli in the myocardium, peripancreatic tissue, and perigastric lymph nodes [<xref ref-type="fig" rid="F3">Figure 3</xref>]. Despite extensive sectioning no primary tumor was found. The immunohistochemistry results showed positivity for pan keratin indicating an epithelial tumor. Tumor cells were negative for CK 7, CK10, TTF 1, and PSA. The final cause of death was given as acute cor pulmonale following pulmonary tumor thrombotic microangiopathy in an unknown primary adenocarcinoma.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Lungs showing presence of florid fibrocellular intimal proliferation with fibrin thrombi of the arterioles and small arteries (H and E, ×100)</p></caption><graphic xlink:href="LI-31-410-g001"/></fig><fig id="F2" position="float"><label>Figure 2</label><caption><p>Lungs showing presence of lymphangiosis carcinomatosis (H and E, ×100)</p></caption><graphic xlink:href="LI-31-410-g002"/></fig><fig id="F3" position="float"><label>Figure 3</label><caption><p>Signet ring cells seen in the lymph nodes (H and E, ×400)</p></caption><graphic xlink:href="LI-31-410-g003"/></fig></sec><sec sec-type="discussion" id="sec1-3"><title>DISCUSSION</title><p>PTTM is characterized by intimal proliferation in pulmonary small arteries and arterioles with or without tumor emboli leading to vascular stenosis and pulmonary hypertension. There is no distinct pulmonary tumour mass or interstitial involvement. It was first described by von Herbay <italic>et al</italic>.[<xref rid="ref1" ref-type="bibr">1</xref>] There are around 80 cases reported in literature so far. In our case also there was no mass lesion or parenchymal involvement by the tumor. There was florid fibrointimal proliferation, fibrin thrombi with some showing occasional tumor emboli and lymphangimatosis carcinomatosis. The tumor cells in this condition invade the pulmonary vascular system occluding the small arteries, arterioles and activate coagulation systems releasing inflammatory mediators, growth factors like serotonin, vascular endothelial growth factor and osteopontin. Some authors have proposed screening with serum VEGF and D - dimer testing for early detection of PTTM.[<xref rid="ref7" ref-type="bibr">7</xref>] The primary sites of malignancy reported are gastrointestinal tract, ovary, breast, pancreas, liver, uterus, gall bladder, and prostate.[<xref rid="ref8" ref-type="bibr">8</xref>] However, rarely there are reported cases where primary has not been detected inspite of extensive search and immunohistochemical stains.[<xref rid="ref4" ref-type="bibr">4</xref>] In our case too despite an extensive search no primary mass was found. Also immunohistochemistry was equivocal with no specific markers positive. Thus, our case belongs to the rare set of PTTM with unknown primary of which only less than 10 cases have been reported.[<xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref5" ref-type="bibr">5</xref>] PTTM is a rare condition with very high mortality. It should be distinguished from ordinary pulmonary thromboembolism and primary pulmonary hypertension and should be considered as a differential diagnosis in patients with acute respiratory symptoms especially in cases with an underlying cancer. There are only occasional case reports of antemortem diagnosis with patient recovering completely, and hence it is important to have a higher clinicopathological awareness of diagnosing this highly fatal condition.[<xref rid="ref9" ref-type="bibr">9</xref>] Role of 2-(F-18)-fluoro-2-deoxy-d-glucose positron emission tomography (FDG-PET) has been described in literature and has been described to be helpful for the diagnosis of PTTM.[<xref rid="ref10" ref-type="bibr">10</xref><xref rid="ref11" ref-type="bibr">11</xref>]</p></sec> |
An extremely unusual presentation of isolated extrathoracic sarcoidosis of submandibular lymph node in a child | <p>A 12-year-old male child presented with left submandibular lymphadenopathy; excision biopsy revealed noncaseating granuloma with numerous Schaumann bodies in histopathology, suggestive of isolated extrathoracic sarcoidosis, which is an extremely rare entity in the pediatric age group.</p> | <contrib contrib-type="author"><name><surname>Agrawal</surname><given-names>Anand</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Singh</surname><given-names>Kulwant</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib><contrib contrib-type="author"><name><surname>Parihar</surname><given-names>Dheeraj</given-names></name><xref ref-type="aff" rid="aff3">2</xref></contrib><contrib contrib-type="author"><name><surname>Chandermani</surname><given-names/></name><xref ref-type="aff" rid="aff1"/></contrib> | Lung India : Official Organ of Indian Chest Society | <sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>A Norwegian dermatologist Caesar Boeck coined the term “sarcoidosis” in 1899.[<xref rid="ref1" ref-type="bibr">1</xref>] Although this disease affects all age groups, it usually develops before the age of 50 years with a peak incidence during the third and fourth decade of life[<xref rid="ref2" ref-type="bibr">2</xref>] and a predilection for the female sex. It is a multisystem disease and is under-reported in our country on account of its similarity to tuberculosis, which is further compounded by the lack of awareness among physicians and pathologists. The frequency of detection of this disorder varies between 61.2/100,000 (Delhi) and 150/100,000 (Kolkata),[<xref rid="ref3" ref-type="bibr">3</xref>] but isolated extrathoracic sarcoidosis in the pediatric age group is rarely reported, which we report here.</p></sec><sec id="sec1-2"><title>CASE REPORT</title><p>A 12-year-old boy reported to the outpatient department with the chief complaint of a painless swelling in the left submandibular region since four years. It was a lymph node swelling which had gradually progressed from its initial size of a pea around 1 cm in diameter to its current size of a small lemon around 5 cm in diameter. The swelling was firm, nontender, nonadherent to skin, cold to the touch, and showed no fluctuation or discharging sinus <italic>in situ</italic>. There was no associated history of fever, weight loss, or any other comorbidity. There was no significant family history of a similar illness or tuberculosis. Systemic examination was unremarkable except for the solitary lymph node swelling. Blood counts were within normal range [hemoglobin (Hb): 12 g%; total leukocyte count (TLC): 6,000; differential leukocyte count (DLC): Polymorphs (P): 67%, lymphocytes (L): 31%, eosinophils (E): 2%, monocytes (M): 1%] with an erythrocyte sedimentation rate (ESR) of 5 mm in the first hour. Renal function tests and liver function tests were within normal range. Serum calcium was within the normal limit, and angiotensin-converting enzyme (ACE) was 52 IU/L just at the higher end. Mantoux test showed an induration of 9 mm diameter after 72 hours (the patient had a history of BCG vaccination). Ultrasonography (USG) of the abdomen showed no abnormality; other systems were also normal. Skiagram of the chest in posteroanterior (PA) view and computed tomography (CT) of the thorax was also normal for the age [<xref ref-type="fig" rid="F1">Figure 1</xref>]. Systemic examination was unremarkable. Fine needle aspiration cytology (FNAC) of the lymph node showed granulomatous inflammation. The lymph node was excised under general anesthesia, and histopathologic study revealed noncaseating granulomatous inflammation [<xref ref-type="fig" rid="F2">Figure 2</xref>] with numerous Schaumann bodies present in the background suggestive of sarcoidosis [<xref ref-type="fig" rid="F3">Figure 3</xref>]. The site of excision was completely healed one week after the excision, and the patient was put on systemic steroid therapy.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Computed tomography of thorax: Normal for age</p></caption><graphic xlink:href="LI-31-413-g001"/></fig><fig id="F2" position="float"><label>Figure 2</label><caption><p>Giant cell noncaseating granuloma</p></caption><graphic xlink:href="LI-31-413-g002"/></fig><fig id="F3" position="float"><label>Figure 3</label><caption><p>Granuloma with Schaumann bodies in multinucleated giant cell: ×400 magnification</p></caption><graphic xlink:href="LI-31-413-g003"/></fig></sec><sec sec-type="discussion" id="sec1-3"><title>DISCUSSION</title><p>Sarcoidosis was first identified by two dermatologists working independently, Dr. Jonathan Hutchinson in England and Dr. Caesar Boeck in Norway. Hence, it was originally called Hutchinson's disease or Boeck's disease.[<xref rid="ref4" ref-type="bibr">4</xref>] Sarcoidosis is less common in children than in adults with an incidence of 0.06 cases per 100,000 in children below four years of age, gradually increasing to 1.02 cases per 100,000 in adults in a Danish study.[<xref rid="ref1" ref-type="bibr">1</xref>] It commonly involves the lung, eyes, and skin, but in 2% cases, isolated extrathoracic sarcoidosis has been observed. It is also reported that extrathoracic sarcoidosis is more common in females than in males (36.7% vs. 28.6%).[<xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref6" ref-type="bibr">6</xref>] Although the etiology of sarcoidosis is unknown, various studies have proven its association with exposure to irritants found in the rural setting like wood-burning, tree pollen, inorganic particles, insecticides, and moldy environment. <italic>Mycobacterium</italic>[<xref rid="ref7" ref-type="bibr">7</xref>] and <italic>Propionibacterium</italic>[<xref rid="ref8" ref-type="bibr">8</xref>] have also been reported as etiological factors. In recent studies, association has also been found between class 1 HLA-B8 (HLA: Human Leukocyte Antigen) and acute sarcoidosis.[<xref rid="ref1" ref-type="bibr">1</xref>]</p><p>Sarcoidosis is characterized by its pathological hallmark: The noncaseating granuloma. A variety of inclusions like crystalline inclusions and colorless refractile crystals composed predominantly of calcium oxalate are frequently found in the giant cells of the granuloma of sarcoidosis. These many serve as nidi for deposition of calcium leading to the formation of Schaumann (conchoidal) bodies.[<xref rid="ref5" ref-type="bibr">5</xref>] Typical laboratory findings of sarcoidosis like ACE, lysozyme, and calcium were nondiagnostic in this patient. USG of the abdomen and CT of the thorax are also important modalities in diagnosis, but the diagnosis was established only by histopathological examination of the excised lesion. The present case study is extremely rare in the context of its isolated extrapulmonary location and involvement of the pediatric age group. Although a few case reports of isolated extrapulmonary sarcoidosis have been reported by clinicians, in the pediatric age group, such a unique case has not been reported to the best of the authors’ knowledge.</p><p>Being a systemic disease, sarcoidosis can affect any organ in the body, but isolated sarcoidosis may sometimes create a diagnostic dilemma. The high prevalence of tuberculosis in our country prevents the specialist from confirming the cause of lymphadenitis by excision biopsy, and the same is responsible for reporting fewer cases of sarcoidosis in this part of the world. The key message delivered by the present case is to perform excision biopsy to rule out other causes of lymphadenitis and to confirm the diagnosis before starting antitubercular therapy on clinical suspicion alone.</p></sec> |
Completely opaque hemithorax | Could not extract abstract | <contrib contrib-type="author"><name><surname>Chawla</surname><given-names>Rakesh K.</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Madan</surname><given-names>Arun</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib><contrib contrib-type="author"><name><surname>Das</surname><given-names>Kamanasish</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Chawla</surname><given-names>Aditya</given-names></name><xref ref-type="aff" rid="aff3">2</xref></contrib> | Lung India : Official Organ of Indian Chest Society | <sec id="sec1-1"><title>CASE REPORT</title><p>A 45-year-old male patient, normotensive, non-diabetic, married with children, auto parts dealer by profession, presented to us with complaints of cough with expectoration, low grade fever and breathlessness from past 15 days. He gave no history of previous diseases or surgical intervention. General examination was unremarkable. On respiratory system examination, the left sided shoulder was drooping; trachea deviated to left and apex beat palpable in fifth intercostal space at left mid axillary line. On percussion, impaired to dull note was observed all over the left side. On auscultation, breath sound were absent in the left axillary and infra axillary areas; decreased intensity breath sounds could be heard in the left inter scapular and infra scapular areas. Occasional crepitations were heard over the right hemithorax. CVS (Cardio-vascular system), CNS (Central nervous system) and per-abdominal examinations revealed no abnormality. TLC (Total leukocyte count) was 12,400/mm<sup>3</sup>, kidney and liver function tests were normal and the Mantoux test was negative. Chest radiograph (PA view) demonstrated opaque left hemithorax with volume loss, ipsilateral mediastinal shift, right sided compensatory hyperinflation and scoliosis of dorsal spine with concavity to the left side [<xref ref-type="fig" rid="F1">Figure 1</xref>]. USG of abdomen was normal. Contrast enhanced CT scans of chest lung window [<xref ref-type="fig" rid="F2">Figure 2</xref>].</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>X-ray chest showing opaque left hemithorax</p></caption><graphic xlink:href="LI-31-416-g001"/></fig><fig id="F2" position="float"><label>Figure 2</label><caption><p>CT scan lung window showing aerated part of left lung</p></caption><graphic xlink:href="LI-31-416-g002"/></fig></sec><sec id="sec1-2"><title>QUESTION</title><p>What is the diagnosis?</p></sec><sec id="sec1-3"><title>ANSWER</title><p>Pulmonary hypoplasia (left).</p></sec><sec sec-type="discussion" id="sec1-4"><title>DISCUSSION</title><p>On CECT chest, trachea and both the bronchi are normal. There is volume loss in the left hemithorax with ipsilateral mediastinal shift, raised left hemidiaphragm and herniation of right lung parenchyma. Only a very small area/volume of the left lung is aerated. Mediastinal window showed displaced mediastinum heart and mediastinal structures and the rudimentary left pulmonary trunk.</p><p>Pulmonary agenesis/aplasia/hypoplasia has been classified by Schneider[<xref rid="ref1" ref-type="bibr">1</xref>] and later modified by Boyden.[<xref rid="ref2" ref-type="bibr">2</xref>] It differentiates between them on the basis of presence or absence of the lung tissue, bronchi or the pulmonary artery [<xref ref-type="table" rid="T1">Table 1</xref>].</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Comparative evaluation of pulmonary hypoplasia/aplasia/agenesis</p></caption><graphic xlink:href="LI-31-416-g003"/></table-wrap><p>Pulmonary hypoplasia is incomplete development of the lungs, resulting in an abnormally low number or size of bronchopulmonary segments or alveoli. Incidence of pulmonary hypoplasia ranges from 9-11 per 10,000 live births.[<xref rid="ref3" ref-type="bibr">3</xref>] The developmental anomalies of the lung between the 4<sup>th</sup> and 24<sup>th</sup> gestational weeks may cause functional damage which is usually discovered in newborns and infants, but it can rarely present in adulthood.[<xref rid="ref4" ref-type="bibr">4</xref>] Practically, earlier the anomaly is present, proportionately, the branching of the tracheal-bronchial tree is reduced.[<xref rid="ref5" ref-type="bibr">5</xref>]</p><p>Contrast enhanced computed tomography (CECT) of the chest revealed that only a very small area/volume of the left lung was aerated [<xref ref-type="fig" rid="F2">Figure 2</xref>]. In 70% of cases, the left lung is affected,[<xref rid="ref6" ref-type="bibr">6</xref>] as was seen in the present patient. Mediastinal window showed displaced mediastinum, heart and mediastinal structures and the rudimentary left pulmonary trunk [<xref ref-type="fig" rid="F3">Figure 3</xref>].</p><fig id="F3" position="float"><label>Figure 3</label><caption><p>CT scan mediastinal window showing left pulmonary artery and rudimentary branch of left pulmonary artery. AA -Ascending Aorta, DA -Descending Aorta, MP -Main Pulmonary Artery, RP -Right Pulmonary Artery, Arrow -Showing left pulmonary artery and its rudimentary branch</p></caption><graphic xlink:href="LI-31-416-g004"/></fig><p>On bronchoscopy, vocal cords were moving normally. Carina was central and sharp, right bronchial tree was well within the normal limits. The left main bronchus was normal up to the first inch, but had a slit like appearance distal to it; bronchoscope could not be negotiated further. Few mucopurulent secretions were seen in the left bronchial stump which were collected and sent for different stains and cultures.</p><p>Differential diagnosis of a completely opaque hemithorax is massive pleural effusion, lobar consolidation and complete collapse of the lung, followed by agenesis/aplasia/hypoplasia of the lung, post-pneumonectomy and large space occupying the lesions (SOL). As the mediastinum was shifted towards the same side and there were evidences of volume loss; effusion, consolidation and SOL were ruled out. CECT chest showed rudimentary left pulmonary artery [<xref ref-type="fig" rid="F3">Figure 3</xref>] and small area of lung parenchyma [<xref ref-type="fig" rid="F2">Figure 2</xref>] in the left lung. As per classification by Boyden[<xref rid="ref2" ref-type="bibr">2</xref>] [<xref ref-type="table" rid="T1">Table 1</xref>], all the 3 components (lung tissue, artery and the bronchus) were present, but in an under-developed form, thus categorizing the present case into pulmonary hypoplasia.</p><p>Pulmonary hypoplasia is usually diagnosed during infancy or early childhood, but many patients remain asymptomatic and the diagnosis may not be made until adulthood.[<xref rid="ref7" ref-type="bibr">7</xref>] It is a common cause of neonatal death.[<xref rid="ref8" ref-type="bibr">8</xref>] Wigglesworth and Desai reported an incidence of 14.5% in a large series of perinatal necropsies.[<xref rid="ref9" ref-type="bibr">9</xref>] Hypoplastic lung, when present, is prone for atelectasis and pulmonary infection and associated congenital anomalies.[<xref rid="ref7" ref-type="bibr">7</xref>]</p><p>The patient was empirically treated for LRTI and discharged in a stable condition. The present case is being reported as pulmonary hypoplasia in itself is rare, it was diagnosed during the adulthood and it is an important element of differential diagnosis of unilateral opaque hemithorax, so the case would be interesting for postgraduate as a radiologic quiz.</p></sec> |
Tracheal compression due to anomalous innominate artery in an adult | <p>A 47-year-old-female presented with dyspnea and unproductive cough for 4 months. General examination revealed pulsatile swelling in the midline below the thyroid cartilage present since childhood. Computed tomography-angiography of the neck showed right innominate artery dilated, elongated and coursing above downward, anterior to the trachea below the thyroid, compressing the trachea and origin of the right subclavian artery higher up. A case of anomalous innominate artery causing symptomatic compression of the trachea in adults is a rare entity.</p> | <contrib contrib-type="author"><name><surname>Gupta</surname><given-names>Krishna B.</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Tyagi</surname><given-names>Diksha</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Prajapat</surname><given-names>Deepak</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Kumar</surname><given-names>Vipul</given-names></name><xref ref-type="aff" rid="aff1"/></contrib> | Lung India : Official Organ of Indian Chest Society | <p>A 47-year-old-female presented in chest Outpatient Department with the chief complaint of unproductive cough and shortness of breath for 4 months that was insidious in onset and gradually progressive in nature. She never smoked and had no history of exposure to indoor air pollution. She was diagnosed to have hypertension and put on treatment 4 months back in the form of monotherapy with amlodipine. She had not given any past history of pulmonary tuberculosis, coronary artery diseases or any significant chronic illness. On physical examination, a pulsatile swelling of size 5.5 cm × 6.6 cm × 5.5 cm was palpable in the midline neck below the thyroid. Her blood pressure was 160/120 mmHg, pulse rate was 86/min, respiratory rate was 24/min, body temperature was 37.0°C and SaO<sub>2</sub> was 96% by pulse oximetry. Systemic examination revealed no abnormality. Laboratory examination revealed hemoglobin 12.9 gm%, TLC 4500 per cu mm, hematocrit 40% and ESR 25 mm in the 1<sup>st</sup> hour. The biochemistry panel revealed no abnormality. Sputum investigations for AFB direct smear and pyogenic culture were negative. Spirometry and echocardiography revealed normal study. Chest radiograph revealed slight mediastinal widening [<xref ref-type="fig" rid="F1">Figure 1</xref>].</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Chest radiograph showing slight mediastinal widening</p></caption><graphic xlink:href="LI-31-419-g001"/></fig><sec id="sec1-1"><title>QUESTION</title><p>What is your diagnosis?</p></sec><sec id="sec1-2"><title>ANSWER</title><p>Color Doppler ultrasonography of the neck in view of neck swelling was performed, revealing that the pulsatile swelling in the midline was the dilated and elongated right innominate artery anomalous course passing anterior to the neck below the thyroid [<xref ref-type="fig" rid="F2">Figure 2</xref>]. To evaluate further, a computed tomography (CT)-angiography of the neck was performed, revealing that the origin of the right subclavian artery from the right innominate artery is higher than the normal origin and the innominate artery is coursing from above downward [Figure <xref ref-type="fig" rid="F3">3a</xref> and <xref ref-type="fig" rid="F3">b</xref>].</p><fig id="F2" position="float"><label>Figure 2</label><caption><p>Ultrasonography of the neck showing the right innominate artery coursing anterior to the trachea</p></caption><graphic xlink:href="LI-31-419-g002"/></fig><fig id="F3" position="float"><label>Figure 3</label><caption><p>(a) Computed tomography (CT)-angiography showing origin of the right innominate artery medial to the superior vena cava. (b) CT-angiography showing the right innominate artery coursing upward in front of the trachea</p></caption><graphic xlink:href="LI-31-419-g003"/></fig></sec><sec id="sec1-3"><title>DIAGNOSIS</title><p>Anomalous innominate artery causing tracheal compression.</p><p>Anomalous innominate artery causing tracheal compression is commonly seen in children less than 2 years, but it is a rare diagnosis in adults<sup>1</sup>. Mostly, the adult patients are asymptomatic and detected incidentally during surgical procedures. Such anomalies should be suspected in a patient presenting with respiratory symptoms with a near-normal chest radiograph. The diagnosis can be easily confirmed by CT angiography. Medical management includes humidified oxygen, steroids and antibiotics, while common surgical approaches include arteriopexy, with suspension of the innominate artery to the sternum or reimplantation at a more proximal site of the ascending aorta.</p></sec> |
Eventration of the right hemidiaphragm with multiple associations: A rare presentation | Could not extract abstract | <contrib contrib-type="author"><name><surname>Mehrotra</surname><given-names>Ashok Kumar</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Vaishnav</surname><given-names>Khyati</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib><contrib contrib-type="author"><name><surname>Gupta</surname><given-names>Prahlad Rai</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Khublani</surname><given-names>Trilok Kumar</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Anupam</surname><given-names/></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Soni</surname><given-names>Shridha</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Feroz</surname><given-names>Asif</given-names></name><xref ref-type="aff" rid="aff1"/></contrib> | Lung India : Official Organ of Indian Chest Society | <p>Sir,</p><p>Eventration of the diaphragm is mostly a congenital anomaly, where it is partly or wholly replaced by a fibrous sheet.[<xref rid="ref1" ref-type="bibr">1</xref>] The reported incidence of the disorder in adults is about 1:10,000. It usually occurs as an isolated entity and is extremely rare to occur bilaterally, almost always presenting in infancy with respiratory compromise, and is associated with a high mortality rate.[<xref rid="ref2" ref-type="bibr">2</xref>] More commonly, it occurs unilaterally, mostly on the left side, and may present at any age and has a male predominance. Complete eventration on the right side should be extremely uncommon in adults as only a few reports are available in the English literature.[<xref rid="ref3" ref-type="bibr">3</xref><xref rid="ref4" ref-type="bibr">4</xref>] We are presenting such a case in an adult male who presented to us with recurrent pneumothorax due to uncontrolled asthma. Further evaluation revealed multiple associations, i.e. lower lobe hypoplasia, a supero–posteriorly placed deformed liver with an aberrant venous drainage and block D4-D5 vertebra, which to the best of our knowledge is the first case of right-sided eventration with multiple associations.</p><p>LR, a 22-year-old male student, presented in the emergency area of this hospital with acute onset of shortness of breath (SOB) and pain chest of 3 h duration. Initial evaluation revealed him to be a known case of bronchial asthma, poorly controlled on oral medication and having two episodes of right-sided pneumothorax, successfully managed with inter-costal chest tube drainage (ICTD), in the past 3 months. He was a normally born child with normal life milestones. There was no history of any infection, trauma or malignancy in the past. He had never smoked, was a teetotaler and never took any drugs, other than the asthma medication. Nothing significant was recorded in the family history.</p><p>The patient was fairly built and adequately nourished. His respiratory rate was 36/min, pulse rate was 120/min and regular, temperature was 99°F, blood pressure was 100/60 mmHg and peripheral oxygen saturation was 80%. Trachea and apex beat were shifted to the left. The chest was hyper-inflated and hyper-resonant on the right side, along with decreased movements, vocal fremitus and breath sounds. Left-sided chest revealed extensive rhonchi. Examination of the other systems revealed nothing abnormal.</p><p>A skiagram chest PA view of the patient was suggestive of right pneumothoax. The patient was given oxygen along with standard asthma therapy and an ICTD was established, after which he improved. A repeat skiagram chest PA view, post ICTD, showed complete expansion of the lung on the right side along with a normally placed trachea and heart, but the right hemidiaphragm was raised up to the level of the 3<sup>rd</sup> intercostal space.</p><p>Routine blood counts, renal functions and liver function tests were within normal limits. Mantoux skin test was negative. Pulmonary function tests revealed a moderate obstructive defect.</p><p>Ultrasonography showed that the right hemidiaphragm had been replaced by a thin, fibrous sheet with reduced excursion, but was continuous and devoid of any paradoxical movements. Sniff test on fluoroscopy in erect position confirmed lack of paradoxical movements. Computerized tomography of the thorax showed that the right hemidiaphragm was thin and grossly elevated [<xref ref-type="fig" rid="F1">Figure 1</xref>], the lower lobe of the right lung was hypoplasic [<xref ref-type="fig" rid="F2">Figure 2</xref>], the liver was deformed [along with an anomalous venous drainage [<xref ref-type="fig" rid="F3">Figure 3</xref>] and the 4<sup>th</sup> and 5<sup>th</sup> vertebral bodies were fused.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Tomogram of the patient, post intercostal chest tube drainage, showing complete expansion of the lung along with raised hemidiaphragm on the right side. The right lower lobe is small in size</p></caption><graphic xlink:href="LI-31-421-g001"/></fig><fig id="F2" position="float"><label>Figure 2</label><caption><p>Computerizd tomography image at the level of carina (lung window) showing very small-sized lower lobe due to hypoplasia of the lung on the right side</p></caption><graphic xlink:href="LI-31-421-g002"/></fig><fig id="F3" position="float"><label>Figure 3</label><caption><p>Contrast-enhanced sagittal computerized tomography image showing the deformed liver with hepatic veins joining each other in the liver parenchyma to form an arc-like vessel (arrow)</p></caption><graphic xlink:href="LI-31-421-g003"/></fig><p>After about 1 week of treatment in the hospital, the patient was discharged on standard asthma medication with the advice to remain under regular follow-up.</p><p>In the present case, the right hemidiaphragm had retained its continuity and attachments to the costal margins, ruling out diaphragmatic hernia,[<xref rid="ref5" ref-type="bibr">5</xref>] and a negative sniff test had ruled out diaphragmatic paralysis.[<xref rid="ref6" ref-type="bibr">6</xref>] Thus, the diagnosis of complete eventration of the right hemidiaphragm was comprehensively established.</p><p>Associated ipsilateral organ anomalies have been described in the medical literature along with left-sided eventration. These include wandering spleen syndrome and gastric volvulus.[<xref rid="ref1" ref-type="bibr">1</xref><xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref8" ref-type="bibr">8</xref><xref rid="ref9" ref-type="bibr">9</xref>] Right-sided dextrogastria and volvulus have also been described in an infant.[<xref rid="ref3" ref-type="bibr">3</xref>] In adults, only a single case report of right-sided eventration has been reported,[<xref rid="ref4" ref-type="bibr">4</xref>] but it was an isolated event. Ours is possibly the first case of right-sided eventration with multiple associations, i.e. lung, liver and vertebral anomalies.</p><p>Why deformation of the organs should occur on the side of eventration is a matter of research. Par <italic>et al</italic>.[<xref rid="ref7" ref-type="bibr">7</xref>] had postulated that wide sub-diaphragmatic space left due to an elevated hemidiaphragm provided adequate space for malformation of the subdiaphragmatic structures to occur, but this fails to explain the multiple associations as were seen in our case. We are reasoning that as a result of eventration, pressure differential across that side of the diaphragm is mostly lost. The altered pressures adversely affect the developmental process, i.e. malformation of the organs, on that side of the chest. Absence or weakness of supporting ligaments may contribute to other associations, i.e. gastric volvulus and wandering spleen.[<xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref8" ref-type="bibr">8</xref><xref rid="ref9" ref-type="bibr">9</xref>]</p><p>Eventration-associated anomalies are mostly of no serious consequence, as was in our case. But, acquired pathologies in these organs may present with atypical clinical manifestations, making its recognition difficult, and prior knowledge of these anomalies should be of great help in such situations.</p> |
A case of recurrent massive right sided pleural effusion-an unusual presentation of carcinoma prostate | Could not extract abstract | <contrib contrib-type="author"><name><surname>Bajpai</surname><given-names>Pranay Ashok</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Joshi</surname><given-names>Prakash</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Joseph</surname><given-names>Dolly</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Bajpai</surname><given-names>Ashok</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib> | Lung India : Official Organ of Indian Chest Society | <p>Sir,</p><p>Carcinoma prostate is the second most common cancer in males worldwide and hematological spread occurs commonly to bones, lung and liver. It has a typical pattern involving axial skeleton and loco regional lymph nodes specially obturator and hypogastric groups. Isolated visceral metastasis in absence of bone metastasis is rare. Pleural involvement is also rare. Pleural effusion as determined on autopsy study ranged from 2.3 to 5%. Herein, we present a case that did not have any urinary symptoms and presented with pleural effusion only.</p><p>An 84-year-old gentleman, presented with generalized weakness, dyspnea on exertion and anorexia for 8 months. He was a non-smoker and a known case of Type 2 Diabetes Mellitus taking oral hypoglycemic agents for last 40 years. He had received empirical anti-tuberculosis treatment (ATT) for the above-mentioned symptoms before presenting to us without any improvement. He had no symptoms pertaining to lower urinary tract. He had the Eastern Cooperative Oncology Group (ECOG) performance status 2 and was hemodynamically stable. On physical examination, he had no pallor, no icterus, clubbing or palpable lymphadenopathy. On evaluation, he was found to have dull breath sounds on mid and lower zone of right chest. His chest X-ray posteroanterior (PA) view [<xref ref-type="fig" rid="F1">Figure 1</xref>] showed right-sided pleural effusion. Contrast-enhanced computed tomography (CECT) chest [<xref ref-type="fig" rid="F2">Figure 2</xref>] and abdomen showed massive right pleural effusion with right lung collapse and prostatic enlargement. There were no obvious lung mass or enlarged mediastinal lymph nodes. Sonography of pelvis showed prostatic enlargement with 150 ml post-void residual urine. Pleural fluid examination showed hemorrhagic fluid, glucose 100 mg/dl, proteins 4.5 mg/dl; total cells were 480 with neutrophils 20% and lymphocyte 80%. Pleural fluid adenosine deaminase (ADA) was 32.35 IU/L. No growth of micro-organism was seen on Gram and Zeil Nelson staining to rule out tuberculosis and no growth was seen after 48 hours of urine culture. Pleural fluid cytology revealed fragments from metastatic adenocarcinoma. His serum prostate-specific antigen (PSA) was found to be>148 ng/dl. An ultrasound guided trans rectal prostate biopsy was done, which showed well-differentiated adenocarcinoma of prostate and a Gleason's Score of 6. He underwent surgical hormonal ablation by bilateral high inguinal orchidectomy and he was started on bicalutamide. After 4 weeks of surgery, he improved symptomatically, massive pleural effusion subsided and his serum PSA came down to 13.48 ng/ml. On last follow-up at three months there was no pleural effusion on chest X-ray and his serum PSA was 4.04 ng/ml. Still there was no bony pain or tenderness.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>X-Ray chest of patient showing massive right-sided effusion</p></caption><graphic xlink:href="LI-31-423-g001"/></fig><fig id="F2" position="float"><label>Figure 2</label><caption><p>CECT chest showing massive right-sided pleural effusion</p></caption><graphic xlink:href="LI-31-423-g002"/></fig><p>The prognosis of patients with carcinoma prostate is determined by the presence or absence of metastasis. The bones of axial skeleton is the frequent site of metastasic spread, spine metastasis precede lung and liver metastases. Pleural involvement without bone involvement is rare. The main cause of metastatic pleural effusion is adenocarcinoma lung, breast, ovary and stomach. In addition lymphoma, primary mesothelioma and sarcomas may cause effusion. This patient has hemorrhagic pleural effusion and is positive for adenocarcinoma cells. We could not find any carcinoma prostate patient in the literature presenting primarily with pleural effusion showing metastatic adenocarcinoma cells and without any urinary symptoms and bone metastasis. Hence, presence of metastatic pleural effusion in elderly males without any urinary symptoms should warn us to do rectal examination, pelvic sonography and serum PSA levels.</p><p>Surgical castration along with hormonal therapy has shown promising results thereby increasing the survival of patients with metastatic adenocarcinoma prostate. Our patient also showed a good response to castration and hormonal therapy and presently after four months there is no pleural effusion and his serum PSA level has dropped markedly up to 4.04 ng/dl. This is a rare occurrence of massive pleural effusion due to local metastasis of carcinoma prostate, emphasizing the need of doing serum PSA and digital rectal examination in patients presenting with malignant pleural effusion.</p> |
Protecting ourselves from tuberculosis. Describing a historic poster printed in italy on 1937 | Could not extract abstract | <contrib contrib-type="author"><name><surname>Inzirillo</surname><given-names>Francesco</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Giorgetta</surname><given-names>Casimiro</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Ravalli</surname><given-names>Eugenio</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Tiberi</surname><given-names>Simon</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib><contrib contrib-type="author"><name><surname>Pona</surname><given-names>Claudio Della</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Robustellini</surname><given-names>Mario</given-names></name><xref ref-type="aff" rid="aff1"/></contrib> | Lung India : Official Organ of Indian Chest Society | <p>Sir,</p><p>Pulmonary TB was the disease that spawned thoracic surgery and pulmonology. During the eighteenth and nineteenth centuries, the population shift to cities and the ensuing overcrowding contributed to TB being the most common cause of death. It was apparent that TB was a communicable disease but medical proof had to await Koch's presentation, entitled “On Tuberculosis” in 1882. Here at last was an enemy (the tubercle bacillus) to which attention could be directed.[<xref rid="ref1" ref-type="bibr">1</xref>] Many folk remedy existed for treatment of the disease. One of the most popular myths was of a change in climate and diet. Ultraviolet light kills acid-fast bacilli in the laboratory, and therefore patients were encouraged to spend time exposed to sunlight. Physical activity in children supposedly conferred resistance. The first sanatorium specific for TB was established at Gobersdorf in Germany, another famous sanatorium was in Davos, Switzerland, the highest town in Europe but, between 1932 and 1940, in the Alps of Northern Italy, at 1000 m above sea level, the biggest sanatorium in Europe (Eugenio Morelli Hospital) was built in Sondalo with 9 wings for each of its 10 floors and covering an area of 350.000 m<sup>2</sup> on a road network of 12.5 km and occupying a volume of 650.000 m<sup>3</sup>. “The Sanatorium City”, with its 3500 beds, cinema, shops and a post office, had a larger population than its nearby towns and was immortalized in a Vittorio De Sica film “A short holiday”. Sondalo is like Davos; two places that share the wonderful landscape of the Alps, the clearness arising from the intense green belt around, the daylight springing over the terraced meadows and the shining sunlight gathered as if in a bright amphitheatre.[<xref rid="ref1" ref-type="bibr">1</xref>] The main activity of these structures was, from the beginning, the fight against the “insidious disease” called TB. High altitude, bed rest, exposure to the environment and good nutrition were pursued as therapeutic options. The Hospital soon became one of the first centers of reference in Italy and Europe for the treatment of TB and other lung diseases. In pre-antibiotic period, therapies against TB were ineffective and a major role was played by the surgery and largely by preventing the disease. Despite the hundreds of sanatoria built around the world, no tangible scientific proof that sanatorium therapy had any influence on the disease exists. Sanatorium therapy, however, concentrated patients in one place and so contributed to the study of the disease. The annals of Morelli Hospital are full of descriptions about surgical operations that were routine and today would just make us cringe. The techniques of thoraco-myoplasties, buckling and iatrogenic pneumothorax are just some examples of how and in what measure the surgeon could “demolish” a patient. The basic idea was valid, reducing the supply of oxygen to the tuberculous cavities to make survival difficult for the obligate aerobes organisms that are mycobacteria. Various methods of collapse therapy, like the “therapeutic pneumothorax of Forlanini” introduced in 1906 and the “intracavitary aspiration of Monaldi”, today are full of historic charm. The development of anti-mycobacterial therapy, in the second half of the last century, was a “godsend” for patients and a professional revolution for Thoracic Surgeon who had already understood the need to “sharpen the blades against cancer”, the new enemy whose incidence was increasing more and more! With the new medical therapy the role of the Surgeon gradually decreased to the treatment of complications of TB: Destroyed lobes and lungs, tuberculous pleural empyema with or without fistula, hemoptysis, etc., bronchoconstriction. Disease prevention was not easy in a period plagued by wars and dominated by poverty and lack of education. One of the sanitary objectives, that the political institutions of the period were trying to achieve, was the defeat of some of the most dangerous infectious diseases including TB. The poster in [<xref ref-type="fig" rid="F1">Figure 1</xref>], printed in 1937 and currently exposed in the Phthisiology Department of Morelli Hospital in Sondalo, represents one of the most practical and fascinating historical examples of the attempt to focus social awareness on the problem of prevention. At the top left you can read: <italic>The messy house, neglected and untidy, away from the air and light, depresses the spirit and hatches germs of the saddest diseases; first and most insidious of all tuberculosis. At the top right you can read: The clean house, tidy, lovingly prepared and open to the pure air and to the beneficial sun, raises the spirit of those who live and keeps infections, contagions, the mortal dangers far away from the body. The text continues: Tuberculosis still kills every year 35,000 Italians. Yet, if it was discovered and treated in time, tuberculosis would have been the most preventable and most curable disease. Tuberculosis is caused by the bacillus discovered by Robert Koch, which, finding favorable conditions in the human body, grow, multiply and destroy organs and tissues. These bacilli that are found in the sputum expelled by coughing tuberculosis patients are really virulent and nefarious, especially when they act on children. When in a house there is a person sick with tuberculosis, we must take these precautions: Room aside, the destruction of the sputum, disinfection of table utensils. With all the forces it is necessary to fight the ridiculous fears that make a poor sick a being to be feared as a cholera victim, a pariah. But above all it is necessary to subtract the children to infection and strengthen their bodies. In fact, while the bacteria that infect a weak body have the fate of a lit match falling on a pile of straw, the bacilli entering a robust body have the fate of a lit match falling on a marble table: Extinguishes. To protect children from tuberculosis, you should: Wash your hands before touching any food; at school, sitting with chest and head erect, so that the apices of the lungs can breathe; to live as much as possible outdoors and do respiratory gymnastics; not spitting and prevent others from spitting on the ground; do not eat foods already touched by others; do at least one bath a week; sleep, when it is not raining and not too moist, with the windows slightly open so that fresh air can enter the room; eat healthy foods and brush your teeth before going to bed. By following these hygienic rules, you can almost always prevent tuberculosis. However, tuberculosis, at the beginning, is almost always curable if treated immediately and with appropriate methods. When the first symptoms, which include slight fever persistent and intermittent, which does not exceed 38 degrees; weight loss; pallor; lack of strength; fixed pain in the chest; coughing light, tough and dry; tenacious disorders of the stomach and intestine; streaks of blood in the sputum, seek immediate medical attention or anti-tuberculosis institutions created by the Fascist Regime, which has made the fight against tuberculosis among the fundamental objectives of its businesses. Let all remember the words of the Duce (tn: Benito Mussolini): “it is necessary that scientists, policymakers, philanthropists constitute a kind of united front to lead the great battle against tuberculosis to a victorious end.”</italic> Today a poster like that described could be perceived as biased because it seems to attribute responsibility for the spread of disease to poverty and therefore to a condition that cannot be changed or that can be changed with extreme difficulty. A closer reading, however, must reveal the merit of emphasizing, in a poor environment, only those aspects that can be changed at low cost. Nowadays, a number of challenges and barriers to good hygiene exist in less developed countries. The greatest of these challenges is the lack of clean water and soap. Hundreds of millions of people do not have access to improved sources of drinking water and worldwide there are 1.6 million deaths per year attributed to diseases spread through unsafe water, poor sanitation, and lack of hygiene.[<xref rid="ref2" ref-type="bibr">2</xref>] The described poster, in that way can be considered current, because, even today, poor sanitations and many diseases are widespread among the lower classes but of course it is not a rule. In conclusion, in our daily activities we can confirm that the “new explosion” of TB, is due mainly to the migratory flows from highly endemic countries in our hospital (mainly countries from Eastern Europe and South Asia). Cases of resistant infections are also increasing. In addition to multi-drug resistant (MDR) and extensively drug-resistant (XDR) bacterial strains, we now must fight against infections that have very few chemotherapeutic options, a kin to the TB we saw in the pre-antibiotic era. The appearance of these resistant strains is due to a selection common to all germs and to an incorrect use of medical therapy (monotherapies, insufficient duration, poor patient compliance, etc). With the resurgence of the disease and the development of resistant strains and more complex patients, the Thoracic Surgeon must learn to deal with the “phoenix reborn” again.[<xref rid="ref3" ref-type="bibr">3</xref>] The battle is increasingly complex and treatment of TB requires a close collaboration between the Physiologist and the Surgeon. Medical therapy of resistant forms must be handled by experts in the field and the corresponding surgical treatments cannot be improvised.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>The poster showing the hygiene rules preventing tuberculosis</p></caption><graphic xlink:href="LI-31-425-g001"/></fig> |
An increasingly breathless patient | Could not extract abstract | <contrib contrib-type="author"><name><surname>Bodington</surname><given-names>Richard S.</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Faruqi</surname><given-names>Shoaib</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib> | Lung India : Official Organ of Indian Chest Society | <p>Sir,</p><p>We read the recent article by Karkhanis and colleagues entitled “Flow volume loops as a diagnostic marker” with great interest.[<xref rid="ref1" ref-type="bibr">1</xref>] The authors describe the case of a young girl who developed exertional dyspnea after a period of mechanical ventilation. Flow-volume loops demonstrated a “box-pattern” suggesting fixed upper airways obstruction. Subsequently, the diagnosis of post-intubation tracheal stenosis was made. We would like to present another case of fixed upper airway obstruction, of a different etiology, reinforcing the importance of the use of simple flow-volume loops.</p><p>A 56-year-old male attended a respiratory clinic appointment for follow-up of a suspected lower respiratory tract infection (LRTI) 6 weeks previously for which he had been admitted to our Medical Assessment Unit. The patient had initially been admitted with complaints of breathlessness and dry cough for the preceding 2 weeks associated with a degree of chest tightness. D-dimer assay performed on his first admission was negative and his chest X-ray [<xref ref-type="fig" rid="F1">Figure 1</xref>] was unremarkable. The presumptive diagnosis of LRTI was made in view of a raised C-reactive protein of 41 mg/L and the patient discharged on antibiotics. Since that time, the patient's symptoms had not improved, and had in fact gradually worsened when reviewed in the respiratory clinic 6 weeks later. The patient described marked breathlessness that had been present for 2 or 3 months only; he had had no symptoms before this time. He did not report any expectoration, hemoptysis, orthopnea, ankle edema or any systemic symptoms. A small goitre was apparent, which the patient reported having noticed for the previous few weeks, but no stridor was audible at this time. Otherwise, the general physical and systemic examination was unremarkable. A simple spirometry performed in the clinic demonstrated an FEV<sub>1</sub>/FVC ratio of 64% and an FEV<sub>1</sub> that was 73% predicted. The peak expiratory flow rate was 290 L/min (56% predicted). The disproportionate decrease in peak expiratory flow as compared with the FEV<sub>1</sub> in conjunction with the clinical history raised a suspicion of upper airway obstruction. Flow-volume loops and a computed tomography (CT) scan were organized. The flow-volume loop demonstrated flattened inspiratory and expiratory curves consistent with fixed extrathoracic airway obstruction. Empey's index and midvital capacity ratios were also in keeping [<xref ref-type="fig" rid="F2">Figure 2</xref> and <xref ref-type="table" rid="T1">Table 1</xref>]. The CT scan demonstrated marked compression of the trachea in the lower neck due to an enlarged thyroid. At its narrowest point, the trachea measured just 0.6 × 1.2 cm in diameter [<xref ref-type="fig" rid="F3">Figure 3</xref>]. A left thyroid lobectomy was subsequently performed, which resulted in prompt amelioration of his breathlessness. Histology subsequently was consistent with a diagnosis of follicular carcinoma of the thyroid. His case was then referred for discussion at the multi-disciplinary team meeting for further management.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>The patient's normal chest X-ray</p></caption><graphic xlink:href="LI-31-427-g001"/></fig><fig id="F2" position="float"><label>Figure 2</label><caption><p>The patient's flow-volume loop. Solid line - patient's results. Dashed line - reference. Initially normal expiration becomes lung volume independent until the terminal portion and inspiration is obstructed in a similar way as expiration, indicative of a fixed extrathoracic obstruction</p></caption><graphic xlink:href="LI-31-427-g002"/></fig><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Spirometry results suggestive of fixed extrathoracic airflow obstruction</p></caption><graphic xlink:href="LI-31-427-g003"/></table-wrap><fig id="F3" position="float"><label>Figure 3</label><caption><p>(a) Sagittal and (b) axial computed tomography images showing the enlarged thyroid compressing the trachea (white arrows)</p></caption><graphic xlink:href="LI-31-427-g004"/></fig><p>Extrathoracic airway obstruction is an uncommon but not a rare cause of breathlessness. Some of the more prevalent causes of upper airway obstruction that present with breathlessness include post-traumatic strictures, bilateral vocal cord paralysis, chronic inflammatory foci and goitres.[<xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref3" ref-type="bibr">3</xref>] Upper airways obstruction is a well-recognized complication of thyroid disease.[<xref rid="ref2" ref-type="bibr">2</xref>] In this context, evidence of airway obstruction on flow-volume loops may well be an incidental finding with no clinical implications. Twenty-five percent of patients with large goitres report the symptom of breathlessness.[<xref rid="ref4" ref-type="bibr">4</xref>] Case series have reported that up to one-third of patients with goitres have upper airway obstruction and that most improve post-thyroidectomy.[<xref rid="ref2" ref-type="bibr">2</xref>]</p><p>The flow-volume loop is a simple non-invasive test and is the best method of evaluating upper airways obstruction and distinguishing it from obstructive pulmonary disease.[<xref rid="ref4" ref-type="bibr">4</xref>] As discussed by Karkhanis <italic>et al</italic>., the Empey's index and ratio of maximal expiratory flow at 50% of vital capacity and maximal inspiratory flow at 50% of the vital capacity (MEF50/MIF50) are also useful indices to help make this diagnosis. A fixed upper airway obstruction will limit maximal flow to the same level regardless of lung volumes.[<xref rid="ref5" ref-type="bibr">5</xref>] The flow will become lung volume independent apart from the initial period of inspiration and end of expiration, where lower lung volumes exert less traction on the small airways allowing the smaller airways to narrow and become the rate-limiting factor in determining flow.[<xref rid="ref5" ref-type="bibr">5</xref>] This gives a clipped or “square box” appearance of the flow-volume loop with flow limited equally in both inspiration and expiration. As discussed by Karkhanis <italic>et al</italic>., variations in this clipping can give a clue as to the cause of upper airway obstruction.[<xref rid="ref1" ref-type="bibr">1</xref>] In addition, flow-volume loops can provide an estimate of the size of the patent airway by changing the diameter of the mouth piece used until it becomes the rate-limiting component.</p><p>However, it should be noted that clinical history predicts obstruction poorly, as exemplified by this case. In one study of patients with large goiters, 29% of patients with goitre symptoms were found to have no airway obstruction on flow-volume loops; conversely, obstruction was demonstrated in 21% of patients with no symptoms.[<xref rid="ref6" ref-type="bibr">6</xref>] Furthermore, clinical assessment of thyroid size demonstrates no relation to likelihood of airway obstruction.[<xref rid="ref4" ref-type="bibr">4</xref>] The retrosternal extent of a goitre is not amenable to clinical examination. Imaging techniques such as a CT scan will both confirm and quantify the presence and extent of airway compromise. If the clinical suspicion is not high, appropriate investigations may not be requested and the diagnosis may remain elusive. Upper airway obstruction presenting as breathlessness is uncommon but should be borne in mind by physicians as the diagnosis can be effectively made with the use of flow-volume loops and imaging and symptoms easily ameliorated with correct treatment.</p> |
Mobile vegetation leading to septic pulmonary embolism | Could not extract abstract | <contrib contrib-type="author"><name><surname>Haider</surname><given-names>Imran</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Gupta</surname><given-names>Raghav</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Song</surname><given-names>Shuang</given-names></name><xref ref-type="aff" rid="aff1"/></contrib> | Lung India : Official Organ of Indian Chest Society | <p>Sir,</p><p>A 42-year-old Caucasian female with a prolonged history of intravenous (IV) methamphetamine abuse presented with high-grade fever, progressively worsening productive cough, dyspnea, myalgia, arthralgia and intermittent confusion for 1 week. The initial laboratory assessment showed leukocytosis, thrombocytopenia, and acute kidney injury. Chest X-ray showed bilateral lower lobe consolidation with small cavities surrounded by focal infiltrates in the right lung [<xref ref-type="fig" rid="F1">Figure 1</xref>]. Computed tomography of the chest confirmed the presence of multiple cavitary pulmonary nodules suggestive of septic embolization [<xref ref-type="fig" rid="F2">Figure 2</xref>]. Because of a high index of suspicion for infective endocarditis, a transthoracic echocardiogram was obtained, which showed a 3.6 cm × 2.6 cm vegetation on the tricuspid valve and 2.5 cm × 1.6 cm vegetation on the tricuspid annulus, accompanied by severe tricuspid regurgitation. Trans-esophageal echocardiogram demonstrated multiple, large multilobulated, mobile vegetation on the tricuspid valve, with the largest being 3.7 cm × 2.5 cm in size [<xref ref-type="fig" rid="F3">Figure 3</xref>]. Blood cultures were positive for methicillin-resistant <italic>Staphylococcus aureus</italic>. The patient was initially treated with vancomycin and ceftriaxone. However, the patient failed to improve clinically and, eventually, underwent tricuspid valve excision, right ventricle/tricuspid valve debridement and tricuspid valve replacement. The post-operative course was uneventful and the patient made a satisfactory recovery.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Chest radiography showing bilateral lower lobe consolidation with small cavities within focal infiltrates in the right lung</p></caption><graphic xlink:href="LI-31-429-g001"/></fig><fig id="F2" position="float"><label>Figure 2</label><caption><p>Computed tomography of the chest showing numerous cavitary pulmonary nodules located peripherally, representing septic pulmonary emboli in the right lung</p></caption><graphic xlink:href="LI-31-429-g002"/></fig><fig id="F3" position="float"><label>Figure 3</label><caption><p>Trans-esophageal echocardiogram with mid position view showing a 3.6 cm × 2.6 cm-sized mobile vegetation in the right atrium attached to the tricuspid valve. RA: Right atrium</p></caption><graphic xlink:href="LI-31-429-g003"/></fig><p>Septic pulmonary embolism (SPE) is a rare and serious disorder that usually presents with non-specific clinical features including fever, pulmonary symptoms and peripheral nodular lung infiltrates with or without cavitation.[<xref rid="ref1" ref-type="bibr">1</xref><xref rid="ref2" ref-type="bibr">2</xref>] Major risk factors are IV drug abuse, indwelling catheters, tricuspid valve endocarditis, head and neck infections and immunocompromised state. Early diagnosis of the infectious source and appropriate use of anti-microbial therapy is critical in the management because untreated SPE can lead to the development of pulmonary abscesses and empyema.[<xref rid="ref3" ref-type="bibr">3</xref>] This vignette highlights how multiple peripheral nodular opacities, often with cavitation, are suggestive of pulmonary septic emboli in the setting of infective endocarditis.</p> |
Relationship between lung function and indoor air pollution | Could not extract abstract | <contrib contrib-type="author"><name><surname>Yasri</surname><given-names>Sora</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Wiwanitkit</surname><given-names>Viroj</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib> | Lung India : Official Organ of Indian Chest Society | <p>Sir,</p><p>The recent report on the, ‘Relationship between lung function and indoor air pollution’ among rural women in the fishing community is very interesting.[<xref rid="ref1" ref-type="bibr">1</xref>] Umoh and Peters noted that ‘chronic exposure to biomass smoke is associated with chronic bronchitis and reduced lung function in women engaged in fish smoking’.[<xref rid="ref1" ref-type="bibr">1</xref>] Of interest, there is another publication by Umoh <italic>et al</italic>., focusing on the problem of chronic bronchitis in the same community and same period, in the Niger delta.[<xref rid="ref2" ref-type="bibr">2</xref>] Umoh <italic>et al</italic>. have further mentioned a relationship between exposure to smoke and anxiety.[<xref rid="ref2" ref-type="bibr">2</xref>] Overall, the number of subjects investigated in both reports (342 VS 342)[<xref rid="ref1" ref-type="bibr">1</xref><xref rid="ref2" ref-type="bibr">2</xref>] is equal, however, the number of the cases with chronic bronchitis is not equal (68 VS 63). This leads to the query on the reliability of the present report. In addition, there is no report on the environment of the community. The main query is whether there is any other pollution in this area, in addition to fish smoking.</p> |
Author's reply | Could not extract abstract | <contrib contrib-type="author"><name><surname>Umoh</surname><given-names>Victor Aniedi</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Petersa</surname><given-names>Etete</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib> | Lung India : Official Organ of Indian Chest Society | <p>Sir</p><p>This is a response to the queries raised in a Letter to the Editor[<xref rid="ref1" ref-type="bibr">1</xref>] concerning the article titled, ‘The relationship between lung function and indoor air pollution among rural women in the Niger Delta region of Nigeria.’ The authors of the letter raised four issues: (1) There are other publications[<xref rid="ref1" ref-type="bibr">1</xref><xref rid="ref2" ref-type="bibr">2</xref>] by Umoh <italic>et al</italic>., focusing on the problem of chronic bronchitis in the same community and same period in the Niger delta; (2) Umoh <italic>et al</italic>. mentioned no further relationship between exposure to smoke and anxiety;[<xref rid="ref2" ref-type="bibr">2</xref>] (3) there is a discrepancy in the number of patients with chronic bronchitis in the two reports;[<xref rid="ref1" ref-type="bibr">1</xref><xref rid="ref2" ref-type="bibr">2</xref>] and (4) whether there is any other pollution in this area in addition to fish smoking.</p><p>
<list list-type="bullet"><list-item><p>The studies were carried out at about the same time, within the same community, by the same investigators. Therefore, the three reports came from the same population. In the article referenced as 1,[<xref rid="ref2" ref-type="bibr">2</xref>] a sample of both males and females were used, while the other two involved only the female population. The index report focused on lung function and exposure. The article referenced as 1[<xref rid="ref2" ref-type="bibr">2</xref>] focused on respiratory symptoms, while the article referenced as 2[<xref rid="ref3" ref-type="bibr">3</xref>] focused on the psychological consequences of indoor air pollution.</p></list-item><list-item><p>In the article referenced as 2[<xref rid="ref3" ref-type="bibr">3</xref>] the authors did mention the relationship between exposure and anxiety in the results section, as well as in the discussion.</p></list-item><list-item><p>Although the reports came from the same population there were slight differences in the samples. The psychiatrists were not satisfied with some responses and those were removed from the analysis and more subjects were subsequently recruited to make up the numbers.[<xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref3" ref-type="bibr">3</xref>]</p></list-item><list-item><p>The study area is a fishing settlement in the Niger delta region of Nigeria. This report evaluated the relationship between biomass smoke and lung function. Other sources of indoor air pollution were not recorded and the authors assumed that the influence of these would not be selective. The only other possible source of outdoor air pollution would have come from gas flaring during crude oil exploration. Although crude oil exploration occurred in the Niger Delta area of Nigeria, the exact locations of these oil wells were far from the study area, thus the authors did not consider that as a factor. Moreover, as the effects of such pollution would not be selective it was unlikely to introduce any bias in the analysis.</p></list-item></list>
</p> |
Bhang - beyond the purview of the narcotic drugs and psychotropic substances act | Could not extract abstract | <contrib contrib-type="author"><name><surname>Balhara</surname><given-names>Yatan Pal Singh</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Mathur</surname><given-names>Shachi</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib> | Lung India : Official Organ of Indian Chest Society | <p>Sir,</p><p>In the publication titled ‘Medical marijuana: A panacea or scourge’ the authors have reviewed an important and widely debated issue.[<xref rid="ref1" ref-type="bibr">1</xref>] The authors have stated that ‘<italic>in India, as per the Narcotic Drugs and Psychotropic Substances Act, 1985, cannabis and its various forms - hashish, ganja, charas, bhang - are banned and their possession is deemed to be unlawful</italic>’. The authors are correct in reporting that cannabis, along with other narcotic and psychotropic substances, comes under the purview of Narcotic Drugs and Psychotropic Substances Act (NDPS Act), 1985. However, interestingly, <italic>bhang</italic> is the only preparation of cannabis that is not covered in this act, and hence, is beyond the purview of this act.</p><p>As per the NDPS Act, 1985, cannabis is defined in Section 2 (iii) of the act. According to this section cannabis (hemp) is defined as: <italic>(a) Charas - a separated resin, in whatever form, whether crude or purified; obtained from the cannabis plant, which also includes the concentrated preparation and resin known as hashish oil or liquid hashish; (b) ganja - the flowering or fruiting tops of the cannabis plant, (excluding the seeds and leaves when not accompanied by the tops), by whatever name they may be known or designated; and (c) any mixture, with or without any natural material, of any of the above forms of cannabis or any drink prepared therefrom.</italic></p><p><italic>Bhang</italic> does not fall within the definition of cannabis (hemp) as defined under Section 2 (iii) of NDPS Act, 1985. This issue has been discussed at length in various judgments of various courts.[<xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref3" ref-type="bibr">3</xref><xref rid="ref4" ref-type="bibr">4</xref>] Hence, the provisions for various narcotic and psychotropic substances under the NDPS Act, 1985, are not applicable to the cannabis in <italic>bhang</italic> form. The National Policy on Narcotics and Psychotropic Substances acknowledges this fact and goes on to mention that the ‘<italic>production and sale of Bhang is permitted by many State Governments</italic>’.[<xref rid="ref5" ref-type="bibr">5</xref>]</p><p>Exclusion of <italic>bhang</italic> from the purview of NDPS Act, 1985, has remained a topic of discussion among medical and legal experts. <italic>Bhang</italic> remains one of the least studied preparations of cannabis. Most of the literature from the west has focused on smoked forms of cannabis (ganja and charas). An earlier report on <italic>bhang</italic> from Pakistan described psychosis, with symptoms of grandiosity, excitement, hostility, disorientation, hallucinations, and thought disorder among 15 patients, who had taken <italic>bhang</italic>. Interestingly, although the authors described <italic>bhang</italic> as ‘<italic>a potent beverage made from an infusion of cannabis leaves and flowering tops</italic>’.[<xref rid="ref6" ref-type="bibr">6</xref>] Technically and legally, <italic>bhang</italic> should not include any other part of the plant except the leaves. Addition of flowering tops or the resin produced from the cannabis plants is not permitted to <italic>bhang</italic> as per the National Policy on Narcotics and Psychotropic Substances.[<xref rid="ref5" ref-type="bibr">5</xref>] Another study reported <italic>bhang</italic>-induced immunotoxicity that could be attributed to a decrease in the fatty acid amide hydrolase (FAAH) protein.[<xref rid="ref7" ref-type="bibr">7</xref>]</p><p>Bhang is generally believed to be a relatively less harmful form of cannabis. Its use has got some sociocultural sanction as well in certain regions of the country. Around 17% of middle-aged adults, seeking treatment for psychoactive substance use–related disorders, report the current use of cannabis in India.[<xref rid="ref8" ref-type="bibr">8</xref>] Individuals seeking treatment for their primary drug of abuse continue to take cannabis.[<xref rid="ref9" ref-type="bibr">9</xref>] With a growing interest in the potential medical role of cannabis it becomes imperative to study different cannabis forms, for their safety profile. However, from a legal point of view bhang remains beyond the purview of the NDPS Act, 1985.</p> |
Author's reply | Could not extract abstract | <contrib contrib-type="author"><name><surname>Kashyap</surname><given-names>Surender</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Kashyap</surname><given-names>Kartikeya</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib> | Lung India : Official Organ of Indian Chest Society | <p>Sir,</p><p>We acknowledge the comments[<xref rid="ref1" ref-type="bibr">1</xref>] regarding our article published in Lung India.[<xref rid="ref2" ref-type="bibr">2</xref>] We agree with the author that <italic>Bhang</italic>, which consists of cannabis leaves, is not included under the purview of the Narcotic Drugs and Psychotropic Substances Act (NDPS Act), 1985, in legal terms. However, the purpose of our review was to shed light on the medicinal use of Marijuana in general. It is also to be noted that, in India, unscrupulous use of <italic>Bhang</italic> is prevalent. <italic>Bhang</italic>, exclusively in its leafy form is sparingly used, because of its frequent mixing with the other constituents of cannabis. It is also our view that <italic>Bhang</italic> should be strongly regulated due to its psychotropic effects, as discussed by the author,[<xref rid="ref1" ref-type="bibr">1</xref>] and therefore, should be included under the NDPS Act.</p><p>The various forms of cannabis are as follows:[<xref rid="ref3" ref-type="bibr">3</xref>]</p><p>
<list list-type="order"><list-item><p>Marijuana: The tetrahydrocannabinol (THC) content 0.5-5%, which further includes two preparations:</p><p>
<list list-type="bullet"><list-item><p><italic>Bhang</italic>-Only dry leaves</p></list-item><list-item><p><italic>Ganja</italic>-Leaves and flowering tops with a higher resin content</p></list-item></list>
</p></list-item><list-item><p>Hashish: Dried resin extracted from flowering tops with a THC content of 2-20%</p></list-item><list-item><p>Hash oil: Liquid extract that contains 15% THC</p></list-item><list-item><p>Sinsemilla: Non-pollinated flowering tops from female cannabis plant, with high content of THC (20%)</p></list-item><list-item><p>Dutch Hemp (Netherweed), with THC concentration as high as 20%</p></list-item></list>
</p><p>There is not much literature on <italic>Bhang</italic> and its effects among its users, so it is prudent to study this form of cannabis in more depth.</p><p>Interestingly, there has been an amendment in the NDPS act in March, 2014.[<xref rid="ref4" ref-type="bibr">4</xref>] The amendment has broadened the objectives of the NDPS Act and promotes the medical and scientific use of narcotic drugs and psychotropic substances. This will also pave the way for more research in the field of chronic pain management and palliative care including the use of medicinal cannabis and its congeners.</p> |
Adenosine deaminase in exudative pleural effusions | Could not extract abstract | <contrib contrib-type="author"><name><surname>Wiwanitkit</surname><given-names>Somsri</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Wiwanitkit</surname><given-names>Viroj</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib> | Lung India : Official Organ of Indian Chest Society | <p>Sir,</p><p>The report on ‘Adenosine Deaminase in Exudative Pleural Effusions’ is very interesting.[<xref rid="ref1" ref-type="bibr">1</xref>] Mehta <italic>et al</italic>. have concluded that ‘pleural fluid adenosine deaminase (ADA) is cost-effective and a good screening test for the diagnosis of tuberculosis (TB)’.[<xref rid="ref1" ref-type="bibr">1</xref>] This conclusion must be carefully considered. First, the reported predictive values of the test, in the present report, are not good.[<xref rid="ref1" ref-type="bibr">1</xref>] This finding is similar to that of a previous report by Khow-Ean <italic>et al</italic>.[<xref rid="ref2" ref-type="bibr">2</xref>] As Islam <italic>et al</italic>. have noted, the test must be used along with the conventional method to get a diagnosis.[<xref rid="ref3" ref-type="bibr">3</xref>] Khan <italic>et al</italic>. have also reported that the use of pleural fluid interferon-gamma can provide a better diagnostic result than ADA.[<xref rid="ref4" ref-type="bibr">4</xref>] Second, Mehta <italic>et al</italic>. have not performed any analysis for cost-effectiveness, hence, the conclusion on cost-effectiveness may not be possible. A good report that has considered the cost-effectiveness analysis of using ADA is the previous report by Sharma <italic>et al</italic>.[<xref rid="ref5" ref-type="bibr">5</xref>]</p> |
Author's reply | Could not extract abstract | <contrib contrib-type="author"><name><surname>Mehta</surname><given-names>Asmita Anilkumar</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Gupta</surname><given-names>Amit Satish</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Ahmed</surname><given-names>Subin</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Venkitakrishnan</surname><given-names>Rajesh</given-names></name><xref ref-type="aff" rid="aff1"/></contrib> | Lung India : Official Organ of Indian Chest Society | <p>We would like to thank Wiwanitkit <italic>et al</italic>., for their interest in our study and their valuable observations.</p><p>In response to the authors’ comments about the reported lower predictive value of the adenosine deaminase (ADA) test in our article,[<xref rid="ref1" ref-type="bibr">1</xref>] we want to clarify that as per our study, ADA is a valuable test to differentiate malignancy from TB. Our study showed that only three (8%) out of 36 cases of malignant pleural effusion had ADA ≥40 U/l.</p><p>We agree with the authors’ comments that ADA must be used along with the conventional method to get a diagnosis, as suggested by Islam <italic>et al</italic>.[<xref rid="ref2" ref-type="bibr">2</xref>]</p><p>Khan <italic>et al</italic>.[<xref rid="ref3" ref-type="bibr">3</xref>] has showed that use of the pleural fluid interferon-gamma can provide a better diagnostic result than ADA, but at a higher cost.[<xref rid="ref3" ref-type="bibr">3</xref>] We would also like to quote from the meta-analysis by Greco <italic>et al</italic>., which showed that the joint sensitivity and specificity of ADA (93%) was similar to IFN-γ (96%) and no significant difference was present in the performance of both the tests in the diagnosis of TB pleurisy.[<xref rid="ref4" ref-type="bibr">4</xref>]</p><p>We accept that we have not conducted a cost-effectiveness analysis for our study,[<xref rid="ref1" ref-type="bibr">1</xref>] and it is improper to state in conclusion that the ADA test is cost-effective. Despite what has just been stated, it has been seen that the ADA test is less expensive than other invasive tests like the thoracoscopy-guided or blind pleural biopsy.</p> |
Comments on non-utilising of rapid onsite evaluation with transbronchial needle aspiration | Could not extract abstract | <contrib contrib-type="author"><name><surname>Sindhwani</surname><given-names>Girish</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Khanduri</surname><given-names>Rakhee</given-names></name><xref ref-type="aff" rid="aff1"/></contrib> | Lung India : Official Organ of Indian Chest Society | <p>Sir,</p><p>This communication is regarding an article e-published in the July-September 2014 issue of your journal. The article is entitled ‘Diagnostic utility of conventional transbronchial needle aspiration without rapid onsite evaluation in patients with lung cancer,’ by Ritika Walia <italic>et al</italic>.</p><p>Our comments are with regard to non-utilization of rapid onsite cytology (ROSE) with transbronchial needle aspiration (TBNA) in your study. It is understandable that despite many studies on the efficacy of ROSE with TBNA, it is largely underutilized in most of the bronchoscopy suites in India, be it a secondary or tertiary care center. This underutilization of ROSE is likely due to factors like non-familiarity with the procedure, lack of cytopathologists for bronchoscopy suites, and so on. However, in a study done at our center, which is a teaching institute in the Himalayan region of north India, it has been proved that ROSE is feasible, cost-effective, and patient-friendly in terms of reduction in the number and repetition of bronchoscopy procedures.[<xref rid="ref1" ref-type="bibr">1</xref>]</p><p>In tertiary care centers with attached teaching institutes, where postgraduate programs for Pathology are being run, there is availability of trainees of Pathology. In our study, we utilized the second and third year residents of Pathology as resource persons and they were able to bring good yield of ROSE. In our study, we have also assessed the cost-effectiveness of ROSE in terms of reduction of repeat procedures in case of negative first results and we found that 45% of the patients would have required a repeat procedure in the absence of ROSE. Similar results have been reported in other studies as well.[<xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref3" ref-type="bibr">3</xref><xref rid="ref4" ref-type="bibr">4</xref>]</p><p>Hence, ROSE should be promoted in bronchoscopy centers in India and studies on TBNA or other procedures used for getting cytological material should be performed with ROSE, so that the message about the feasibility of ROSE is passed on to the pulmonologists of our country.</p> |
Quantum magnetic phase transition in square-octagon lattice | <p>Quantum magnetic phase transition in square-octagon lattice was investigated by cellular dynamical mean field theory combining with continuous time quantum Monte Carlo algorithm. Based on the systematic calculation on the density of states, the double occupancy and the Fermi surface evolution of square-octagon lattice, we presented the phase diagrams of this splendid many particle system. The competition between the temperature and the on-site repulsive interaction in the isotropic square-octagon lattice has shown that both antiferromagnetic and paramagnetic order can be found not only in the metal phase, but also in the insulating phase. Antiferromagnetic metal phase disappeared in the phase diagram that consists of the anisotropic parameter <italic>λ</italic> and the on-site repulsive interaction U while the other phases still can be detected at T = 0.17. The results found in this work may contribute to understand well the properties of some consuming systems that have square-octagon structure, quasi square-octagon structure, such as ZnO.</p> | <contrib contrib-type="author"><name><surname>Bao</surname><given-names>An</given-names></name><xref ref-type="aff" rid="a1">1</xref><xref ref-type="aff" rid="a2">2</xref></contrib><contrib contrib-type="author"><name><surname>Tao</surname><given-names>Hong-Shuai</given-names></name><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Liu</surname><given-names>Hai-Di</given-names></name><xref ref-type="aff" rid="a3">3</xref></contrib><contrib contrib-type="author"><name><surname>Zhang</surname><given-names>XiaoZhong</given-names></name><xref ref-type="aff" rid="a1">1</xref></contrib><contrib contrib-type="author"><name><surname>Liu</surname><given-names>Wu-Ming</given-names></name><xref ref-type="corresp" rid="c1">a</xref><xref ref-type="aff" rid="a3">3</xref></contrib><aff id="a1"><label>1</label><institution>Laboratory of Advanced Materials, Department of Materials Science and Engineering, Tsinghua University</institution>, Beijing 100084, <country>China</country></aff><aff id="a2"><label>2</label><institution>School of Mathematics, Physics and Biological Engineering, Inner Mongolia University of Science and Technology</institution>, Baotou 014010, <country>China</country></aff><aff id="a3"><label>3</label><institution>Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Sciences</institution>, Beijing 100190, <country>China</country></aff> | Scientific Reports | <p>The discovery and classification of quantum phases of matters and the transition between these distinctive phases have been recurring theme in condensed matter physics for many years and still wheel the researchers' extensive interests<xref ref-type="bibr" rid="b1">1</xref><xref ref-type="bibr" rid="b2">2</xref><xref ref-type="bibr" rid="b3">3</xref><xref ref-type="bibr" rid="b4">4</xref><xref ref-type="bibr" rid="b5">5</xref><xref ref-type="bibr" rid="b6">6</xref><xref ref-type="bibr" rid="b7">7</xref><xref ref-type="bibr" rid="b8">8</xref><xref ref-type="bibr" rid="b9">9</xref><xref ref-type="bibr" rid="b10">10</xref><xref ref-type="bibr" rid="b11">11</xref><xref ref-type="bibr" rid="b12">12</xref><xref ref-type="bibr" rid="b13">13</xref><xref ref-type="bibr" rid="b14">14</xref><xref ref-type="bibr" rid="b15">15</xref>. Notable quantum phases, such as super-conductivity, quantum hall effect, Mott insulating phase and topological phase, have great significance in theoretical investigations and promising potential in applications. These exotic phases have been found in many quantum systems with quite common structure, such as the honeycomb lattice, the triangular lattice, the decorated honeycomb lattice, the kagomé lattice and so forth<xref ref-type="bibr" rid="b16">16</xref><xref ref-type="bibr" rid="b17">17</xref><xref ref-type="bibr" rid="b18">18</xref><xref ref-type="bibr" rid="b19">19</xref><xref ref-type="bibr" rid="b20">20</xref><xref ref-type="bibr" rid="b21">21</xref><xref ref-type="bibr" rid="b22">22</xref><xref ref-type="bibr" rid="b23">23</xref><xref ref-type="bibr" rid="b24">24</xref><xref ref-type="bibr" rid="b25">25</xref><xref ref-type="bibr" rid="b26">26</xref><xref ref-type="bibr" rid="b27">27</xref><xref ref-type="bibr" rid="b28">28</xref><xref ref-type="bibr" rid="b29">29</xref><xref ref-type="bibr" rid="b30">30</xref><xref ref-type="bibr" rid="b31">31</xref>. Recently a unique quantum many particle lattice system named square-octagon lattice have been investigated in theoretical way intensively and a plenty of meaningful results have been presented. Researchers have found topological phases and the transitions between these novel phases in the square-octagon lattice that 1/4 and 3/4 filled with fermions under the framework of the tight binding model through considering the spin-orbit coupling fermions<xref ref-type="bibr" rid="b32">32</xref>. Another one theoretical joy models named Fully packed loop model also has been adopted to investigate the square-octagon lattice<xref ref-type="bibr" rid="b33">33</xref>. Additionally, researchers have found quasi square-octagon structure in (<inline-formula id="m9"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e166" xlink:href="srep06918-m9.jpg"/></inline-formula>) surface of functional material ZnO by first principle calculations and aberration-corrected transmission electron microscopy (ACTEM) observation experimentally during its pressure induced phase transition process<xref ref-type="bibr" rid="b34">34</xref>.</p><p>However, few of the previous work related to the square-octagon lattice considered the particles' on-site repulsive interactions that have crucial effect on the properties of the systems. Therefore in this work, the celebrated Hubbard model<xref ref-type="bibr" rid="b35">35</xref><xref ref-type="bibr" rid="b36">36</xref> was used to describe this strongly correlated many particle systems for the purpose of understanding well the influences of interaction on the properties of the square-octagon lattice with fermions. The cellular dynamical mean field theory (CDMFT)<xref ref-type="bibr" rid="b37">37</xref><xref ref-type="bibr" rid="b38">38</xref><xref ref-type="bibr" rid="b39">39</xref><xref ref-type="bibr" rid="b40">40</xref><xref ref-type="bibr" rid="b41">41</xref><xref ref-type="bibr" rid="b42">42</xref>, which maps the lattice to a self-consistent embedded cluster in real space, was adopted to deal with the Hubbard model and the continuous time quantum Monte Carlo (CTQMC)<xref ref-type="bibr" rid="b47">47</xref> algorithm was used as a impurity solver to deal with the mean field equations. The CDMFT is proved to be more successful than the dynamical mean field theory and the CTQMC is more accurate than the general quantum Monte Carlo method. Based on the single-particle Greens function given by the CDMFT and CTQMC, the single-particle density of states and the double occupancy which play critical role in the identification of Mott metal-insulator transition have been calculated. The phase diagram which composed of the on-site interaction and the energy gap, the relationship between the interaction and magnetic properties of the systems also have investigated through defining the magnetic order parameter. We also presented phase diagram which consists of the competition between the temperature and on-site repulsive interaction for isotropic square-octagon lattice and the the competition between the anisotropy and on-site repulsive interaction.</p><sec disp-level="1" sec-type="results"><title>Results</title><sec disp-level="2"><title>Strongly correlated square-octagon lattice system</title><p>The square-octagon lattice is a bipartite lattice that can be thought of as a square lattice in which each vertex has been decorated with a tilted square, as shown in <xref ref-type="fig" rid="f1">Fig. 1 (a)</xref> and its first Brillouin zone in <xref ref-type="fig" rid="f1">Fig. 1 (b)</xref>. It has the same coordination number as the honeycomb lattice has and its boundary shapes armchair. It enjoys the symmetry of the square lattice and symmetrically it satisfies C<sub>4</sub> point group.</p><p>The standard Hubbard model is adopted to investigate the square-octagon lattice and the Hamiltonian can be written as follows, <disp-formula id="m1"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e199" xlink:href="srep06918-m1.jpg"/></disp-formula>where <inline-formula id="m10"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e202" xlink:href="srep06918-m10.jpg"/></inline-formula> and <italic>c<sub>iσ</sub></italic> represent creation and annihilation operator of fermions with spin <italic>σ</italic> on site i respectively, while <inline-formula id="m11"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e213" xlink:href="srep06918-m11.jpg"/></inline-formula> denote the particle number operator on lattice site i. The value of spin index <italic>σ</italic> is spin up or spin down. The first two terms in this Hamiltonian account for the kinetic energy of the system, which is characterized by the coefficient factor t<sub>1</sub> and t<sub>2</sub>. t<sub>1</sub> represents the hopping between the nearest neighboring sites in the same square lattice and t<sub>2</sub> is the hopping between the endpoints of the liking line of the two nearest neighboring square lattice. The third term describes the on-site repulsive interaction (U > 0) between fermions with opposite spin. Here we set <italic>t</italic><sub>1</sub> as energy unit (<italic>t</italic><sub>1</sub> = 1). <italic>μ</italic> is chemical potential and in order to reach half filled case <italic>μ</italic> should equals zero for this lattice system. We also defined an anisotropic parameter <italic>λ</italic> which equals to the ration <italic>t</italic><sub>1</sub>/<italic>t</italic><sub>2</sub> (<italic>λ</italic> = <italic>t</italic><sub>1</sub>/<italic>t</italic><sub>2</sub>).</p><p>For the case of U = 0 and <italic>μ</italic> = 0, the Hubbard model transmits to the tight binding model and the Hamiltonian in the momentum space is <inline-formula id="m12"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e282" xlink:href="srep06918-m12.jpg"/></inline-formula>, in which Ψ<italic><sub>k</sub></italic> = (<italic>c</italic><sub>1<italic>k</italic>↑</sub>, <italic>c</italic><sub>2<italic>k</italic>↑</sub>, <italic>c</italic><sub>3<italic>k</italic>↑</sub>, <italic>c</italic><sub>4<italic>k</italic>↑</sub>, <italic>c</italic><sub>1<italic>k</italic>↓</sub>, <italic>c</italic><sub>2<italic>k</italic>↓</sub>, <italic>c</italic><sub>3<italic>k</italic>↓</sub>, <italic>c</italic><sub>4<italic>k</italic>↓</sub>)<sup>T</sup>. The index i = 1, 2, 3,4 in creation and annihilation operators represent the four sites in each unit cell as illustrated in <xref ref-type="fig" rid="f1">Fig. 1 (a)</xref> and k is the locations in the first Brillouin zone. ↑ and ↓ hint the spin-up and spin-down states respectively. <inline-formula id="m13"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e362" xlink:href="srep06918-m13.jpg"/></inline-formula> takes the following form <disp-formula id="m2"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e365" xlink:href="srep06918-m2.jpg"/></disp-formula></p><p>Since <italic>H</italic><sub>0</sub> is decoupled in spin states, so <inline-formula id="m14"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e374" xlink:href="srep06918-m14.jpg"/></inline-formula> is block diagonalized, i.e. two blocks representing spin-up and spin-down electrons are the same. The energy band of the first Brillouin zone of the square-lattice under the frame of the tight biding model has been obtained through diagonalizing <inline-formula id="m15"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e377" xlink:href="srep06918-m15.jpg"/></inline-formula> and shown in <xref ref-type="fig" rid="f1">Fig. 1 (c)</xref>. The density of states of the square-octagon lattice half filled with fermions without interaction at T = 0.2 for different anisotropic parameter <italic>λ</italic> in <xref ref-type="fig" rid="f1">Fig. 1 (d)</xref>.</p><p>In order to get the effect of anisotropic parameter <italic>λ</italic> and the value of hopping term <italic>t</italic><sub>1</sub> and <italic>t</italic><sub>2</sub> on the phase transitions, we presented the energy band along the line between the high symmetric points in the first Brillouin zone in <xref ref-type="fig" rid="f2">Fig. 2</xref> even has shown the 3-dimensional energy band in the first Brillouin zone in <xref ref-type="fig" rid="f1">Fig. 1 (c)</xref>. The energy band Ek<sub>2</sub> and Ek<sub>3</sub> touch at Γ point and M point for <italic>λ</italic> = 2.0 in <xref ref-type="fig" rid="f2">Fig. 2 (a)</xref>. Energy band Ek<sub>2</sub>, Ek<sub>3</sub> and Ek<sub>4</sub> cross at Γ point while Ek<sub>1</sub>, Ek<sub>2</sub> and Ek<sub>3</sub> cross at M point for <italic>λ</italic> = 1, the system is in metallic states. It can be seen that with the decreasing of anisotropic parameter <italic>λ</italic>, Ek<sub>2</sub> and Ek<sub>3</sub> separate and meanwhile Ek<sub>1</sub> and Ek<sub>2</sub> contact at M point, Ek<sub>3</sub> and Ek<sub>4</sub> contact at Γ point while <italic>λ</italic> = 0.83. The system is still in metallic states. As <xref ref-type="fig" rid="f2">Fig. 2 (d)</xref> shows that energy band Ek<sub>2</sub> and Ek<sub>3</sub> completely separated by the Fermi energy level while <italic>λ</italic> = 0.5 and the system turns into insulating states.</p></sec><sec disp-level="2"><title>Phase diagrams of the square-octagon lattice</title><p>With the increase of on-site repulsive interaction U the the probability of more than one fermions occupying the same lattice site will reduce and eventually only one fermion confined in per lattice site at certain large value of U. The confinement of fermion in one lattice site is described by double occupancy (Docc)<xref ref-type="bibr" rid="b43">43</xref> which is an important quantity that used to characterize the critical point in Mott phase transitions and indicates the transition order, and also can be used to describe the localization of the electrons in strongly correlated electron systems. The formula of double occupancy is <inline-formula id="m16"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e491" xlink:href="srep06918-m16.jpg"/></inline-formula>, where <italic>F</italic> is free energy. The double occupancy of isotropic square-octagon lattice as a function of interaction for fixed temperature and as a function of temperature for fixed interaction have been shown in outer part and inner part of <xref ref-type="fig" rid="f3">Fig. 3</xref> respectively. It can be seen in the outer part of <xref ref-type="fig" rid="f3">Fig. 3</xref> that Docc decreases as the interaction increases due to the suppressing of the itinerancy of the atoms. When the interaction is stronger than the critical interaction of the Mott transition, the effect of the temperature on Docc is weakened and Docc for different temperatures consistently trend to zero, which shows the temperature does not affect the double occupancy distinctly. The continuity of the evolution of the double occupancy by interaction shows that it is a second-order transition. We also have shown the relation between Docc and the temperature at different interaction in inner part of <xref ref-type="fig" rid="f3">Fig. 3</xref>. From the inner part of <xref ref-type="fig" rid="f3">Fig. 3</xref> we can find that the double occupancy decreases with the increase of the temperature for fixed on-site repulsive interaction.</p><p>The density of states is one of the most important quantities in the characterization of the Mott metal-insulator phase transition of Hubbard model. For the purpose of investigating the Mott metal-insulator phase transition as the evolution of single particle spectral<xref ref-type="bibr" rid="b44">44</xref>, we defined Density of states, the formula is <disp-formula id="m3"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e514" xlink:href="srep06918-m3.jpg"/></disp-formula>where <italic>i</italic> is the lattice points index in the cluster. The Density of states can be derived from the imaginary time Greens function <italic>G</italic>(<italic>τ</italic>) by using the maximum entropy method<xref ref-type="bibr" rid="b45">45</xref>. <xref ref-type="fig" rid="f4">Fig. 4 (a) and (b)</xref> respectively shows the density of states of isotropic square-octagon lattice for different temperature at <italic>U</italic>/<italic>t</italic><sub>1</sub> = 6 and the density of states for different repulsive interactions while <italic>T</italic>/<italic>t</italic><sub>1</sub> = 0.5. The inner part of <xref ref-type="fig" rid="f4">Fig. 4 (a)</xref> is the density of states of system for <italic>U</italic>/<italic>t</italic><sub>1</sub> = 0 and <italic>T</italic>/<italic>t</italic><sub>1</sub> = 0.17. It can be evidently seen in <xref ref-type="fig" rid="f3">Fig. 3</xref> that the systems will change from metal state to Mott insulating state which characterized by the opened gap at <italic>ω</italic> = 0 with the increase of the repulsive interaction for fixed temperature and the decrease of the temperature for the fixed repulsive interactions. However, the evolution shape of the density of states with the change of frequency in this two cases is much different from each other. The critical point between paramagnetic metal state and Mott insulating state is (<italic>T</italic>/<italic>t</italic><sub>1</sub> = 0.17, <italic>U</italic>/<italic>t</italic><sub>1</sub> = 6), (<italic>T</italic>/<italic>t</italic><sub>1</sub> = 0.25, <italic>U</italic>/<italic>t</italic><sub>1</sub> = 7) and (<italic>T</italic>/<italic>t</italic><sub>1</sub> = 0.5, <italic>U</italic>/<italic>t</italic><sub>1</sub> = 8).</p><p>In order to describe the Fermi surface evolution, we defined the spectral function <inline-formula id="m17"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e628" xlink:href="srep06918-m17.jpg"/></inline-formula>. A linear extrapolation of the first two Matsubara frequencies is used to estimate the self-energy to zero frequency. The Fermi surface of isotropic square-octagon lattice half filled with fermions for different interaction <italic>U</italic>/<italic>t</italic><sub>1</sub> at fixed temperature <italic>T</italic>/<italic>t</italic><sub>1</sub> = 0.1 is shown in <xref ref-type="fig" rid="f5">Fig. 5 (a_ 2), (b_ 2) and (c_ 2)</xref>. We also have shown the Fermi surface of anisotropic square-octagon lattice in <xref ref-type="fig" rid="f5">Fig. 5</xref> for <italic>U</italic>/<italic>t</italic><sub>1</sub> = 4, 6, 8 while <italic>T</italic>/<italic>t</italic><sub>1</sub> = 0.1. With the decreasing of the <italic>λ</italic> for fixed interaction the amplitude of the spectral weight becomes bigger due to the localization of particles.</p><p>Based on the systematic calculations on the quantities mentioned above, we have presented the T - U phase diagram of isotropic square-octagon lattice and the competition between anisotropic parameter <italic>λ</italic> and the on-site repulsive interaction (U) for fixed low temperature <italic>T</italic>/<italic>t</italic><sub>1</sub> = 0.17. We also studied the magnetic properties of each phase in the square-octagon lattice by using the magnetic order parameter <inline-formula id="m18"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e687" xlink:href="srep06918-m18.jpg"/></inline-formula>, where 〈<italic>n<sub>iσ</sub></italic>〉 is the electron density at lattice site <italic>i</italic> with spin index <italic>σ</italic> and sign(i) = 1 if i = 1, 3 and sign(i) = −1 if i = 2, 4 as shown in <xref ref-type="fig" rid="f1">Fig. 1(a)</xref>. From the definition of magnetic order it can be known that <italic>m</italic> = 0 correspond to paramagnetic phase while <italic>m</italic> ≠ 0 represents antiferromagnetic phase. Both paramagnetic and anti-ferromagnetic order as shown in <xref ref-type="fig" rid="f6">Fig. 6 (a) and (b)</xref> have been not only found in insulating state but also in the metal state in the T - U phase diagram of isotropic square-octagon lattice. Fermi surface evolution of isotropic square-octagon lattice in paramagnetic metal state in <xref ref-type="fig" rid="f6">Fig. 6 (c)</xref> and in antiferromagnetic metal state in <xref ref-type="fig" rid="f6">Fig. 6 (d)</xref> for for U = 5.5 and T = 0.17. As shown in <xref ref-type="fig" rid="f6">Fig. 6 (e)</xref> that only at low enough temperature or weak enough on-site repulsive scale the systems can transform to antiferromagnetic metal state. The narrow antiferromagnetic metal state region in <xref ref-type="fig" rid="f6">Fig. 6 (e)</xref> means this state is sensitive to the temperature and the on-site repulsive interaction. This results have been confirmed further by the relation between the energy gap and on-site repulsive interaction and the magnetic order parameter m and the on-site repulsive interaction in <xref ref-type="fig" rid="f7">Fig. 7</xref>. The anti-ferromagnetic metal state disappeared in the competition of anisotropic parameter <italic>λ</italic> and interaction diagram while other phases still exist at T = 0.17.</p></sec></sec><sec disp-level="1" sec-type="discussion"><title>Discussion</title><p>In this work, we use standard Hubbard model to describe the square-octagon lattice and present the quantum magnetic phases and the transition between these novel phases in this many particle systems. We have investigated not only the effect of on-site repulsive interaction of particles with the opposite direction spin on the same site, but also shown the influence of the Kinetic energy of the systems on the phase transitions. We also have studied the magnetic properties of the square-octagon lattice through defining the magnetic order parameter <italic>m</italic>. We hope the results found in this study can be useful for understanding the property of this lattice and the real materials with this structure, even can be helpful for the research on the functional material ZnO with quasi square-octagon lattice.</p></sec><sec disp-level="1" sec-type="methods"><title>Methods</title><sec disp-level="2"><title>Cluster dynamical mean-field theory</title><p>The cellular dynamical mean-field theory (CDMFT) was used to investigate this many particle square-octagon lattice. In comparison to the general dynamical mean field theory, the cellular dynamical mean field theory gives much more reliable simulation results for low-dimensional system with strong quantum fluctuations due to its efficient consideration of the nonlocal effect. In our case, the cellular dynamical mean field theory maps the original square-octagon lattice onto a 4-site effective cluster embedded in a self-consistent bath field, as shown in <xref ref-type="fig" rid="f1">Fig. 1 (a)</xref>. At the beginning of the self consistent calculation process, we guess a mini self-energy Σ(<italic>iω</italic>) which is independent of momentum<xref ref-type="bibr" rid="b46">46</xref> and the Weiss field <italic>G</italic><sub>0</sub>(<italic>iω</italic>) can be obtained by the coarse-grained Dyson equation: <disp-formula id="m4"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e766" xlink:href="srep06918-m4.jpg"/></disp-formula>where <italic>ω</italic> is Matsubara frequency, <italic>μ</italic> is the chemical potential, Σ<bold><sub>K</sub></bold> is the summation all over the reduced Brillouin zone of the super-lattice. <italic>t</italic>(<bold>K</bold>) is 4 dimensional hopping matrix of super-lattice which drawn from the square-octagon lattice under the framework of cluster dynamical mean field theory.</p></sec><sec disp-level="2"><title>Continuous-time quantum Monte-Carlo algorithm</title><p>The continuous-time quantum Monte-Carlo (CTQMC) algorithm was used as impurity solver. The CTQMC is based on a series expansion for the partition function in the powers of interaction and the partition function is <disp-formula id="m5"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e790" xlink:href="srep06918-m5.jpg"/></disp-formula>where <italic>T<sub>τ</sub></italic> is time-ordering operator, <inline-formula id="m19"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e798" xlink:href="srep06918-m19.jpg"/></inline-formula> and <italic>H</italic><sub>1</sub> is Hamiltonian in interaction picture, <inline-formula id="m20"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e806" xlink:href="srep06918-m20.jpg"/></inline-formula> is the partition function for the unperturbed term. Through inserting <italic>H</italic><sub>1</sub> = <italic>U</italic>Σ<italic><sub>i</sub>n<sub>i</sub></italic><sub>↑</sub><italic>n<sub>i</sub></italic><sub>↓</sub> into the partition function and using Wick's theorem further to reform ordering operators in partition functions. The ordering operators can be expressed by the determinants of matrix which consist of the non-interacting Green functions <italic>G</italic><sup>0</sup>. The new self-energy Σ(<italic>iω</italic>) is recalculated by the Dyson equation: <disp-formula id="m6"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e842" xlink:href="srep06918-m6.jpg"/></disp-formula></p><p>The cluster Green's function <italic>G</italic>(<italic>iω</italic>) can be obtained by CTQMC and 1 × 10<sup>6</sup> QMC sweeps are carried through for each CDMFT loop<xref ref-type="bibr" rid="b47">47</xref>. The cluster Green's function both in imaginary time and at Matsubara frequencies: <disp-formula id="m7"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e857" xlink:href="srep06918-m7.jpg"/></disp-formula><disp-formula id="m8"><inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" id="d33e859" xlink:href="srep06918-m8.jpg"/></disp-formula>where <italic>G</italic><sub>0</sub>(<italic>iω</italic>) is a bare Green's function and <italic>M<sub>i</sub></italic><sub>,<italic>j</italic></sub> is the elements of inverse matrix of matrix that composed of non-interacting Green's functions. The more details about CTQMC can be found in the reference herein<xref ref-type="bibr" rid="b47">47</xref>.</p></sec></sec><sec disp-level="1"><title>Author Contributions</title><p>A.B. performed calculations. A.B., H.S.T., H.D.L., X.Z.Z. and W.M.L. analyzed numerical results. A.B., X.Z.Z. and W.M.L. contributed in completing the paper.</p></sec> |
A time of change | Could not extract abstract | <contrib contrib-type="author"><name><surname>Kumar</surname><given-names>Santosh</given-names></name><xref ref-type="aff" rid="aff1"/></contrib> | Indian Journal of Urology : IJU : Journal of the Urological Society of India | <p>We are going through a time of change. Apart from the ones affecting the journal and national politics, about 3 years back, there was a paradigm shift in the government policy on medical education with an emphasis on specialist training. This resulted in a sudden increase of post-graduate training positions in surgical specialties, including Urology. Without a simultaneous increase in infrastructure or operating time in most departments, it can be argued that such an increase has possibly diluted urological training.</p><p>However, this increase in number of post-graduate trainees has some obvious benefits. The greater number of urologists will hopefully translate into more of them migrating to smaller towns to set up their practice. The number of members of the Urological Society of India is also increasing rapidly, with a proportionate increase in the number of papers submitted for presentation in its zonal and national meetings. It is interesting to note that there are more prospective trials being conducted and a greater number of research protocols being submitted to Institutional Review Boards for approval. Unfortunately, most of these trials are still not being registered with clinical trial registries. It is also not clear if the increasing number of submissions for conferences is being translated into publications. Registration of trials ensures that data of negative or equivocal studies that are not published are also are available thus improving transparency, accountability and accessibility of clinical trials. While a number of journals make trial registry a precondition to publication, it is still not the norm. Academic institutions must take a lead to ensure that trials on human subjects are registered with these registries before enrollment of the first participant.</p><p>In this the last issue for the year 2014, we have focused on prostatic cancer. With increasing life expectancy and more widespread availability of prostate-specific antigen testing, there is an increase in the detection of prostate cancer, particularly in urban India. This trend is expected to continue.[<xref rid="ref1" ref-type="bibr">1</xref>] The number of patients who present with metastatic disease continues to be large. In this issue of the journal, Gagan Gautam writes a provocative article on the evidence for radical prostatectomy in men with metastatic prostate cancer.[<xref rid="ref2" ref-type="bibr">2</xref>] However outrageous it may sound, a select group may be benefited. Associate Editor, Arabind Panda, provides a counter-argument.[<xref rid="ref3" ref-type="bibr">3</xref>] The quality of evidence is low and should be interpreted accordingly. Two original articles on radical prostatectomy highlight the changes in prostate cancer occurring around the world. The first from the Weill Cornell Medical College, New York, looks at the change in pathological outcomes and operative trends with robot-assisted laparoscopic radical prostatectomy at their center, where they noted a “reverse stage migration” with increasing proportion of men undergoing surgery for extracapsular disease, while the second from India looks at continence outcomes in early-stage disease.</p><p>Continuing on the theme of prostate cancer is the symposium on newer trends in radical prostatectomy, guest edited by Professor Narmada P. Gupta. He has roped in a team of experts on the subject who have vast original experience of their own and have covered contemporary topics from neurovascular anatomy and its preservation, surgery in high-risk disease, the challenges of salvage surgery and handling difficult anastomosis with management issues of erectile dysfunction and positive surgical margins. If the increasing number of installations of the robotic systems in the country is any indication, this may become the focus of a lot of articles in the journal in the future.</p> |
Is it truly outrageous to consider radical prostatectomy for men with metastatic prostate cancer? | <p>Radical prostatectomy is a leading form of treatment for non metastatic prostate cancer. It has been shown to improve survival in the long term as well as delay or prevent the onset of metastatic disease. However, as the treatment of metastatic prostate cancer has evolved in the last few years with the introduction of newer agents, the possible role of ‘cytoreductive’ radical prostatectomy is now being explored. Preliminary evidence suggests that radical prostatectomy may have a future role in this clinical scenario with a potential to improve quality and quantity of life in selected patients with metastatic disease.</p> | <contrib contrib-type="author"><name><surname>Gautam</surname><given-names>Gagan</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib> | Indian Journal of Urology : IJU : Journal of the Urological Society of India | <p>Radical prostatectomy (RP) is a leading treatment option for clinically organ confined prostate cancer (CaP) in men with a life expectancy greater than 10-15 years.[<xref rid="ref1" ref-type="bibr">1</xref>] Of late, there is growing evidence to suggest that patients with high-risk and/or locally advanced disease may benefit from RP and have prolonged disease free and overall survival (OS), especially when treated with a multimodal approach in combination with androgen deprivation therapy (ADT) and/or radiation therapy.[<xref rid="ref2" ref-type="bibr">2</xref>] It is probably not just pure coincidence, that these newer data have been accompanied by a growing body of evidence of a lack of benefit of RP in patients with low risk disease, especially in older men.[<xref rid="ref3" ref-type="bibr">3</xref>] All clinical guidelines now make it mandatory to counsel men with low risk organ confined CaP about the option of active surveillance vis a vis immediate treatment as a means of reducing morbidity without compromising survival.[<xref rid="ref1" ref-type="bibr">1</xref>] As the RP pendulum shifts from low risk to high-risk and locally advanced disease, is it now time to actually go to the other extreme and offer RP to patients with metastatic prostate cancer? Is it possible that in the years to come RP + ADT would be a frontline treatment option for otherwise healthy men with metastatic CaP? As provocative and outrageous this may seem at present, there is some recent data to suggest that this statement may not be far from the truth.</p><p>A recent study by Culp <italic>et al</italic>. has evaluated the role of definitive local treatment (RP or brachytherapy [BT]) in patients with metastatic CaP.[<xref rid="ref4" ref-type="bibr">4</xref>] In this retrospective analysis, the authors identified 8185 men with metastatic CaP over a 7 year period (2004-2010) from the surveillance epidemiology and end results (SEER) database and comparatively evaluated those who underwent definitive treatment of the primary tumor, with the rest, who did not receive local treatment. A comparative analysis was performed between patients undergoing RP (<italic>n</italic> = 245) or BT (<italic>n</italic> = 129) and those not receiving surgery or radiation (NSR, <italic>n</italic> = 7811). They found that men with metastatic disease who underwent RP or BT had a significantly better disease specific survival (DSS) and OS when compared to the NSR group (5 years DSS: 75.8% vs. 61.3% vs. 48.7%, respectively, <italic>P</italic> < 0.001; 5 years OS: 67.4% vs. 52.6% vs. 22.5%, respectively, <italic>P</italic> < 0.001). There was no significant intergroup difference in the survival of patients dying from nonprostate cancer causes thereby lending credence to the argument that there was a true beneficial effect of the local intervention and that the observed advantage cannot completely be attributed to a selection bias. Although survival benefits were maintained even in patients with prostate-specific antigen (PSA) >20 ng/ml or those above 70 years of age, the authors did identify certain factors associated with an improved response to local treatment. These included age <70 years, clinical stage ≤T3, Gleason score ≤ 7, PSA <20 ng/ml and an absence of pelvic lymphadenopathy.</p><p>Although, this particular study is fraught with potential shortcomings due to its retrospective nonrandomized design, inherent selection bias due to unmeasured variables, lack of standardized follow-up and a lack of information regarding adjuvant and salvage treatments, There is little doubt that it does ask some provocative questions and implores us to look at the biological role of definitive primary treatment in the setting of metastatic prostate cancer. While hypothetical explanations for this observed benefit can be many (decreased tumor burden, immune modulation, improved response to secondary treatment, avoidance of secondary complications attributable to local tumor growth), it still remains to be seen whether these findings can be replicated in prospective trials.</p><p>The impact of prior local treatment in patients being treated for metastatic CaP has been studied earlier. In a secondary analysis of the SWOG 8894 study, the authors found that patients who had undergone RP and then subsequently developed metastatic disease, had a significantly better survival with a hazard ratio of 0.77 (95% confidence interval 0.53-0.89) when compared to those who never received the procedure.[<xref rid="ref5" ref-type="bibr">5</xref>] This survival advantage was however, not replicated in a similar study published later.[<xref rid="ref6" ref-type="bibr">6</xref>] Indirect evidence also exists in the form of a positive impact of RP in patients with locally advanced disease and metastatic lymph node involvement. In a recent analysis of patients from the Munich cancer registry, Engel <italic>et al</italic>. demonstrated a survival advantage for patients who underwent RP in spite of an intraoperative detection of positive lymph nodes when compared to those in whom the procedure was abandoned.[<xref rid="ref7" ref-type="bibr">7</xref>] In this study, encompassing 938 lymph node positive patients, those who proceeded with RP (<italic>n</italic> = 688) had a significantly better 5 and 10 year OS (84% and 64%) when compared to those in whom RP was abandoned (60% and 28%). The estimated 5 and 10 years DSS was also better in the former group (95% and 85%) as compared to the latter (70% and 40%). The quality of evidence presented in these studies, however, is far from ideal. All these analyses suffer from selection bias inherent to their retrospective design and, short of a prospective randomized trial, continue to be debatable.</p><p>Another major factor to be studied is the quality-of-life after a palliative surgery performed in the setting of metastatic prostate cancer. The proponents of this approach would cite potential benefits of removing the malignant prostate and avoiding the complications of local tumor growth, including hematuria, urinary tract infections, outflow obstruction, involvement of ureteric orifices and consequent upper tract deterioration requiring multiple surgical procedures and their consequent morbidity. These quality-of-life issues tend to blur the lines between a palliative and a definitive surgical intervention in prostate cancer.</p><p>Although, as of now, we may not have the evidence to offer RP to all men who present with metastatic prostate cancer, the day may not be far when this approach may become standard for highly selected patients. At the very least, the urology community needs to debate and evaluate this topic, and lay the ground for future prospective studies to clarify it further. After all, literature abounds with the beneficial effect of the treatment of the primary tumor in other metastatic malignancies such as breast, colon, ovary and closer home-renal cell carcinoma.[<xref rid="ref8" ref-type="bibr">8</xref>] The era of “cytoreductive prostatectomy” may be upon us, sooner than we think!</p> |
Commenatry on Gautam G. Is it truly outrageous to consider radical prostatectomy for men with metastatic prostate cancer? Indian J Urol 2014;30:366-7 | Could not extract abstract | <contrib contrib-type="author"><name><surname>Panda</surname><given-names>Arabind</given-names></name><xref ref-type="aff" rid="aff1"/></contrib> | Indian Journal of Urology : IJU : Journal of the Urological Society of India | <p>The concept of radical prostatectomy for metastatic prostate cancer is interesting. The rationale of debulking in metastatic cancer is derived from the experience in gastric and ovarian carcinomas where debulking and systemic chemotherapy have shown a survival benefit. The essential difference between adenocarcinoma prostate and these cell types is the absence of an equally effective chemotherapeutic regimen for prostate cancer. Hormonal ablation alone invariably leads to castration resistant disease in the course of time.</p><p>Patients who received local therapy in the SEER study could be a highly selected group. A retrospective analysis, particularly from a registry, has disadvantages. The reasons for selecting a treatment may not be adequately documented. Further, the comparisons would be biased if the groups (without surgery or radiation therapy) were not adequately matched. In fact, in this study, they are unmatched. The prostate specific antigen (PSA) levels were >30 ng/dl in 61.6% of patients of the no surgery or radiation group but only 13 % of patients in the radical prostatectomy group had similar levels of PSA. The grade and exact TNM stage was unknown in a significant number of patients in the no surgery group while the documentation was much better in the local therapy group, perhaps an example of selection bias.[<xref rid="ref1" ref-type="bibr">1</xref>]</p><p>The paper using data from the Munich cancer registry also suffers from similar issues. The groups and unmatched and <italic>per se</italic> the patients who underwent radical prostatectomy had lesser disease bulk than those who did not.[<xref rid="ref2" ref-type="bibr">2</xref>] The message that comes out of this research is that there appears to be a role for lymphadenectomy in radical prostatectomy and the subset of patients who may have disease that has not spread beyond the regional lymph nodes may have the greatest benefit from this approach.[<xref rid="ref3" ref-type="bibr">3</xref>]</p><p>Cytoreductive surgery has been tried in renal cell carcinoma (along with targeted therapy with tyrosine kinase inhibitors), with reports of improved survival in a retrospective series. However, the benefit was minimal in poor risk disease.[<xref rid="ref4" ref-type="bibr">4</xref>] The question for renal cell carcinoma is likely to be answered with the multinational, prospective, randomized trial that is underway (CARMENA: NCT00930033).</p><p>For a similar trial to be attempted in carcinoma prostate, it will have to be powered to detect differences in outcome in the subset analysis as benefit, if any, is likely to be seen in a highly selected group. At the moment, based on current evidence, cytoreductive prostatectomy in metastatic disease remains investigational.</p> |
Painful varicoceles: Role of varicocelectomy | <p>The incidence of varicocele in the general population is up to 15%. It is estimated that the prevalence of pain with varicoceles is around 2-10%. Till the year 2000, only two studies evaluated efficacy of varicocelectomy in painful varicoceles with conflicting results. Over the past decade many other studies have addressed this issue and reported on the treatment outcome and predictors of success. We critically appraised studies published from March 2000 to May 2013 evaluating surgical management in painful varicoceles to provide an evidence based review of effectiveness of varicocelectomy in relieving pain in patients with symptomatic painful varicoceles. The association between varicoceles and pain is not clearly established. Conservative treatment is warranted as the first line of treatment in men with painful clinical varicoceles. In carefully selected men with clinically palpable varicoceles and associated characteristic chronic dull ache, dragging or throbbing pain who do not respond to conservative therapy, varicocelectomy is warranted and is associated with approximately 80% success. However, surgical success does not always translate into resolution of pain and pain might persist even when no varicoceles are detected postoperatively.</p> | <contrib contrib-type="author"><name><surname>Abrol</surname><given-names>Nitin</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Panda</surname><given-names>Arabind</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Kekre</surname><given-names>Nitin S.</given-names></name><xref ref-type="aff" rid="aff1"/></contrib> | Indian Journal of Urology : IJU : Journal of the Urological Society of India | <sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Varicoceles are detectable in approximately 15% of general adult male population.[<xref rid="ref1" ref-type="bibr">1</xref>] While pain was the earlier indication for varicocelectomy and its role in infertility was recognized later, there is more literature on the varicocelectomy for male infertility than for pain. The role of varicoceles in causation of testicular pain is less well-defined.</p><p>In his review of 4470 patients with varicoceles, Ivanissevich stated that varicoceles are rarely symptomatic.[<xref rid="ref2" ref-type="bibr">2</xref>] Tulloch described a case of 27-year-old male with azoospermia and bilateral varicoceles. Three months after varicocele treatment spermatozoa appeared in his semen and his wife conceived after 9 months.[<xref rid="ref3" ref-type="bibr">3</xref>] Following this report research mainly focused on varicoceles in subfertile men and new surgical techniques evolved over next few decades. Studies have shown that varicoceles are present in 2-10% men with orchialgia.[<xref rid="ref4" ref-type="bibr">4</xref>] Biggers <italic>et al</italic>. published the first study evaluating surgical treatment for painful varicoceles in 1981.[<xref rid="ref5" ref-type="bibr">5</xref>] Till year 2000 only two studies evaluated outcome of surgery in painful varicoceles.[<xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref6" ref-type="bibr">6</xref>] While Biggers <italic>et al</italic>. reported complete resolution of pain in 48%; Peterson <italic>et al</italic>. reported success in 86%.[<xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref5" ref-type="bibr">5</xref>] The purpose of this article is to review the role of surgery and predictors of success in treatment of painful varicoceles.</p><p>A Pubmed/Medline search was performed in June 2013 with key words “varicocele,” “pain,” and “varicocelectomy.” Of 93 search results, the majority of studies were done in subfertile men and data for pain resolution were culled from this population. For this review we focused on studies specifically evaluating pain resolution after varicocelectomy published after year 2000. Critical appraisal of 13 studies from March 2000 to May 2013 was performed and an algorithm based on quantitative assessment is presented for guiding management of painful varicoceles [<xref ref-type="fig" rid="F1">Figure 1</xref>].</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Algorithm for management of painful varicoceles</p></caption><graphic xlink:href="IJU-30-369-g001"/></fig></sec><sec id="sec1-2"><title>STUDY DESIGNS</title><p>There is no randomized controlled trial evaluating efficacy of varicocelectomy for painful varicoceles. Most studies are retrospective case series and include small number of cases [<xref ref-type="table" rid="T1">Table 1</xref>]. No case control study is published till date.</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Summary of various studies on treatment of painful varicoceles</p></caption><graphic xlink:href="IJU-30-369-g002"/></table-wrap></sec><sec id="sec1-3"><title>CONSERVATIVE TREATMENT</title><p>In all studies, conservative treatment was tried before surgery for a variable duration of time. Conservative treatment included scrotal support, oral non-steroidal anti-inflammatory medicine, and limitation of physical activity like lifting weights and strenuous activity. While no study has reported on efficacy and acceptance of conservative treatment; in a study by Yaman <italic>et al</italic>. five of the 119 patients had their pain resolved after conservative treatment.[<xref rid="ref6" ref-type="bibr">6</xref>] In another study 15/99 (15.2%) men experienced resolution of pain after 4 weeks of conservative treatment.[<xref rid="ref7" ref-type="bibr">7</xref>] Limitation of physical activity may not be acceptable in military recruits, athletes, and policemen.</p></sec><sec id="sec1-4"><title>SURGICAL APPROACH</title><p>No study has compared the efficacy of different techniques of varicocelectomy for pain resolution. There is a trend toward microsurgical varicocelectomy. Maghraby reported 84.5% complete response rate and 10.3% partial response with only 3/58 patients having persistent symptoms after laparoscopic varicocelectomy. One patient developed hydrocele and two (3.4%) had persistent varicoceles.[<xref rid="ref8" ref-type="bibr">8</xref>] Contemporary series of microsurgical inguinal/subinguinal approach have shown similar results.[<xref rid="ref9" ref-type="bibr">9</xref>] Kim <italic>et al</italic>. operated 114 patients of painful varicoceles with microsurgical inguinal approach. Overall response was 91.2% while 8.8% had pain postoperatively.[<xref rid="ref10" ref-type="bibr">10</xref>] In another study, 72.4% responded while 27.6 did not respond to microsurgical subinguinal varicocelectomy.[<xref rid="ref7" ref-type="bibr">7</xref>] Park <italic>et al</italic>. used microsurgical inguinal or subinguinal approach and reported complete, partial, and no response in 52.8, 41.5, and 5.7%, respectively.[<xref rid="ref11" ref-type="bibr">11</xref>] Overall response with non-microscopic open technique has also been similar with slightly more partial response than complete.[<xref rid="ref12" ref-type="bibr">12</xref><xref rid="ref13" ref-type="bibr">13</xref><xref rid="ref14" ref-type="bibr">14</xref>]</p></sec><sec id="sec1-5"><title>PREDICTORS OF SUCCESS</title><p>Most patients with chronic orchialgia do not have varicoceles and majority of patients with varicoceles are asymptomatic. The presence of pain in men with varicocele may be related to some other pathology. Careful evaluation is recommended to rule out epididymitis, hydrocele, inguinal hernia, testicular tumor, and epididymal cyst etc.[<xref rid="ref9" ref-type="bibr">9</xref>] In the absence of any clinical finding, some men may have idiopathic orchialgia. Due to absence of a clear cause - effect relationship between varicoceles and orchialgia, many predictors of successful outcome have been studied.</p><sec id="sec2-1"><title/><sec id="sec3-1"><title>Grade of varicocele</title><p>All studies accept the varicocele grading system defined by Lyon <italic>et al</italic>.[<xref rid="ref15" ref-type="bibr">15</xref>] The American Urologic Association and European Association of Urology guidelines recommend that only clinically palpable varicoceles (Grade I, II, and III) should be considered for treatment. All studies have excluded men with subclinical varicoceles.</p><p>Yaman <italic>et al</italic>. reported outcome in 82 patients with at least 3 months follow up after varicocelectomy. 88% (72/82) had complete response, 11% did not respond (9/82), and 1% (1/82) had epididymal discomfort which resolved after conservative management. Among non responders varicocele was grade III in 6.1% (5/82), grade II in 3.6% (3/82), and grade I in 1.2% (1/82).[<xref rid="ref6" ref-type="bibr">6</xref>] However, they included four patients with partial response in their analysis and did not comment on statistical significance of their results. Others did not find significant difference in varicocele grade between responders and non-responders.[<xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref10" ref-type="bibr">10</xref><xref rid="ref11" ref-type="bibr">11</xref><xref rid="ref16" ref-type="bibr">16</xref>] In study by Chen <italic>et al</italic>. percentage of responders vs. non-responders in grade I, II, and III was 21.8% vs. 23.8%, 50.9% vs. 47.6%, and 27.3% vs. 28.6%, respectively.[<xref rid="ref7" ref-type="bibr">7</xref>]</p></sec><sec id="sec3-2"><title>Character of pain</title><p>Pain due to varicoceles is characteristically dull, aching, or dragging in nature. Pain is worsened by physical activity and on prolonged standing. Peterson <italic>et al</italic>. described that for successful outcome of surgery, pain must be dull aching and throbbing without any sharp component or radiation.[<xref rid="ref4" ref-type="bibr">4</xref>] Kim <italic>et al</italic>. on multivariate analysis found that quality of pain is an independent predictor of success. They reported 100% success when pain was dull in nature. Of 114 treated patients ten (8.8%) did not respond to treatment. Of ten non-responders eight had aching pain while two had dragging pain. None of the patient had sharp component in their series.[<xref rid="ref10" ref-type="bibr">10</xref>] Others did not find significant relation between pain character and response to surgery.[<xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref11" ref-type="bibr">11</xref><xref rid="ref16" ref-type="bibr">16</xref>] No patient with sharp pain failed treatment in study by Park <italic>et al</italic>.[<xref rid="ref11" ref-type="bibr">11</xref>]</p><p>Character of pain is essentially a subjective parameter. Different patients may describe their pain differently. This might be the reason for variable results in literature with respect to relation between pain character and treatment outcome.</p></sec><sec id="sec3-3"><title>Duration of pain</title><p>Studies have used varying cut-off criteria for pain duration.[<xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref10" ref-type="bibr">10</xref><xref rid="ref11" ref-type="bibr">11</xref><xref rid="ref16" ref-type="bibr">16</xref>] In one study, mean duration of pain in whole group and in non-responders was 13.7 months and 4.2 months, respectively.[<xref rid="ref10" ref-type="bibr">10</xref>] However, on multivariate analysis this difference was not significant. This study did not use cut off limit to divide groups according to duration of pain. Other studies using cut off of 3, 6, and 9 months found significant difference in response rate. Patients with longer duration of pain before surgery had better outcome.[<xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref11" ref-type="bibr">11</xref><xref rid="ref16" ref-type="bibr">16</xref>] Success rate was 98.6% in group who had pain for more than 3 months while 17.7% with duration less than 3 months failed treatment.[<xref rid="ref16" ref-type="bibr">16</xref>]</p><p>This highlights the importance of carefully selecting patients for surgery. There are many other causes for orchialgia. Given time, pain due to other reasons might resolve. However, if surgery is considered early these cases are likely to fail.</p></sec><sec id="sec3-4"><title>Intensity of preoperative pain</title><p>Severity of pain has been evaluated in few studies. Altunoluk <italic>et al</italic>. did not find any difference in intensity of pain in responders and non-responders.[<xref rid="ref16" ref-type="bibr">16</xref>] The same study did not find any significance of quality of pain. It is important to note that authors did not quantify pain. Kim <italic>et al</italic>. used 11-point numeric rating scale (NRS) for pain quantification.[<xref rid="ref10" ref-type="bibr">10</xref>] Degree of pain resolution was related in multivariate analysis to low pain score (<7). Non-responders had higher preoperative NRS scale (mean 9; range 7-10). In contrast, study using 10 cm visual analogue scale (VAS) showed that responders had greater preoperative pain score (>6).[<xref rid="ref7" ref-type="bibr">7</xref>] There was significant overlap in VAS pain score between two groups (Mean; 95% CI Responders: Non-responders = 6; 2.8-9.3:4; 1.4-6.6).[<xref rid="ref7" ref-type="bibr">7</xref>]</p><p>VAS and NRS provide almost identical results and are equally sensitive in assessing intensity of acute pain after surgery.[<xref rid="ref17" ref-type="bibr">17</xref>] NRS with numbers from 0 to 10 is more easily understood by patients than VAS.[<xref rid="ref10" ref-type="bibr">10</xref><xref rid="ref17" ref-type="bibr">17</xref>] These are the best tools for assessment of patients’ subjective feeling of pain intensity. Pain memory is often not accurate and this limits the use of these scales for assessment of chronic pain. This, combined with different follow up duration, makes any conclusion difficult. Quantifying pain using NRS at initial evaluation and during follow up visits; while patient is given trial of conservative treatment; may be more representative than assessment made once at the time of first visit.</p></sec><sec id="sec3-5"><title>Recurrence of varicocele</title><p>In general, persistence of pain after surgery is poorly correlated with persistence of varicoceles. In one study, varicocele recurrence was detected in 1/19 non-responders; 17 of them had pain duration of less than 3 months.[<xref rid="ref17" ref-type="bibr">17</xref>] In another study none of patients with recurrent pain had varicocele when tested with Doppler ultrasound.[<xref rid="ref11" ref-type="bibr">11</xref>] Pain in these patients probably was not due to varicocele. This highlights the importance of careful patient selection for surgery.</p></sec><sec id="sec3-6"><title>External spermatic vein ligation</title><p>Chawla <italic>et al</italic>. selected 11 men with failed varicocelectomy who had persistent pain after surgery.[<xref rid="ref18" ref-type="bibr">18</xref>] All had non-microsurgical repair previously. Authors performed microsurgical subinguinal varicocelectomy and ligated gubernacular as well as ESV. Ten men experienced significant improvement in pain. In other study 92.4% who underwent ESV ligation experienced complete response, while only one patient had complete response when ESV was not ligated.[<xref rid="ref13" ref-type="bibr">13</xref>]</p></sec><sec id="sec3-7"><title>Body mass index</title><p>Association between BMI and pain response was first reported by Park <italic>et al</italic>.[<xref rid="ref11" ref-type="bibr">11</xref>] All failures were in patients with BMI > 22 Kg/m<sup>2</sup>. In other study BMI in responders and non-responders was 21.3 and 22.1 Kg/m<sup>2</sup>, respectively though difference was not statistically significant.[<xref rid="ref7" ref-type="bibr">7</xref>] In multivariate analysis BMI was not significant predictor of response.[<xref rid="ref10" ref-type="bibr">10</xref>]</p></sec><sec id="sec3-8"><title>Other predictors</title><p>Sperm quality, semen pH, testicular volume, semen volume, FSH, LH, testosterone, maximum vein diameter, peak retrograde flow on Doppler, distance from renal hilum to center of scrotum, and scrotal temperature have been evaluated as predictors of success but none has reached statistic significance.[<xref rid="ref7" ref-type="bibr">7</xref>]</p></sec></sec></sec><sec id="sec1-6"><title>SYSTEMATIC APPROACH TO MANAGEMENT</title><p>Based on our review, we suggest a painful varicocele score (PVS) factoring in duration of pain, character, and intensity of pain [<xref ref-type="fig" rid="F1">Figure 1</xref>]. This assessment should be done at initial visit and repeated at 3 and 6 months while patient is on conservative management. Decision to operate varicocele can be taken if PVS is 6 or more.</p></sec><sec sec-type="conclusion" id="sec1-7"><title>CONCLUSION</title><p>The presence of varicocele in men with orchialgia is a diagnostic and treatment challenge. While subclinical varicoceles are insignificant, grade of palpable varicocele has poor correlation with symptoms and treatment outcome. Careful history and examination are mandatory to rule out other causes of pain. Each evaluation of painful varicocele must include a checklist of characteristics (Duration, Character, and Intensity of pain) with metrics or grading system. Conservative treatment should always be the first line of treatment.</p><p>Varicocelectomy in carefully selected men with clinically palpable varicoceles is associated with approximately 80% success. Surgical success does not always translate into resolution of pain. Pain might persist even when no varicoceles are detected postoperatively. Failures may be due to a wrong diagnosis. Although the literature suggests that microsurgical varicocelectomy has fewer complications and recurrences, the literature on surgical treatment of painful varicoceles is limited and it is not possible to make a surgical recommendation. Continued research and randomized studies with longer follow up are needed to solve the question of which patient will benefit from surgery for painful varicoceles. Till further studies are available PVS can be used to evaluate the level of pain for each patient and to guide management.</p></sec> |
Continence outcomes following robotic radical prostatectomy: Our experience from 150 consecutive patients | <sec id="st1"><title>Introduction:</title><p>Urinary continence is an important outcome parameter after robot assisted radical prostatectomy (RARP). We evaluated the continence outcomes following RARP using a double-layered urethrovesical reconstruction.</p></sec><sec id="st2"><title>Materials and Methods:</title><p>One hundred fifty consecutive patients undergoing RARP and double-layered urethrovesical reconstruction were prospectively studied for preoperative, intraoperative and post operative parameters. Key points followed during surgery were: Minimal dissection of sphincteric complex, preservation of puboprostatic ligament, selective ligation of deep venous complex and both posterior and anterior reconstruction using the Von Velthoven stitch. Intraoperative bladder fill test was done at the end of anastomosis to rule out urine leak. Check cystogram was done prior to catheter removal in the outpatient department. Patients were subsequently followed at regular intervals regarding the status of urinary continence. All patients irrespective of adjuvant therapy were included in the analysis.</p></sec><sec id="st3"><title>Results:</title><p>The mean age was 64 years (standard deviation ± 6.88), and mean serum PSA was 20.2 ng/ml. The mean BMI was 25.6 (SD: ±3.84). The mean prostate weight was 44.09 gm (range 18-103 gm, SD: ±15.59). Median days to catheter removal after surgery was 7 (range 4-14 days) days. Cystographically determined urinary leaks were seen in two patients. Urine leak was managed by delaying catheter removal for 1 week. Minimum 6 month follow up was available in 126 patients. ‘No pad’ status at 1 week, 1 month, 3 months, 6 months and 1 year was 15.1%, 54.9%, 78%, 90.5% and 94.1%, respectively.</p></sec><sec id="st4"><title>Conclusion:</title><p>Excellent continence outcomes are observed in patients undergoing double-layered urethrovesical reconstruction.</p></sec> | <contrib contrib-type="author"><name><surname>Gupta</surname><given-names>Narmada P.</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Yadav</surname><given-names>Rajiv</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Akpo</surname><given-names>Emmanuel E.</given-names></name><xref ref-type="aff" rid="aff1"/></contrib> | Indian Journal of Urology : IJU : Journal of the Urological Society of India | <sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Prostate cancer is the most common cancer diagnosed in men and the second most common cause of death from cancer in industrialized countries. In India, the incidence of prostate cancer is second among all the cancers in men and a rising mean annual percentage change of 0.14-8.6.[<xref rid="ref1" ref-type="bibr">1</xref>] More Indian men with prostate cancer are being diagnosed with a localized prostate cancer as a result of awareness, screening and improved diagnostic armamentaria. Robot-assisted radical prostatectomy (RARP) has become the most preferred surgical modality for the treatment of localized prostate cancer.[<xref rid="ref2" ref-type="bibr">2</xref>] However, postoperative urinary incontinence remains one of the most bothersome complications with reported incidence of 7-40%.[<xref rid="ref3" ref-type="bibr">3</xref>] Significant improvement in continence results following RARP has been reported in recent series. Several technical modifications proposed and put into practice over the years have contributed to improved overall continence rates as well as faster return to continence. The underlying basic concept of all these techniques is to maintain normal anatomical and functional structures in the pelvis or to restore the anatomy to the best possible extent. Some of the surgical modifications to improve early continence outcomes described in the literature include (but not limited to): Sparing of the puboprostatic ligaments,[<xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref6" ref-type="bibr">6</xref>] bladder neck preservation,[<xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref8" ref-type="bibr">8</xref><xref rid="ref9" ref-type="bibr">9</xref>] posterior reconstruction of Denonvilliers’ musculofascial plate,[<xref rid="ref10" ref-type="bibr">10</xref><xref rid="ref11" ref-type="bibr">11</xref><xref rid="ref12" ref-type="bibr">12</xref><xref rid="ref13" ref-type="bibr">13</xref>] restoration of the puboprostatic collar,[<xref rid="ref14" ref-type="bibr">14</xref>] and complete reconstruction of the periurethral supportive tissues.[<xref rid="ref15" ref-type="bibr">15</xref><xref rid="ref16" ref-type="bibr">16</xref>] However, the technique of RARP is a evolutionary continuum and improved continence result is the additive effect of all the individual steps. We performed double-layered urethrovesical anastomosis using two, monofilament Van Velthoven stitches in 150 consecutive patients. The aim of this study is to evaluate continence outcomes in our patients.</p></sec><sec sec-type="materials|methods" id="sec1-2"><title>MATERIALS AND METHODS</title><p>Between April 2010 and August 2013, clinical and pathologic data were prospectively recorded of all cases who underwent RARP and total anatomical reconstruction. All the patients underwent RARP through transperitoneal approach using 4 arm da Vinci surgical system. Key points followed during prostatic dissection were: Minimal dissection of sphincteric complex with preservation of puboprostatic ligament, nerve sparing surgery whenever indicated, selective ligation of deep venous complex, preservation of maximal urethral length. Reconstruction consisted of double-layered anastomosis done by reapproximation of Denonvillier's fascia and posterior rhabdosphincter, as well as reapproximation of the puboprostatic ligaments to the anterior pubovesical collar.[<xref rid="ref15" ref-type="bibr">15</xref>] Two, 3-0 double-armed monofilament sutures were used. The first suture was started at 5’0 clock position for posterior reconstruction and creation of posterior plate by approximating posterior rhabdosphincter (on urethral side) to posterior layer of Denonvilliers fascia (on bladder side). Using the second suture, the urethrovesical anastomosis (inner layer) was then completed all around by performing mucosa to mucosa approximation. Finally, the outer layer was completed by approximating the puboprostatic ligament to the anterior pubovesical collar. Bladder fill test with 120 ml saline was done after completion of anastomosis. Any significant leak noted during bladder fill test was repaired by interrupted sutures. Indwelling Foley catheter was kept in all the cases. Check cystogram was done prior to catheter removal in the outpatient department on Day 7 of surgery. Catheter removal was done if no leak was noted on cystogram. Patients were instructed to perform ‘Kegel exercises’ starting 3 days after catheter removal.</p><p>Follow up: Demographic, operative and follow-up data were retrieved from the electronic database in the hospital's electronic medical records. Patients were followed at regular intervals (at 1 week, 4 weeks, 3 months, 6 months and 12 months post catheter removal) in the outpatient clinic or contacted on phone/email regarding the status of urinary continence and number of pad used. Continence was defined as using ‘no pad’ or just a security liner. All patients irrespective of adjuvant therapy were included in the analysis.</p><p>Data collection and follow-up correspondence were conducted in accordance with the hospital's ethical guidelines.</p></sec><sec sec-type="results" id="sec1-3"><title>RESULTS</title><p>From April 2010 to August 2013, 153 patients underwent RALP by the two surgeons in a single institution. Of these patients three international patients were excluded from analysis due to non-availability of follow up data. Clinical and pathologic characteristics of the patients are shown in <xref ref-type="table" rid="T1">Table 1</xref>. The mean age was 64 years (standard deviation ± 6.88), and the mean serum PSA was 20.2 ng/ml. The mean BMI was 25.6 (SD: ±3.84), and the mean prostate weight (on final specimen) was 44.09 gm (range 18-103 gm, SD: ±15.59). Median postoperative hospital stay was 3 days. Median days to catheter removal after surgery were 7 (range 4-14 days) days. Cystographically determined urinary leaks were seen in two patients. Urine leak was managed by delaying the catheter removal for another 1 week. At least 6 month follow up was available in 126 patients. ‘No pad’ status at 0 week, 1 month, 3 months, 6 months and 1 year was 15.1%, 54.9%, 78%, 90.5% and 94.1%, respectively [<xref ref-type="table" rid="T2">Table 2</xref> and <xref ref-type="fig" rid="F1">Figure 1</xref>]. No significant correlation was found on multivariate logistic regression analysis in rate of continence and prostate size, nerve sparing status or age of patient. However, trend toward early continence was noted in younger patients.</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Clinical and pathologic characteristics of the study population</p></caption><graphic xlink:href="IJU-30-374-g001"/></table-wrap><table-wrap id="T2" position="float"><label>Table 2</label><caption><p>Status of urinary continence on follow up</p></caption><graphic xlink:href="IJU-30-374-g002"/></table-wrap><fig id="F1" position="float"><label>Figure 1</label><caption><p>Graph showing percentage of patients achieving continence during follow up</p></caption><graphic xlink:href="IJU-30-374-g003"/></fig></sec><sec sec-type="discussion" id="sec1-4"><title>DISCUSSION</title><p>The focus of treatment for localized prostate cancer is not only better cancer control but also to maintain the quality of life. Postoperative urinary incontinence (UI) following radical prostatectomy (RP) remains a bothersome issue. Large contemporary series have reported continence rates ranging from 84% to 97% at 1 year.[<xref rid="ref17" ref-type="bibr">17</xref>] Thus, even though most of the patients regain their continence at 1 year, patients do suffer the psychological trauma of UI for varying period in the post operative phase. Over the years, several technical modifications and collective experience of open and conventional laparoscopic RP have contributed to the improvement seen in early and overall continence rates. Both posterior[<xref rid="ref10" ref-type="bibr">10</xref>] and anterior reconstructions[<xref rid="ref14" ref-type="bibr">14</xref>] have been independently shown to be helpful in improving early continence. In our patient we have taken advantage of both the approaches and noted that approximately 55% of our patients are fully continent just after 1 month of surgery. By 6 months 90% of our cohort regained continence. The results are comparable to other large series who have taken the similar reconstructive approach.[<xref rid="ref17" ref-type="bibr">17</xref><xref rid="ref18" ref-type="bibr">18</xref><xref rid="ref19" ref-type="bibr">19</xref><xref rid="ref20" ref-type="bibr">20</xref>]</p><p>Technical modifications allowing rapid return of urinary continence after surgery relies on maintaining anatomical and functional structures in the pelvis to the greatest possible extent.[<xref rid="ref21" ref-type="bibr">21</xref>] The major components of the pelvic supporting system in males are Denonvilliers’ fascia, puboprostatic ligament, endopelvic fascia, levator ani muscle and arcus tendineus fascia pelvis. Thus, not only preservation, but also reconstruction of these structures play a potential role in improving the recovery of urinary incontinence.[<xref rid="ref21" ref-type="bibr">21</xref>] Based on this concept, three steps have been advocated intra-operatively to preserve post-prostatectomy continence: (1) Preservation of bladder neck, nerves, puboprostatic ligament, pubovesical complex and urethral length; (2) reconstruction of the rhabdosphincter posteriorly, anterior retropubic suspension, reattachment of the arcus tendineus to the bladder neck and total reconstruction of the vesicourethral junction and (3) reinforcement of the bladder neck by plication and bladder neck sling suspension.[<xref rid="ref21" ref-type="bibr">21</xref>]</p><p>In our patients we made no special attempt to preserve the bladder neck as the majority of cases were high risk with large volume of tumor. However, we did attempt to preserve the puboprostatic ligament and preserve maximal urethral length. During reconstruction, posterior reconstruction helps in restoring the anatomic and functional length of the rhabdosphincter and by providing posterior support to the urethra.[<xref rid="ref10" ref-type="bibr">10</xref>] Although the delayed continence rate achieved at 1 year may be the same in patients with or without posterior reconstruction, several authors have reported achievement of faster continence (early continence) with this method.[<xref rid="ref10" ref-type="bibr">10</xref><xref rid="ref11" ref-type="bibr">11</xref><xref rid="ref12" ref-type="bibr">12</xref><xref rid="ref15" ref-type="bibr">15</xref>] We observed that more than 90% of our patients achieve continence by 6 months. Another potential advantage is that along with anterior reconstruction, it also takes away the tension from the actual urethrovesical anastomosis and makes it easier to perform (especially in difficult cases). Sammon <italic>et al</italic>.[<xref rid="ref20" ref-type="bibr">20</xref>] have also reported significantly lower rates of cystographically determined anastomotic leak rate with double-layer anastomosis (none versus 10% when compared to single layered anastomosis). We have also noted a very low leak rate (<1%) in our patients.</p><p>We realize that our study has certain shortcomings, chiefly being the non-objective way of defining continence leak based upon usage of number of pads and not the urine leakage weight. However, the effect on results is minimal as our definition of continence was use of either ‘no pad’ or just a ‘security liner’. No health-related quality of life score assessment was done during the study which could have added another parameter to the analysis. Also, all our patients underwent double-layered urethrovesical anastomosis therefore it is not possible for us to gauge the difference in outcomes between single-layered versus double-layered anastomosis (if at all).</p><p>In conclusion, double-layered urethrovesical anastomosis combines the benefits of both posterior and anterior reconstructions and provides excellent continence outcomes.</p></sec> |
Changes in pathologic outcomes and operative trends with robot-assisted laparoscopic radical prostatectomy | <sec id="st1"><title>Introduction:</title><p>We hypothesized that there is a reverse stage migration, or a shift toward operating on higher-risk prostate cancer, in patients undergoing robot-assisted laparoscopic prostatectomy (RALP). We therefore evaluated the stage of disease at the time of surgery for patients with prostate cancer at a large tertiary academic medical center.</p></sec><sec id="st2"><title>Materials and Methods:</title><p>After institutional review board approval, we reviewed all patients that had undergone robotic prostatectomy. These patients were separated into three categories: An early era of 2005-2008, intermediate era of 2009-2010, and a current era of 2011-2012.</p></sec><sec id="st3"><title>Results:</title><p>A total of 3451 patients underwent robotic prostatectomy from 2005 to 2012. The proportion men with clinical T1 tumors declined from 88.3% in the early era to 72.2% in the current era (<italic>P</italic> < 0.0001). Men with preoperative biopsy Gleason 6 disease decreased from the early to the current era (<italic>P</italic> < 0.0001), while men with preoperative biopsy Gleason ≥ 8 showed the opposite trend, increasing from the early to the current era (<italic>P</italic> = 0.0002). From the early to the current era, the proportion of patients with National Comprehensive Cancer Network (NCCN) low risk prostate cancer decreased, while those with NCCN intermediate and high-risk disease increased. The proportion of pathologic T3 disease increased from 15.5% in the early to 30.6% in the current era (<italic>P</italic> < 0.0001). On the other hand, the proportion of pathologic T2/+ SMS (surgical margin status) decreased from 6.6% in the early era to 3.1% in the current era (<italic>P</italic> = 0.0002).</p></sec><sec id="st4"><title>Conclusions:</title><p>We have demonstrated a reverse stage migration in men undergoing robotic prostatectomy. Despite the increasing proportion of men with extra-capsular disease undergoing RALP, the surgical margin status has remained similar. This could reflect both the changing dynamics of the population opting for surgery as well as the learning curve of the surgeons.</p></sec> | <contrib contrib-type="author"><name><surname>Bernie</surname><given-names>Aaron</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Ramasamy</surname><given-names>Ranjith</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Ali</surname><given-names>Adnan</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Tewari</surname><given-names>Ashutosh K.</given-names></name><xref ref-type="aff" rid="aff1"/></contrib> | Indian Journal of Urology : IJU : Journal of the Urological Society of India | <sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Although Prostate-specific antigen (PSA) testing for prostate cancer screening has lowered death rates due to prostate cancer,[<xref rid="ref1" ref-type="bibr">1</xref>] there is growing concern that clinically insignificant prostate cancer would be detected in many men in the population, and also at an earlier age, leading to therapies that otherwise would have not changed the course of the disease.[<xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref3" ref-type="bibr">3</xref>] Studies have demonstrated a general trend of downward stage and grade migration after institution of PSA testing.[<xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref6" ref-type="bibr">6</xref>] The overtreatment of low-grade disease in prostate cancer in the population is largely due to stage migration,[<xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref8" ref-type="bibr">8</xref>] and approximately 80% of American men had organ-confined disease after radical prostatectomy in 2001.[<xref rid="ref9" ref-type="bibr">9</xref>]</p><p>To sum up, PSA screening has played a major role in the over-diagnosis and over-treatment of clinically insignificant prostate cancer.[<xref rid="ref10" ref-type="bibr">10</xref><xref rid="ref11" ref-type="bibr">11</xref>] The objective of our analysis was to evaluate our series for a shift in the operative volume on low and high-risk prostate cancer as well as the pathologic changes seen over time in patients undergoing robot-assisted laparoscopic prostatectomy (RALP).</p></sec><sec sec-type="materials|methods" id="sec1-2"><title>MATERIALS AND METHODS</title><p>We performed an institutional review board-approved, retrospective review of 3451 consecutive patients who underwent robot-assisted prostatectomy by a single surgeon from 2005 to 2012 for localized prostate cancer. Patients who had received preoperative radiation therapy or androgen-deprivation therapy were excluded from the analysis. Patient data were collected and entered into a prospective prostate cancer database. The seventh edition of the American Joint Committee on Cancer tumor-lymph node (LN)-metastasis classification was used to define clinical stage, and histopathologic grading was done according to the Gleason system. Biopsies performed at referring facilities were reviewed by dedicated genitourinary pathologists at our institution. All patients underwent robot-assisted radical prostatectomy and pelvic LN dissection in this series.</p><p>Patients were stratified according to year of operation based on surgeon experience, 2005-2008, 2009-2010, 2011-2012 (early, intermediate, and current groups for robot-assisted prostatectomy). These categories were determined on volume and surgeon learning curve. Patients also were stratified according to National Comprehensive Cancer Network (NCCN) guidelines into the following risk categories: Low risk (PSA ≤10 ng/mL, ≤T2a, and Gleason score ≤6), intermediate risk (PSA 10-20 ng/mL, or T2b-T2c, or Gleason score 7), or high risk (PSA >20 ng/mL, or ≥T3a, or Gleason score ≥8).[<xref rid="ref12" ref-type="bibr">12</xref>]</p><p>Postoperative pathologic reports were identified for all patients, and high-risk characteristics of extracapsular extension (ECE), positive nodal status (N1), and positive surgical margins (PSM) (+SMS) were determined and sorted by the above year stratification system.</p><p>Microsoft Excel 2013 (Microsoft Corporation, Seattle WA) and GraphPad Prism 5 (Graph-Pad Software Inc., La Jolla, CA) software were used to perform all statistical calculations with <italic>P</italic> < 0.05 considered as statistically significant. Two analyses were used to compare factors between the different eras.</p></sec><sec sec-type="results" id="sec1-3"><title>RESULTS</title><p>RALP for prostate cancer was performed on a total of 3451 patients all of whom met the inclusion criteria and had preoperative characteristics available from 2005 to 2012. Baseline characteristics are listed in <xref ref-type="table" rid="T1">Table 1</xref>.</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Baseline characteristics of the sample categorized by era</p></caption><graphic xlink:href="IJU-30-378-g001"/></table-wrap><sec id="sec2-1"><title/><sec id="sec3-1"><title>Preoperative markers</title><p>PSA levels did not demonstrate any significant changes over time in either the total cohort or the individual era in which RALP was performed. The proportion of clinical T1 tumors in the operative cohorts declined from the early to current eras (88.3% of patients were clinical T1 in the early group, 72.2% were T1 in the current group; <italic>P</italic> < 0.0001).</p><p>Similarly, the percentage of patients in each era that represented Gleason 6 disease at biopsy was statistically decreased from the early to the current group (63.5% of cases in the early group, 38.7% of cases in the current group; <italic>P</italic> < 0.0001). Preoperative biopsy Gleason 7 disease showed the opposite trend to that of Gleason 6 disease, and the proportion of Gleason 7 disease was also statistically significantly higher from the early (30.1%) to the current group (50.5%) (<italic>P</italic> < 0.0001). Preoperative biopsy Gleason 8 disease showed a similar trend to that of Gleason 7, and there was a statistically significant difference between the early (6.4%) and current group (10.8%) (<italic>P</italic> = 0.0002).</p></sec><sec id="sec3-2"><title>Stratification by National Comprehensive Cancer Network Category</title><p>Figures <xref ref-type="fig" rid="F1">1</xref> and <xref ref-type="fig" rid="F2">2</xref> demonstrate the trends seen from the early, intermediate and current groups with respect to NCCN classification. A downward trend is seen in the proportion of NCCN low risk patients undergoing RALP from the early (55.9%) to current groups (32.0%) (<italic>P</italic> < 0.0001) [<xref ref-type="fig" rid="F1">Figure 1</xref>]. An upward trend is seen in both the NCCN intermediate risk patients from the early (36.6%) to high-risk patients (55.1%) (<italic>P</italic> < 0.0001) and high-risk patients from early (7.5%) to current groups (12.9%) (<italic>P</italic> < 0.0001) [<xref ref-type="fig" rid="F2">Figure 2</xref>].</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Percentage of sample categorized as National Comprehensive Cancer Network low risk categorized by era</p></caption><graphic xlink:href="IJU-30-378-g002"/></fig><fig id="F2" position="float"><label>Figure 2</label><caption><p>Percentage of sample categorized as National Comprehensive Cancer Network high-risk categorized by era</p></caption><graphic xlink:href="IJU-30-378-g003"/></fig></sec><sec id="sec3-3"><title>Pathologic markers</title><p>Pathologic characteristics from men in this cohort are shown in Tables <xref ref-type="table" rid="T2">2</xref> and <xref ref-type="table" rid="T3">3</xref>. Pathologic T3 disease increased from the early to current groups (from 15.5% in 2005-2008 to 30.6% in 2011-2012; <italic>P</italic> < 0.0001) [<xref ref-type="fig" rid="F3">Figure 3</xref>]. Pathologic surgical margin status remained similar from the early to current groups (from 10.6% in 2005-2008 to 8.8% in 2011-2012; <italic>P</italic> = 176). On the other hand, the proportion of T2/+SMS findings on pathologic specimens decreased from the early to current groups (from 6.6% in 2005-2008-3.1% in 2011-2012; <italic>P</italic> = 0.0002) [<xref ref-type="fig" rid="F4">Figure 4</xref>].</p><table-wrap id="T2" position="float"><label>Table 2</label><caption><p>Pathologic characteristics of the sample categorized by era</p></caption><graphic xlink:href="IJU-30-378-g004"/></table-wrap><table-wrap id="T3" position="float"><label>Table 3</label><caption><p>Breakdown of pathologic Gleason scores by era</p></caption><graphic xlink:href="IJU-30-378-g005"/></table-wrap><fig id="F3" position="float"><label>Figure 3</label><caption><p>Percentage of sample with pathologic T3 disease categorized by era</p></caption><graphic xlink:href="IJU-30-378-g006"/></fig><fig id="F4" position="float"><label>Figure 4</label><caption><p>Percentage of sample with pathologic T2/positive surgical margin disease categorized by era</p></caption><graphic xlink:href="IJU-30-378-g007"/></fig></sec></sec></sec><sec sec-type="discussion" id="sec1-4"><title>DISCUSSION</title><p>In this study, we examined the preoperative baseline characteristics and stages of risk as well as the postoperative pathologic rates of ECE as well as LN and SMS positive rates in RALP patients over an 8-year period in men with localized prostate cancer. From the early to current groups of RALP, we found an increase in the proportion of preoperative high-risk patients undergoing surgery, as well as an increase in the high-risk pathologic characteristics. These trends suggest a shift with operative emphasis on higher risk disease as well as the learning skills gained as RALP became an established practice. The increasing trend toward operating on higher risk patients could also be explained by increasing use of active surveillance as a treatment option for low risk prostate cancer. Active surveillance could lead to patients being upstaged as a result of repeat biopsies performed.</p><p>The advent of PSA-based screening has led to a significant shift in the presentation and treatment of prostate cancer. PSA screening has led to patients presenting with prostate cancer at a significantly earlier age[<xref rid="ref13" ref-type="bibr">13</xref>] and with lower-risk disease.[<xref rid="ref14" ref-type="bibr">14</xref>] In line with this, the pathological makeup of prostate cancer specimens from early RALP showed a trend of lower-stage disease. This is largely due to the high increase in surgical intervention for lower-risk prostate cancers with the intent to cure all prostate cancer disease,[<xref rid="ref15" ref-type="bibr">15</xref>] despite the fact that many of these patients with low risk cancer are unlikely to benefit from surgical intervention.</p><p>It has been previously demonstrated that ECE serves as an excellent marker for the likelihood of tumor progression because of its lack of variation with surgeon experience or skill,[<xref rid="ref16" ref-type="bibr">16</xref>] and that rates of ECE significantly declined after the advent of PSA testing, in line with the increase in operative pathology suggesting more low risk disease after radical prostatectomy.[<xref rid="ref17" ref-type="bibr">17</xref><xref rid="ref18" ref-type="bibr">18</xref>] The fact that ECE declined so dramatically in these studies between the pre and current PSA testing era widely suggested that there was in fact a stage migration toward operating on more low-risk and potentially clinically insignificant prostate cancer.</p><p>Using this same concept, we were able to demonstrate an increase in the rate of high-risk preoperative and pathologic factors from groups of early to current RALP. The use of AS, alternative therapies for low risk prostate cancer and the learning curve with skills gained after the initial experience of RALP would explain a shift, or reverse stage migration, from the surgical treatment of low risk and potentially clinically insignificant prostate cancer to the treatment of only high-risk prostate disease. This shift suggests that PSA and prostate cancer screening are used diligently at our center, with operative intervention only on those cancers that impose a potentially significant health risk to the patient.</p><p>Furthermore, we used two more pathologic markers as well as high-risk preoperative factors to demonstrate our point. The rate of N1 disease, another clinical marker for potential tumor progression,[<xref rid="ref16" ref-type="bibr">16</xref>] demonstrated similar trending to that of ECE, rising at a statistically significant rate from the early to current RALP era. The rate of PSM status initially rose from the early to intermediate era and then again fell in the current era, which would be expected as surgeons performing the procedure have become more skilled with RALP and are achieving similar SMS rates to that seen with initial RALP despite the fact that they are now operating on a higher grade disease. Similarly, there was a trend seen with NCCN guideline criteria of operating on higher risk disease from the early to the current era.</p><p>We are limited in our analysis by several variables. As this study was performed at a tertiary care center and many of these patients return to their local urologists for follow-up, the biochemical recurrence rate of these patients cannot be assessed. Furthermore, several genitourinary pathologists were involved in interpretation of the specimens and staging and could have potentially contributed to the variation identified. Similarly, this could have led to a skewing of the groups of patients presenting, in that those with low risk disease are much less likely to come for consultation at our institution as they have already been educated about low risk disease in a community setting.</p></sec><sec sec-type="conclusions" id="sec1-5"><title>CONCLUSIONS</title><p>We have demonstrated an increase in favorable pathologic outcomes with a decrease in operative intervention on low risk prostate cancer in men who opted to undergo robot-assisted radical prostatectomy. Despite the increasing proportion of men with extra-capsular disease undergoing RALP, the surgical margin status has remained similar. This could reflect both the changing dynamics of the population opting for surgery as well as the learning curve of the surgeons.</p></sec> |
Practical approach to screen vesicoureteral reflux after a first urinary tract infection | <sec id="st1"><title>Introduction:</title><p>Vesicoureteral reflux (VUR) is a common pediatric urologic disorder. After the first urinary tract infection (UTI), imaging studies are recommended, starting with a renal ultrasound (RUS). Voiding cystourethrography (VCUG) and dimercaptosuccinic acid (DMSA) scan are the other main radiologic studies used to detect VUR. We evaluated the use of RUS as a screening method for VUR in children below 2 years of age, in order to avoid unnecessary VCUG.</p></sec><sec id="st2"><title>Materials and Methods:</title><p>Medical records and imaging studies of infants (<2 years) who had their first UTI in a 6 year period were retrospectively reviewed. We evaluated the sensitivity, specificity, and negative predictive values of RUS and DMSA for diagnosing VUR.</p></sec><sec id="st3"><title>Results:</title><p>Among 155 children (51% males) with their first UTI, 148 RUS were performed, 128 VCUG and 29 DMSA. VUR was detected in 21% patients; 14.5% low grade and 6.5% high grade. One hundred and twenty-one patients underwent both RUS and VCUG, 101 RUS were normal and 20 abnormal. Of the normal RUS 98% had no or low grade VUR. Among those with an abnormality on RUS 30% had high grade VUR (<italic>P</italic> < 0.001).</p></sec><sec id="st4"><title>Conclusions:</title><p>After the first UTI in infants (<2 years) RUS is a good screening method for VUR. Among such shildren with a normal RUS, we do not recommend VCUG or DMSA. In our opinion, VCUG should be performed only in patients with abnormal findings in RUS or in recurrent UTI.</p></sec> | <contrib contrib-type="author"><name><surname>Fuente</surname><given-names>María Álvarez</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Costa</surname><given-names>Talía Sainz</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>García</surname><given-names>Begoña Santiago</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Serrano</surname><given-names>Marcelina Algar</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Alonso</surname><given-names>Manuel Sosa</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Luján</surname><given-names>Esther Aleo</given-names></name><xref ref-type="aff" rid="aff1"/></contrib> | Indian Journal of Urology : IJU : Journal of the Urological Society of India | <sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Urinary tract infection is a very common condition in pediatric patients, affecting up to 2% of children in their first year of life.[<xref rid="ref1" ref-type="bibr">1</xref>] On an average 30-40% of children with their first UTI episode have associated VUR).[<xref rid="ref1" ref-type="bibr">1</xref><xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref3" ref-type="bibr">3</xref>] This relationship is stronger in infants and lesser in newborns and continent children.[<xref rid="ref4" ref-type="bibr">4</xref>] High grade VUR (defined as Grades IV and V following the International Reflux Study in Children classification[<xref rid="ref5" ref-type="bibr">5</xref>]) has been associated with renal scars, which cause hypertension and renal failure at adult age.[<xref rid="ref6" ref-type="bibr">6</xref>] On an average, 20-25% of children above the age of 15 years who are included in transplant and dialysis programs have renal scars due to UTI, often related to VUR.[<xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref8" ref-type="bibr">8</xref><xref rid="ref9" ref-type="bibr">9</xref><xref rid="ref10" ref-type="bibr">10</xref><xref rid="ref11" ref-type="bibr">11</xref><xref rid="ref12" ref-type="bibr">12</xref>] Guidelines agree that after the first UTI, imaging the child's urinary tract is recommended to search for VUR.[<xref rid="ref10" ref-type="bibr">10</xref><xref rid="ref11" ref-type="bibr">11</xref>]</p><p>VCUG is considered the gold standard investigation for VUR.[<xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref13" ref-type="bibr">13</xref>] Unfortunately, this technique has drawbacks. It requires urethral catheterization, which causes pain, risk of infection, and radiation. DMSA is the ideal technique to image the renal parenchyma, diagnose acute pyelonephritis (APN) and to identify the presence of renal scars.</p><p>All these techniques are used to optimize the management of children after their first UTI. Patients with VUR grades III-V could require surgical treatment, while those with low grade reflux (I and II) require only a clinical follow-up.[<xref rid="ref14" ref-type="bibr">14</xref>] Therefore, it is important to distinguish between these two groups. Guidelines, such as the one proposed by the American Academy of Pediatrics,[<xref rid="ref3" ref-type="bibr">3</xref>] recommend that after the first UTI, imaging should start with an RUS.[<xref rid="ref15" ref-type="bibr">15</xref><xref rid="ref16" ref-type="bibr">16</xref><xref rid="ref17" ref-type="bibr">17</xref><xref rid="ref18" ref-type="bibr">18</xref>] Some studies suggest RUS as the only imaging test, other combine it with DMSA or VCUG.[<xref rid="ref19" ref-type="bibr">19</xref><xref rid="ref20" ref-type="bibr">20</xref>] A common practice is to replace VCUG with DMSA.[<xref rid="ref21" ref-type="bibr">21</xref>] Some studies suggest that DMSA cannot replace VCUG in the diagnosis of VUR.[<xref rid="ref19" ref-type="bibr">19</xref>] NICE guidelines recommend imaging the urinary tract only in patients with atypical UTI, poor responses to treatment, or recurrent UTI.[<xref rid="ref23" ref-type="bibr">23</xref>] Till date, there is no consensus on how to proceed in this matter.</p><p>Our study analyzes the negative predictive value of RUS for VUR, in order to propose a more practical approach in the diagnosis of high grade VUR. Among this background, the objective of our study is to avoid all unnecessary diagnostic tests.</p></sec><sec sec-type="materials|methods" id="sec1-2"><title>MATERIALS AND METHODS</title><p>Over a 6 year period between January 2003 and December 2008, we selected a cohort of children, between 1 day and 2-years of age, admitted in our hospital with their first UTI episode, defined as positive urine culture by urethral catheterization (>100,000 bacterial colonies). We excluded patients already diagnosed with urinary tract pathology and those with previous UTI diagnosis.</p><p>The study variables were sex, date of admission, age at admission (in months), urine culture, maximum temperature during the UTI (°C), C-reactive protein (CRP) (mg/dL), creatinine, and imaging diagnostic procedures, which were all done by two experienced pediatric radiologists.</p><p>We considered an RUS as abnormal when it had anatomical changes such as hydronephrosis, dilated ureter, ureterocele or changes in the renal parenchyma. We considered high grade VUR when at least one of the two urinary tracts had grade III or higher reflux (even though Grade III is not considered high grade, the risk of renal damage is greater than in grades I and II).[<xref rid="ref8" ref-type="bibr">8</xref><xref rid="ref10" ref-type="bibr">10</xref><xref rid="ref14" ref-type="bibr">14</xref>] Based on the existing protocols of UTI management during the study years, VCUG was performed in many patients as routine after a first UTI and DMSA was subsequently performed in children with VUR of any grade. The timing of each imaging study was also recorded.</p><p>Our data were analyzed with IBM SPSS 15.0 Statistics software. We compared the contributions of different imaging techniques depending on the timing and compared the clinical variables in the high degree VUR group versus the low or no VUR group.</p></sec><sec sec-type="results" id="sec1-3"><title>RESULTS</title><p>155 patients were included in the study, 51.2% boys (none circumcised), median age was 3 months (interquartile range [IQR] =2-7 months). The most frequent infectious agent was <italic>Echerichia coli</italic> (76%), followed by <italic>Klebsiella</italic> spp. (5.4%) and <italic>Proteus</italic> spp. (3.6%). The maximum temperature during the UTI was 38.4°C, (standard deviation [SD] =0.965). The CRP median was 4.13 mg/dL (IQR = 1.37-8.45). No difference was observed between boys and girls.</p><p>The mean creatinine was 0.4 mg/dl (SD = 0.1), no patient had altered renal function and the hydration status was normal. Among 155 children with their first UTI, 148 RUS were performed, 128 VCUG and 29 DMSA. VUR was detected in 21% (36): 14.5% (25) low Grade (I-II) and 6.5% (11) high Grade (III-V), we distinguished these two groups regarding the dilation of the urinary tract.</p><p>Among the one hundred and twenty-one children who underwent both RUS and VCUG, 101 RUS were normal (83.5%) and 20 were abnormal (16.5%). 98% of children with a normal RUS had either no or low grade VUR. Two patients with normal RUS had high grade VUR on VCUG (2%). Of the 20 abnormal RUS, 6 patients (30%) had high grade VUR, the other 14 had a low grade VUR or none. The presence of VUR in patients with an altered ultrasound is 15.15 times more frequent (confidence interval [CI] =3.291-69.736) than in patients with a normal RUS (<italic>P</italic> < 0.001). RUS predicted VUR with a sensitivity of 83% (CI 95% =74-91%), specificity of 88% (CI 95% =87-89%) and a negative predictive value of 98% (CI 95% =97-99%).</p><p>During the study years, the imaging study protocols changed and the number of DMSA performed is limited. Both DMSA and VCUG were performed in only 27 patients. Of these 27, two patients had renal scars (7.4%), both with a high degree VUR. Of the 25 patients with normal DMSA (92.6%), 5 (20%) had a high degree VUR and 8 low grade. The risk of high degree VUR in patients with altered DMSA is 0.2 times higher than in those patients with a normal DMSA (CI: 0.091-0.438) (<italic>P</italic> = 0.013).</p><p>The timing of each of the image techniques was also analyzed. Ninety-one RUS were performed in the first 3 weeks of the UTI, and 57 between 3 weeks and 3 months after the UTI episode. No statistical difference was seen between these two groups; therefore, we found no difference in the timing of performing the RUS. All VCUG were performed 4 weeks after the UTI episode and all DMSA were performed between 4 and 6 months after the UTI in order to look for renal scars. DMSA was not used as a diagnostic method to diagnose APN.</p><p>Of the other variables that were analyzed, the infectious agent and the CRP could identify patients at risk of high grade VUR. UTI caused by microorganisms other than <italic>E. coli</italic> had 5 times more risk of having high grade VUR (<italic>P</italic> = 0.011). The median CRP in patients without high grade VUR was 4.00 mg/dL, and with high grade VUR was 7.20 mg/dL (<italic>P</italic> = 0.026). Temperature was also higher in the high grade VUR group, 39.04°C versus 38.35°C (<italic>P</italic> = 0.06). When analyzing the receiver-operating characteristics curves for CRP and temperature, no optimal cut point was achieved.</p></sec><sec sec-type="discussion" id="sec1-4"><title>DISCUSSION</title><p>In an infant with his first UTI, the aim is to start the correct treatment as soon as possible in order to lower the risk and damage of APN and its sequelae. Once the acute infection is solved, investigation into underlying pathologies are begun. The actual recommendations are to perform a RUS in all infants after their first UTI.[<xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref16" ref-type="bibr">16</xref><xref rid="ref18" ref-type="bibr">18</xref>] Our results favor this recommendation, we consider RUS the first imaging study after a first UTI in infants, being a safe and inexpensive technique. In our study group RUS accurately predicted the presence of VUR and therefore, we consider it a good screening method for VUR, in agreement with the American Academy of Pediatrics and other study groups.[<xref rid="ref3" ref-type="bibr">3</xref>]</p><p>VUR and renal scars can originate during the fetal period, related with prenatal hydronephrosis, or can appear postnataly, in the context of an UTI.[<xref rid="ref13" ref-type="bibr">13</xref>] In a systematic review by Shaikh <italic>et al</italic>., children with VUR appeared to have 1.5 times more risk of having findings consistent with APN evidence by technetium-99 DMSA.[<xref rid="ref12" ref-type="bibr">12</xref>] Furthermore, children with VUR had prevalence of renal scarring of 2.6 times higher that children with no VUR, being higher in VUR grades III–V. There is also evidence of renal scars in the absence of VUR, consequence to VUR suffered in the prenatal period or VUR with spontaneous resolution.[<xref rid="ref9" ref-type="bibr">9</xref>]</p><p>Many study groups recommend a DMSA in the acute phase of UTI to optimize the screening of APN, optimize treatment, and minimize the damage that APN can do to the kidney.[<xref rid="ref22" ref-type="bibr">22</xref><xref rid="ref23" ref-type="bibr">23</xref>] Considering that DMSA is not available in all centers and that it cannot always be done as part of the initial diagnosis of a febrile infant, we argue that the lack of DMSA in the initial diagnosis should not be a handicap at the time of initiating adequate treatment. In addition, we believe that in many cases, DMSA is not required to initiate more aggressive treatment. In an infant with high fever and a suspicion of APN, a normal DMSA would not change our practical approach. In such cases, we would treat the patient as an APN, having in mind that infections in this age can extend easily and become an APN or even sepsis.</p><p>There is no agreement on the he follow-up after a first UTI. We believe that the next step should rely on an imaging technique that can make a difference in the treatment and follow-up. Given the good correlation of RUS with high grade VUR, we suggest continuing investigations in only those patients with abnormal findings on the RUS. Some studies have shown a low sensitivity, specificity, and negative predictive value for RUS,[<xref rid="ref23" ref-type="bibr">23</xref>] These results may have been influenced by radiologist experience which is a an important factor in order to perform a valid RUS. Furthermore, additional factors like CRP, recurrent UTI or bacteria other than <italic>E. coli</italic>, can indicate a high risk UTI, which may require VCUG.</p><p>We propose that if an abnormal RUS is found, patients should undergo a VCUG, irrespective of sex of the patient. An abnormal VCUG with high-grade VUR would require close follow-up, antibiotic prophylaxis, or surgery.[<xref rid="ref24" ref-type="bibr">24</xref>] Further, these patients with high grade VUR have a higher risk of renal scars, andshould undergo a DMSA[<xref rid="ref3" ref-type="bibr">3</xref><xref rid="ref25" ref-type="bibr">25</xref>]. In our cohort, despite the low number of patients in which both DMSA and VCUG was performed, a high percentage of patients with high grade VUR had a normal DMSA (25/27). In our cohort, the performance of RUS by expert sonographers was sufficient to identify patients for a VCUG, missing only two patients (2%). One of these 2 pattients was a 2-month-old male with VUR Grade III, 40°C fever, <italic>E. coli</italic> infection and a CRP of 23 mg/dL. These clinical parameters could indicate the risk of high grade VUR. Subsequently, this patient did not have any more UTI episodes, the DMSA was normal and the VUR resolved spontaneously. The other misdiagnosed patient with RUS was a 2-year-old female with a positive culture for <italic>E. coli</italic> and a CRP of 15 mg/dL. This patient had recurrent UTI episodes, but did not require surgical nor medical treatment because the VUR decreased to low grade. Both cases did not require additional intervention.</p><p>Other variables such as high fever, infectious agent or procalcitonin, should be considered as indicators of high grade VUR.[<xref rid="ref26" ref-type="bibr">26</xref>] In our cohort, high fever and microorganism different than <italic>E. coli</italic> were associated with high grade VUR.[<xref rid="ref27" ref-type="bibr">27</xref>].</p><p>Renal scarring after UTI is a fact, but we consider that our objective is to avoid the kidney from getting new scars. These new lesions can appear in the presence of VUR or with new UTI episodes. Therefore, diagnosing VUR would make a difference in the management.</p><p>The main limitation of our study is that this is a retrospective study; therefore, the protocols on imaging techniques were not uniform. However, we have a large amount of RUS which makes the results more reliable.</p></sec><sec sec-type="conclusions" id="sec1-5"><title>CONCLUSIONS</title><p>We support RUS as a screening method for high grade VUR in infants under 2 years of age after their first UTI. Given the high negative predictive value, we consider normal RUS enough to stop further imaging after a first UTI, postponing further investigations for recurrent UTI episodes. If the RUS is abnormal, we propose performing VCUG as the next step instead of a DMSA, because it can change our treatment. DMSA should be performed only for those patients with high grade VUR in order to complete the renal study and optimize therapy.</p></sec> |
Efficacy of nifedipine and alfuzosin in the management of distal ureteric stones: A randomized, controlled study | <sec id="st1"><title>Introduction:</title><p>Stone disease is a significant and world-wide health problem. Recently, certain drugs have been used as a supplement to observation alone in an effort to improve spontaneous stone expulsion. We evaluated the efficacy of nifedipine and alfuzosin in the medical treatment of symptomatic, uncomplicated distal ureteral stones.</p></sec><sec id="st2"><title>Materials and Methods:</title><p>This was a randomized controlled prospective study to determine the efficacy of alfuzosin and nifedipine as an adjunctive medical therapy, to increases the stone-expulsion rates in distal ureteric calculus of size ≤10 mm. Investigators and patients were blinded to the randomization scheme. Patients were randomly divided into three equal groups of 35 patients each. Patients in Group I received tablet nifedipine 30 mg/day, Group II received alfuzosin 10 mg/day and Group III was the control group received tablet diclofenac sodium. The patient blood pressure, stone position on imaging, number of pain attacks, time of stone-expulsion, hospital re-admission and any adverse events were assessed. Patients were followed-up weekly and continued until the patient was rendered stone free or up to 28 days. Statistical analysis was performed and <italic>P</italic> < 0.05 was considered to be significant.</p></sec><sec id="st3"><title>Results:</title><p>Stone-expulsion was observed in 60%, 85.7% and 20% patients in Group I, II and III respectively. A statistically significant difference was noted in between Groups I versus III, Groups II versus III and Groups I versus II (<italic>P</italic> < 0.0001, <italic>P</italic> < 0.0001, and <italic>P</italic> < 0.0315 respectively). The mean number of pain attacks was 2.91 ± 1.01 for Group I, 1.8 ± 0.83 for Group II, and 2.82 ± 1.12 for Group III, which is statistical significant in Groups II versus III, and Groups I versus II (<italic>P</italic> < 0.001 and <italic>P</italic> < 0.001). Hospital re-admission rate was less in treatment groups when compare to control group (<italic>P</italic> < 0.0001).</p></sec><sec id="st4"><title>Conclusion:</title><p>The use of alfuzosin and nifedipine as a medical expulsive therapy for distal ureteric stones proved to be safe and effective in term of increased stone-expulsion rate, reduced pain attacks and decrease hospital re-admissions.</p></sec> | <contrib contrib-type="author"><name><surname>Sameer</surname><given-names/></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Lal</surname><given-names>Shyam</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Charak</surname><given-names>K. S.</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Chakravarti</surname><given-names>Sumit</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Kohli</surname><given-names>Supreeti</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib><contrib contrib-type="author"><name><surname>Ahmad</surname><given-names>Shamshad</given-names></name><xref ref-type="aff" rid="aff3">2</xref></contrib> | Indian Journal of Urology : IJU : Journal of the Urological Society of India | <sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Urinary stone disease is a common condition affecting up to 12% of the world population. A large number of patients presenting in surgical emergency have symptoms and signs of ureteric colic. Many of these patients suffer from distal ureteric stones (70%). Spontaneous expulsion of distal ureteral stones of ≤10 mm diameter occurs in 25-53% of cases.[<xref rid="ref1" ref-type="bibr">1</xref>] The watchful waiting approach can result in complications, such as infection of the urinary tract, hydronephrosis, and deranged renal function.[<xref rid="ref2" ref-type="bibr">2</xref>] Ureteric stones have been treated traditionally with interventional techniques like ureteroscopy or open surgery. In recent years, medical expulsive therapy (MET) has been used in the management of distal ureteric stones as a supplement to conservative treatment. Drugs such as α1 receptor blockers, calcium channel blockers, prostaglandins synthesis inhibitors, antihistamines, glyceryl trinitrate, and corticosteroids are used for MET. Factors influencing the spontaneous passage of ureteral stones are stone size, configuration, location, smooth muscle spasm, sub-mucosal edema, and anatomy.[<xref rid="ref3" ref-type="bibr">3</xref><xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref5" ref-type="bibr">5</xref>]</p><p>Alpha blockers, calcium channel blockers are the most commonly used drugs for MET. Tamsulosin has been the most commonly studied α−1 blocker; however, alfuzosin is a combined α−1 A and α−1 D selective adrenergic antagonist resulting in relaxation of distal ureteric smooth muscles to facilitate passage of stone, and relieving pain. It is easily available and has less cardiac and ejaculatory side effects. Nifedipine is a calcium channel blocker, which acts by relieving the smooth muscle spasm in the ureter without interfering with its peristaltic activity. It is effective in stone-expulsion and relieving pain.[<xref rid="ref4" ref-type="bibr">4</xref>] Therefore, we performed a comparative study to evaluate the efficacy of nifedipine and alfuzosin in the medical management of symptomatic, uncomplicated distal ureteral stones.</p></sec><sec sec-type="materials|methods" id="sec1-2"><title>MATERIALS AND METHODS</title><p>Between November 2008 -and October 2010, patients presenting with renal colic were enrolled for this double-blind, randomized, controlled study. Patients were assessed with history, physical examination and investigated with complete blood count, blood urea, serum creatinine, routine urine analysis, X-rays kidney, ureter, and bladder (KUB), ultrasonography, intravenous urography and helical computed tomography (CT) whenever was necessary. Patients ≥8 years of either sex with a single, unilateral ureteral stone of ≤10 mm were included. The distal ureter was defined as the segment from the lower border of the sacroiliac joint to the vesico-ureteric junction. Patients having previous surgery on the ipsilateral ureter, bilateral ureteric stones, multiple stones, solitary kidney, urinary tract infection, moderate or severe hydronephrosis, contraindications for non-steroidal anti-inflammatory drugs (e.g. gastritis), known allergy to tamsulosin or alfuzosin, renal insufficiency, or currently on α-blocker therapy, and pregnant or lactating women were excluded. The study was approved by the ethical committee of hospital.</p><sec id="sec2-1"><title/><sec id="sec3-1"><title>Sample size calculation and randomization</title><p>Spontaneous expulsion rate for lower ureteric stones ranges between 25% and 60%. We took an average rate of spontaneous stone-expulsion as 40% and also considered an expected increase of this rate by 35% from baseline after MET. Taking significance level α at 95% and power of study 80%, the sample size was calculated by using formula [<italic>N</italic> = 2 ×{z<sub>1−α</sub> + z<sub>1−β</sub>/<italic>d</italic><sub>−</sub>δ<sub>0</sub>}<sup>2</sup> × <italic>p</italic> (1 − <italic>p</italic>), where <italic>N</italic> = size/group; <italic>P</italic> = spontaneous expulsion rate; Z<sub>α</sub> = standard normal deviate for one sided test; <italic>d</italic> = expected difference: δ<sub>0</sub> = clinically acceptable margin (taken as 0.05). The sample size for each group was fixed at 35 and total for three groups was 105. Block randomization was used to achieve balance in the allocation of patients to different treatment groups. To get 35 patients in each group, 7 blocks of sample size 15 were created by a statistician. The details of blocks were given to the pharmacist. Packaging of drugs was done in this order to achieve blinding. After taking written informed consent, patients received tablet nifedipine 30 mg/day in Group I, tablet alfuzosin 10 mg/day in Group II and tablet diclofenac sodium in Group III as a control group. Patients in all 3 groups received tablet diclofenac sodium (50 mg) every 12 hourly for 1 week and injection diclofenac sodium (75 mg) as needed. An intramuscular injection Tramadol hydrochloride 100 mg was given for persistent pain.</p><p>Patients were followed-up weekly up to 28 days; history of stone passage, number of pain episodes were recorded and investigated with routine urine examination, kidney function test and X-ray KUB. Patients were advised to watch for stone-expulsion. Medications were stopped after spontaneous stone-expulsion, any intervention before the end of the study, and adverse effect. For patients with a stone-free ureter on the last imaging, but no documented stone-expulsion, the last date of positive stone status was recorded. Abdominal CT was performed for patients with radiolucent stones if the stone was not expelled by the end of the study. Patients underwent ureteroscopic stone removal for persistent stones after 28 days. Patients having uncontrollable pain were readmitted for injectable analgesics and medication was continued. The blood pressure, stone position on imaging, number of pain attacks, time of stone-expulsion, hospital re-admission and any adverse events were recorded.</p></sec><sec id="sec3-2"><title>Statistical analysis</title><p>Data analysis was performed using Statistical Package for the Social Sciences trial version 17.1 statistical software. Student's <italic>t</italic>-test, ANOVA, Chi-square, and Fisher's exact test were applied as required. The power of the study was 0.80, and the level of significance was 95%.</p></sec></sec></sec><sec sec-type="results" id="sec1-3"><title>RESULTS</title><p>A total of 246 patients with symptoms of ureteric colic were assessed for inclusion, of which 102 patients were excluded as they did not have distal urolithiasis. Of the remaining 146 patients, 105 were eligible and included for the study. [<xref ref-type="fig" rid="F1">Figure 1</xref>]. Group I had 19 male and 16 female (mean age: 32.74 ± 9.58 years), Group II had of 26 male and 9 female (mean age: 30.82 ± 7.85 years), and Group III had 23 male and 12 female (mean age: 33.06 ± 8.76 years). The mean stone size was 6.5 ± 1.78 mm for Group I, 6.26 ± 1.85 mm for Group II, and 6.37 ± 1.85 mm for Group III. There were no statistically significant differences between the groups in terms of sex, age, or stone size (<italic>P</italic> > 0.05).</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Trial profile all randomized patients completed trial</p></caption><graphic xlink:href="IJU-30-387-g001"/></fig><p>A statistical significant difference was observed for stone-expulsion rate between Group I versus Group II (60% vs. 85.7%, <italic>P</italic> < 0.0315), Group I versus Group III (60% vs. 20%, <italic>P</italic> < 0.000) and Group II versus Group III (85.7% vs. 20%, <italic>P</italic> < 0.000). Average time for stone-expulsion was 12.6 ± 6.69 days in Group I, 12.0 ± 6.67 days in Group II, and 12.29 ± 9.46 days in Group III. No statistically significant difference was observed in time for stone-expulsion between these groups.</p><p>Patients taking alfuzosin had fewer pain attacks compared with others. The average number of pain attacks was 2.91 ± 1.01 for Group I, 1.8 ± 0.83 for Group II, and 2.82 ± 1.12 for Group III patients. A significant statistical difference was observed between Groups II versus III, and Groups I versus II (<italic>P</italic> < 0.001 and <italic>P</italic> < 0.001, respectively).</p><p>Hospital re-admissions due to uncontrollable pain occurred in 43 patients: 11 patient (31.4%) in Group I, five patients (14.3%) in Group II, and 27 patients (77.1%) in Group III. The difference was statistically significant (<italic>P</italic> < 0.0001) in Group I versus Group II and Group II versus Group III [<xref ref-type="table" rid="T1">Table 1</xref>].</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Demographic data of all 3 groups</p></caption><graphic xlink:href="IJU-30-387-g002"/></table-wrap></sec><sec sec-type="discussion" id="sec1-4"><title>DISCUSSION</title><p>Recent advances in ureteric stone management have allowed these to be treated using minimally invasive techniques, which have increased success rates and decreased treatment related morbidity. These advances include shock wave lithotripsy and ureteroscopic lithotripsy. Although these approaches are less invasive than traditional open surgical methods, they are expensive and have inherent risks. Consequently, observation has been advocated for small ureteral stones which have a high probability to pass spontaneously. Observation can be supplemented by using MET. The factors influencing expulsion of calculi include stone size, shape, and location, ureteric edema, and convolutions. (Of these, the location of the calculus and its size are the most important factors.[<xref rid="ref1" ref-type="bibr">1</xref>]</p><p>Malin <italic>et al</italic>. first demonstrated the presence of alpha - adrenergic receptors in the human ureter in 1970.[<xref rid="ref6" ref-type="bibr">6</xref>] Recently, Sigala <italic>et al</italic>.[<xref rid="ref7" ref-type="bibr">7</xref>] studied the presence of α−1 adrenergic receptor gene and protein expression in the proximal, middle, and distal ureter. Characteristic distribution of α−1 receptor subtypes in distal, middle, and proximal segments is α 1D > α 1A > α 1B.[<xref rid="ref8" ref-type="bibr">8</xref>] α−1D was the most common receptor present in all portions of the ureter. It has the strongest effect on the contractions of distal ureter and bladder detrusor, especially for the ureter-bladder wall section, and therefore plays an important role in the distal ureter. α−1 receptor blockers relax ureteral smooth muscle, reduce peristalsis frequency and amplitude, decrease intraluminal pressure of the ureter, enhance transportation capability and pulses of urine. Moreover, they establish pressure gradient surrounding calculi by increasing the pressure above calculi, relax smooth muscles of the bladder neck and urethra, and finally form one thrust to expel calculi. α1-receptor blockers can also affect the C-type fast fiber of the spinal cord and the sympathetic post-ganglionic neuron; block the pain transmission pathway to central nervous system and reduce renal colic during the process of calculi expulsion.[<xref rid="ref9" ref-type="bibr">9</xref>] Hellstrom and Sikka[<xref rid="ref10" ref-type="bibr">10</xref>] studied the effect of tamsulosin and alfuzosin on ejaculatory function and concluded that marked decrease in ejaculatory volume in almost 90% of subjects and anejaculation in approximately 35% of individuals taking tamsulosin. Thus, we chose to study alfuzosin to avoid ejaculatory disturbance.</p><p>The basic functional unit of the ureter is the smooth muscle cell, which reacts to calcium concentration change: contraction with an increase in calcium concentration and diastole when calcium concentration decreases. Ideal MET drugs should inhibit incongruous contraction but without influencing the slow peristalsis. Previous studies indicated that calcium antagonist could inhibit the fast contraction phase of animal and human ureter, but without any effect on the tonic activities.[<xref rid="ref11" ref-type="bibr">11</xref><xref rid="ref12" ref-type="bibr">12</xref>] Therefore, this kind of drug was thought to have potential use for calculi expulsion. Nifedipine is one potential calcium channel antagonist with lows adverse effect. In an article in 1994 Borghi <italic>et al</italic>.[<xref rid="ref13" ref-type="bibr">13</xref>] demonstrated the efficacy of calcium channel blockers in the treatment of ureteral stones. They enrolled 86 patients to receive methylprednisolone with placebo or nifedipine. Patients receiving nifedipine had a significantly higher rate of stone passage compared with the placebo group, 87% versus 65%.</p><p>A meta-analysis compared stone passage rates in patients who were given calcium channel blockers or α1-adrenergic receptor antagonist versus controls who did not receive these medications. They demonstrated a 65% greater chance of passing a ureteral stone in patients who received either medication.[<xref rid="ref14" ref-type="bibr">14</xref>] Another study also showed that calcium channel antagonist could promote distal calculi expulsion and reduce analgesia requirement, renal colic rate, and hospitalization rate.[<xref rid="ref15" ref-type="bibr">15</xref>]</p><p>The comparison of alfuzosin and tamsulosin in the management of lower ureteral calculi has shown that both drugs increase the stone-expulsion rate (82.3% vs. 70.5%), decrease expulsion time (12.3 versus 14.5) and reduce the need for analgesics[<xref rid="ref16" ref-type="bibr">16</xref>] We chose Alfuzosin because it is easily available and like tamsulosin has comparatively less cardiac side effects than other alpha1 blockers like doxazosin or terazosin. Moreover, it is a combined α1 A and α1 D – selective adrenergic antagonist. Pedro <italic>et al</italic>.[<xref rid="ref17" ref-type="bibr">17</xref>] conducted a double-blinded placebo-controlled trial on 76 patients to test the efficacy of alfuzosin for distal ureteral stones, and concluded that alfuzosin improves patient comfort associated with stone passage and decreases the time to distal ureteral stone passage but does not increase the rate of spontaneous stone passage (77.1% for placebo and 73.5% for alfuzosin, <italic>P</italic> = 0.83). Whereas in our study statistically significant difference was noted in alfuzosin (85.7%) and nifedipine (60%) for stone-expulsion rates (<italic>P</italic> < 0.05) when compared with control group (20%). In another randomized placebo-controlled trial study done by Pirzada <italic>et al</italic>.[<xref rid="ref18" ref-type="bibr">18</xref>] on 60 patients to test the efficacy of alfuzosin in kidney stones which are fragmented by extracorporeal shock-wave lithotripsy and authors concluded that stone-expulsion was significantly higher in alfuzosin group when compared to placebo (76.7% and 46.7% respectively, <italic>P</italic> = 0.01).</p><p>Our results confirmed the efficacy of nifedipine and alfuzosin for distal ureteric stones. Nearly60% of patient taking nifedipine and 85.7% of patients taking alfuzosin were able to expel their stones at the end of study compared to 20% in the control group. Alfuzosin was found to be significantly better in term of stone-expulsion compared to nifedipine and control group (<italic>P</italic> < 0.05). Moreover, both nifedipine and alfuzosin groups hade significantly less hospital re-admission rate as compared to the control group (<italic>P</italic> < 0.0001).</p><p>Nifedipine and alfuzosin also decreased the frequency of pain attacks. Patients taking alfuzosin had significantly less pain as compared to nifedipine and placebo group (<italic>P</italic> < 0.001). Use of α-blockers for expulsion of ureteral stones probably decreases the analgesic requirement in two ways: Expulsion of stones, leading to longer stone free periods, and blockade of C-fibers. It is difficult to assess which of these may be primarily responsible for decreasing the analgesic requirement, because α-blockers are known to be associated with both.</p><p>The most frequently reported adverse event with α blockers was transient hypotension[<xref rid="ref19" ref-type="bibr">19</xref>] Pedro <italic>et al</italic>. reported 12% adverse events in the alfuzosin group compared with 0% in the placebo group.[<xref rid="ref17" ref-type="bibr">17</xref>] whereas Yilmaz <italic>et al</italic>. have reported no serious adverse events.[<xref rid="ref20" ref-type="bibr">20</xref>] In the present study, MET related side effects were observed in four patients (three patients taking alfuzosin developed retrograde ejaculation and one patient taking nifedipine developed an episode of hypotension), but they were able to complete the study. No patients developed serious side effects during the study period. Patients who were not stone free after the 4 week follow-up were successfully treated with ureteroscopy. This study demonstrated that neither watchful waiting nor medical therapy seems to negatively affect the success rate of stone removal.</p></sec><sec sec-type="conclusions" id="sec1-5"><title>CONCLUSIONS</title><p>Medical expulsion therapy is a useful adjunct to observation in the conservative management of ureteral stones. The use of alfuzosin and nifedipine for uncomplicated distal ureteric stones is safe and effective in term of increased stone-expulsion rate, reduced pain attacks and decrease hospital re-admissions. Alfuzosin was found to be significantly better in terms of stone-expulsion rate and pain attacks as compared to nifedipine.</p></sec> |
Outcome of tubularized incised plate urethroplasty with spongioplasty alone as additional tissue cover: A prospective study | <sec id="st1"><title>Introduction:</title><p>Additional cover after neourethra formation to decrease the fistula rate, has been described using the dartos, tunica, denuded skin and corpus spongiosum. The use of corpus spongiosum alone to cover the neourethra is infrequent. The objective of this study was to evaluate the efficacy of spongioplasty alone as an intervening layer in the prevention of urethral fistula following <italic>tubularized incised plate</italic> urethroplasty (TIPU).</p></sec><sec id="st2"><title>Materials and Methods:</title><p>A prospective study was performed including 113 primary hypospadias cases undergoing TIPU with spongioplasty from June 2010 to March 2012. Correction of chordee was carried out by penile degloving alone in 5, mobilization of urethral plate with spongiosum in 22 and combination of both in 45 cases. Intra-operatively, spongiosum was taken to be poorly developed if it was thin and fibrous, moderate if good spongiosal tissue with good vascularization and well-developed if healthy robust spongiosum, which became bulkier than native spongiosum after tubularisation. Spongioplasty was done in a single layer after mobilization of spongiosum, starting just proximal to the native meatus and into the glans distally.</p></sec><sec id="st3"><title>Results:</title><p>The mean age of the patients was 11.53 years. The type of hypospadias was distal, mid and proximal in 81, 12 and 20 cases respectively. Spongiosum was poorly developed in 13, moderate in 53 and well-developed in 47 cases. The mean hospital stay was 8-10 days and follow-up ranged from 6 months to 2 years. Urethral fistula was seen in six patients (11.3%) with moderate spongiosum (distal 1, mid 1 and proximal 4), and three (23.03%) with poorly developed spongiosum (one each in distal, mid and proximal) with an overall 7.96% fistula rate. None of the patients with well-developed spongiosum developed a fistula. Poorer spongiosum correlated with a greater number of complications (<italic>P</italic> = 0.011). Five out of thirteen cases with poor spongiosum (38.46%) had proximal hypospadias, i.e. more proximal was the hypospadias, poorer was the development of the spongiosum (<italic>P</italic> = 0.05). Meatal stenosis was seen in two patients (1.76%) with proximal hypospadias, one with moderate and the other with poorly developed spongiosum. More proximal was the hypospadias, greater were the number of complications (<italic>P</italic> = 0.0019).</p></sec><sec id="st4"><title>Conclusion:</title><p>TIPU with spongioplasty reconstructs a near normal urethra with low complications. Better developed and thicker spongiosum results in lower incidence of fistula and meatal stenosis. More proximal hypospadias is associated with poorer spongiosum. We recommend spongioplasty to be incorporated as an essential step in all patients undergoing tubularized incised-plate repair for hypospadias.</p></sec> | <contrib contrib-type="author"><name><surname>Bhat</surname><given-names>Amilal</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Sabharwal</surname><given-names>Karamveer</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Bhat</surname><given-names>Mahakshit</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib><contrib contrib-type="author"><name><surname>Saran</surname><given-names>Ramakishan</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Singla</surname><given-names>Manish</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Kumar</surname><given-names>Vinay</given-names></name><xref ref-type="aff" rid="aff1"/></contrib> | Indian Journal of Urology : IJU : Journal of the Urological Society of India | <sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>The tubularized incised plate urethroplasty (TIPU) repair is the procedure of choice for distal and mid-shaft hypospadias and is increasingly being used for proximal hypospadias and redo repairs.[<xref rid="ref1" ref-type="bibr">1</xref><xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref3" ref-type="bibr">3</xref><xref rid="ref4" ref-type="bibr">4</xref>] It has excellent functional and cosmetic outcomes with minimal complications. Post-operatively, urethrocutaneous fistula is the most common complication.[<xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref7" ref-type="bibr">7</xref>] To decrease fistula rate, an additional tissue cover after neourethra reconstruction has been described using transverse island dorsal subcutaneous flap,[<xref rid="ref8" ref-type="bibr">8</xref>] de-epithelialized skin flaps,[<xref rid="ref9" ref-type="bibr">9</xref><xref rid="ref10" ref-type="bibr">10</xref>] lateral dartos flap,[<xref rid="ref11" ref-type="bibr">11</xref>] double dartos flaps,[<xref rid="ref12" ref-type="bibr">12</xref><xref rid="ref13" ref-type="bibr">13</xref>] double breasted de-epithelialized penile skin flap[<xref rid="ref14" ref-type="bibr">14</xref>], tunica vaginalis[<xref rid="ref15" ref-type="bibr">15</xref><xref rid="ref16" ref-type="bibr">16</xref>], and corpus spongiosum.[<xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref17" ref-type="bibr">17</xref><xref rid="ref18" ref-type="bibr">18</xref>] The use of corpus spongiosum as an intervening layer over the neourethra is less frequent. We evaluated the efficacy of spongioplasty alone after mobilization of corpus spongiosum in prevention of urethral fistula in patients undergoing TIP repair for hypospadias.</p></sec><sec sec-type="materials|methods" id="sec1-2"><title>MATERIALS AND METHODS</title><p>A prospective clinical study was carried out at our institution between June 2010 and March 2012. We included 113 cases of hypospadias undergoing TIPU repair with spongioplasty. All patients presenting with primary hypospadias were included. Re-operative repairs, intersex state and ambiguous genitalia were excluded. The parents (for minors) and patients were informed about the merits and complications of the procedure and a written informed consent was obtained. Approval was sought from the hospital ethical committee. All patients were operated upon by a single surgeon(ALB). Intra-operatively, we divided the corpus spongiosum into three categories depending on the appearance and vascularity.</p><p>Poorly developed-Thin spongiosal tissue with decreased vascularity; the diameter of the neourethra covered by spongiosum after spongioplasty was less than the proximal healthy urethra [<xref ref-type="fig" rid="F1">Figure 1</xref>].</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Poorly developed corpus spongiosum (a) Mobilized spongiosum on left side with minimal spongiosum tissue (b) Mobilized spongiosum on right with minimal spongiosum tissue (c) Hypoplastic urethra with mobilized spongiosum on both sides with minimal spongiosum tissue (d) after completion of spongioplasty, proximal normal urethra bulkier than distal neourethra with spongioplasty</p></caption><graphic xlink:href="IJU-30-392-g001"/></fig><p>Moderately developed-Average thickness and vascularity of spongiosal tissue; the diameter of the neourethra covered by spongiosum after spongioplasty was almost equal to that of the proximal healthy urethra [<xref ref-type="fig" rid="F2">Figure 2</xref>].</p><fig id="F2" position="float"><label>Figure 2</label><caption><p>Moderately developed corpus spongiosum (a and b) Both side mobilized spongiosum with moderate spongiosum tissue (c and d) Spongioplasty of mobilized spongiosum</p></caption><graphic xlink:href="IJU-30-392-g002"/></fig><p>Well-developed-Robust, thick spongiosum with good vascularity; the diameter of the neourethra covered by spongiosum was greater than that of the proximal healthy urethra [<xref ref-type="fig" rid="F3">Figure 3</xref>].</p><fig id="F3" position="float"><label>Figure 3</label><caption><p>Well-developed corpus spongiosum (a-c) Both side mobilized spongiosum with robust healthy spongiosum tissue (d) Spongioplasty of mobilized spongiosum</p></caption><graphic xlink:href="IJU-30-392-g003"/></fig><sec id="sec2-1"><title/><sec id="sec3-1"><title>Surgical technique</title><p>An U-shaped incision is made encircling the meatus up to the corona, preserving the urethral plate and then extended circumferentially around the corona. Penile de-gloving is done when required, up to the root of the penis by creating a plane at the level of Buck's fascia. A Gittes test is carried out to evaluate the chordee after penile de-gloving. Starting well proximal to the hypospadiac meatus, a plane is created between the Buck's fascia and tunica albuginea [<xref ref-type="fig" rid="F4">Figure 4</xref>] and the dissection is carried out on the penile shaft just lateral to the margin of the corpus spongiosum. From lateral to medial, the spongiosum is dissected off the underlying corpora cavernosa. Mobilization is done well beneath the true urethral plate on both sides, lifting off the midline spongiosum and urethral plate [<xref ref-type="fig" rid="F5">Figure 5</xref>] from the corpora cavernosa when required, to correct chordee. Dissection is performed with care, avoiding damage to the corpus spongiosum or corpus cavernosum. The proximal urethra is mobilized according to the severity of ventral curvature up to the bulbar region in case of persistent chordee. Adequacy of curvature correction is confirmed by Gittes test.[<xref rid="ref17" ref-type="bibr">17</xref>] Since the spongiosal pillars spread beneath the glans wings on each side, care must be taken while mobilizing the glans wings distally with an oblique incision at about 45 degrees, which leaves healthy, thick glans wings yet intact spongiosum when done correctly [<xref ref-type="fig" rid="F6">Figure 6</xref>]. Glanular chordee correction is checked by Gittes test. The urethral plate is then tubularized with a subcuticular 7-0 polydioxanone (PDS) suture after a dorsal incision to create a new urethra. The mobilized spongiosal pillars are then approximated in the midline with 7-0 PDS continuous sutures covering the entire neourethra right up to the glans [<xref ref-type="fig" rid="F2">Figure 2d</xref>]. A 6-to 10 Fr urethral catheter, depending on patient age is left <italic>in situ</italic>. No additional cover of dartos, tunica vaginalis or de-epithelialized skin was used. Skin is sutured with 7-0 PDS interrupted suture.</p><fig id="F4" position="float"><label>Figure 4</label><caption><p>Showing creation of plane proximal to meatus between tunica and Buck's fascia to mobilize the corpus spongiosum</p></caption><graphic xlink:href="IJU-30-392-g004"/></fig><fig id="F5" position="float"><label>Figure 5</label><caption><p>Mobilization of the corpus spongiosum and urethral plate</p></caption><graphic xlink:href="IJU-30-392-g005"/></fig><fig id="F6" position="float"><label>Figure 6</label><caption><p>Mobilization of the corpus spongiosum and urethral plate into the glans</p></caption><graphic xlink:href="IJU-30-392-g006"/></fig><p>Cephalosporins with analgesic and anti-inflammatory drugs are given for 7-to 10 days till the catheter is <italic>in situ</italic>. Patients were evaluated at follow-up visits after 1, 3, 6, 12 months and then yearly. Results were evaluated in terms of patients’, parents’ and surgeon's satisfaction, keeping in mind the stream, cosmesis and lack of complications. If at 3 months follow-up the stream was narrow, we did a meatal/urethral calibration with a 6-10 Fr infant feeding tube.</p></sec></sec></sec><sec sec-type="results" id="sec1-3"><title>RESULTS</title><p>The age range of the patients was 4 months to 38 years (mean 11.53 years). The hospital stay ranged from 8-10 days and period of follow-up was 6 months to 2 years. The hypospadias was distal, mid and proximal in 81, 12, and 20 cases respectively. Correction of chordee was carried out by penile de-gloving alone in 5, mobilization of urethral plate with spongiosum in 22 and combination of both in 45 cases.</p><p>On correlating the type of hypospadias with development of spongiosum; more proximal hypospadias was associated with poorer development of the spongiosum (<italic>P</italic> = 0.05) [<xref ref-type="table" rid="T1">Table 1</xref>]. Two patients had meatal stenosis with fistula and seven patients had urethral fistula (Clavien-Dindo grade 3b)[<xref rid="ref19" ref-type="bibr">19</xref>] alone with an overall complication rate of 7.96%. Fistulas occurred in 2 cases each of distal and mid hypospadias and 5 cases of proximal hypospadias. Both patients who developed meatal stenosis had proximal hypospadias. Complications were higher in proximal hypospadias as compared to distal (<italic>P</italic> = 0.0019) [<xref ref-type="table" rid="T1">Table 1</xref>].</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Correlation of type of hypospadias, development of spongiosum and complications</p></caption><graphic xlink:href="IJU-30-392-g007"/></table-wrap><p>Three (one each in distal, mid and proximal) out of thirteen (23.03%) cases with poorly developed spongiosum and six (distal 1, mid 1 and proximal 4) out of 53 (11.32%) cases with moderately developed spongiosum developed a fistula while none of the cases with well-developed spongiosum developed a fistula. One out of 53 (1.88%) cases with moderately developed spongiosum and 1/13 (7.69%) cases with poorly developed spongiosum developed meatal stenosis. Thus, poorer spongiosum was associated with greater number of complications (<italic>P</italic> = 0.011) [<xref ref-type="table" rid="T1">Table 1</xref>].</p><p>None of the patients developed a stricture urethra and all patients/parents were satisfied with cosmesis. All nine patients with urethral fistula were operated upon 6-12 months later and the fistulae repaired with no further sequelae.</p></sec><sec sec-type="discussion" id="sec1-4"><title>DISCUSSION</title><p>The male urethra develops from the urethral folds as they fuse in the midline after the 6<sup>th</sup> week of fetal life. The endodermally derived distal solid urethral plate must canalize to form the distal most glanular part of the urethra. The urethra forms sequentially after the proximal urethral folds fuse and the mesenchyme within the urethral folds forms the corpus spongiosum. According to the classic theory, embryologically, the last step in urethra formation is the joining of the main endodermal urethral channel with the ectoderm as it invades the glans.[<xref rid="ref20" ref-type="bibr">20</xref>] The normal and hypospadiac penises differ in the development of glans and urethra, especially their vascularity. In hypospadias, there is failed urethral fold fusion and ectodermal intrusion, histologically characterized by an extremely vascular area with large endothelial lined sinuses around the abortive urethral spongiosum and abnormal glans.[<xref rid="ref21" ref-type="bibr">21</xref>] The degree of hypoplasia of corpus spongiosum depends upon the severity of hypospadias and proliferation of mesodermal tissue.</p><p>We classified the spongiosum according to its development and vascularity as poorly developed, moderately developed and well-developed. No such classification has been reported earlier in literature. This classification is a good parameter to predict the outcome. Further, it may act as a guide to new surgeons attempting hypospadias about the feasibility of spongioplasty in a particular case and whether an additional layer may be required. None of the patients with well-developed spongiosum in distal hypospadias had any complication. Complications in proximal hypospadias with well-developed spongiosum were also very low as compared to poorly developed spongiosum cases. Most importantly, poorer was the spongiosum, greater was the number of complications (<italic>P</italic> = 0.011). On analysis of our results, we found that more proximal meatus was associated with poorer development of the spongiosum (<italic>P</italic> = 0.05) and proximal hypospadias was associated with greater incidence of complications (<italic>P</italic> = 0.0019). However, not all hypospadias with poorly developed spongiosum had complications, suggesting that other factors like development and width of urethral plate also play a role in the outcome of the repair. Since none of the patients of distal hypospadias with well-developed spongiosum had fistula, dorsal dartos cover in such patients may be omitted and the prepuce can be utilized for preputioplasty. However in patients with poorly developed spongiosa, an additional interposing layer like dartos or tunica should be used.</p><p>Since its first description by Snodgrass[<xref rid="ref1" ref-type="bibr">1</xref>] for repair of hypospadias in 1994, TIP has gained widespread acceptance due to its versatility, low complication rate and reliable creation of vertically oriented meatus, with excellent cosmetic outcome. Some authors recommend TIPU as the technique to be considered for primary and re-operative repair of distal and some midshaft hypospadias cases with selected proximal ones.[<xref rid="ref22" ref-type="bibr">22</xref><xref rid="ref23" ref-type="bibr">23</xref>] The common complications reported after TIPU repair include fistulae, urethral stricture, meatal stenosis, persistent chordee, infections and wound dehiscence[<xref rid="ref14" ref-type="bibr">14</xref>] with development of urethrocutaneous fistula being the most common. The fistula rate in large series of hypospadias repairs varies from 0% to 33%.[<xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref14" ref-type="bibr">14</xref><xref rid="ref24" ref-type="bibr">24</xref>] The causes of fistulae are local infection, ischemia, an inadequate procedure, poor tissue healing and distal obstruction due to meatal stenosis/encrustation which promotes fistulae formation.[<xref rid="ref25" ref-type="bibr">25</xref>]</p><p>To decrease the incidence of post-operative fistulae, many modifications in the TIP repair have been tried. However, no single surgical technique can be considered to be a clear winner to prevent the formation of fistulae. One of the most important factors in reducing fistula formation with the TIP technique is the introduction of a protective intermediate layer between the neourethra and the skin. Dorsal,[<xref rid="ref3" ref-type="bibr">3</xref>] lateral, single or double dartos flaps, ventral based dartos flap, scrotal dartos, de-epithelized local penile skin, preputial flap, paraurethral tissue, spongioplasty, or tunica vaginalis flaps have all been used for the same. Out of all these, the dorsal dartos flap is used by most surgeons, but there is still no consensus over the ideal interposing tissue in TIPU. Because of edema, necrosis of skin, hematoma and torque with mobilization of the dorsal dartos flap, its role as an interposing tissue has been questioned. The corpus spongiosum provides a well-vascularized, spongy protective covering to the normal urethra. It may aid in the natural propulsion of urine and semen. Using this natural, locally available tissue to cover the neourethra in hypospadias seems to make sense. Spongioplasty also decreases the tension on the suture line of urethroplasty in the midline, especially during erections.</p><p>The use of spongiosal tissue as an intermediate layer between the urethra and skin was described in 2000 by Yerkes[<xref rid="ref26" ref-type="bibr">26</xref>] and Beaudoin[<xref rid="ref27" ref-type="bibr">27</xref>] in separate studies. Yerkes reported no fistulae in any of her patients for whom spongioplasty was implemented in a number of urethral repair techniques, whereas Beaudoin described the anatomical characteristics of the spongiosal layer in the hypospadic penis and implemented spongioplasty in patients who underwent urethral tubularization.</p><p>In 2003, Dodat <italic>et al</italic>.[<xref rid="ref28" ref-type="bibr">28</xref>] showed no fistula formation in any of the 51 patients who underwent TIPU and spongioplasty. El-Sherbiny <italic>et al</italic>.[<xref rid="ref5" ref-type="bibr">5</xref>] reported that covering the neourethra with a flap or spongioplasty minimized the risk of fistula formation. In a series of 500 cases, Sarhan <italic>et al</italic>.[<xref rid="ref29" ref-type="bibr">29</xref>] used a dartos flap, spongioplasty, or a combination of these techniques in addition to TIP. They observed a statistically significant decrease in fistula formation in cases in which spongioplasty was implemented compared with cases in which it was not. In the present series we had an acceptable overall fistula rate of 7.96% with no complications in distal hypospadias with well-developed spongiosum where we did not use dorsal/ventral dartos or any other tissue in addition with spongioplasty. These results are comparable with other studies where dorsal dartos has been used with or without spongioplasty. Since the results of well-developed spongioplasty alone in distal hypospadias were very good, additional cover with dorsal dartos can be omitted. Kassaby <italic>et al</italic>.[<xref rid="ref17" ref-type="bibr">17</xref>] report that various studies using dartos based flaps, tunica vaginalis and subcutaneous tissue flaps have a fistula rate of 2-33%. When spongioplasty alone was used as an interposing layer, in various series complications ranged from 4% to 40% with 14.2% complication rate in 197 cases whereas in the present series they were seen in 7.96 % cases [<xref ref-type="table" rid="T2">Table 2</xref>].</p><table-wrap id="T2" position="float"><label>Table 2</label><caption><p>Series using spongioplasty alone for hypospadias repair</p></caption><graphic xlink:href="IJU-30-392-g008"/></table-wrap><p>Our technique of spongioplasty is similar to those mentioned earlier in that we mobilize the urethral plate with the spongiosum completely off the corpora cavernosa starting laterally and moving medially. Furthermore we mobilized the spongiosal pillars into the glans obliquely at an angle of 45° almost up to the tip of the glans. The mobilization of urethral plate with spongiosum have added advantages of correction of torsion and ventral curvature, urethral plate can be tubularized without tension on suture line and many a times without incising the urethral plate.</p><p>Complete spongioplasty if done properly as outlined above restores a near normal urethra and provides good support. It is a healthy, vascular cover which probably decreases tension on the midline sutures especially during erections. Better is the spongiosum, lesser are the chances of post-operative fistulae formation.</p><p>The strengths of our study are that it is prospective, cases belonged to similar socio-economic status, procedures were performed by a single surgeon in similar circumstances and with adequate follow-up. The weaknesses are that the criteria for development of spongiosum are subjective so there are chances of error in judgment, especially in well and moderately developed spongiosum. To the best of our knowledge, this is the only series in current literature with such a large number of patients undergoing spongioplasty alone with TIP repair.</p></sec><sec sec-type="conclusions" id="sec1-5"><title>CONCLUSIONS</title><p>A more proximal hypospadias is associated with poorer spongiosum and this is likely to increase the chances of complications. Better developed and thicker spongiosum results in lower incidence of fistula formation after TIPU. Spongioplasty reconstructs a near normal urethra as per anatomical principles of surgery with least complications. Finally, it adds an extra layer of locally available healthy vascular tissue avoiding dissection for local flaps. We recommend spongioplasty be incorporated as an essential step in all patients undergoing TIP repair for hypospadias.</p></sec> |
Current concepts in robotic radical prostatectomy | Could not extract abstract | <contrib contrib-type="author"><name><surname>Gupta</surname><given-names>Narmada P</given-names></name><xref ref-type="aff" rid="aff1"/></contrib> | Indian Journal of Urology : IJU : Journal of the Urological Society of India | <p>The pioneering contributions of Dr Patrick Walsh on the anatomic dissection for preservation of the neurovascular bundles (NVBs) remain one of the most significant landmarks in urological history. He also described “Anatomical Radical Prostatectomy” on the basis of the venous drainage, arterial and nerve supply and fascial relations of the prostate. As a result, radical prostatectomy (RP) has become the treatment of choice for patients with clinically localized prostate cancer and life expectancy of more than 10 years. With the development of minimally invasive surgery came laparoscopic radical prostatectomy (LRP) in 1994 and robotic-assisted laparoscopic radical prostatectomy (RALP) in 2000. Because of the technical difficulties and a steep learning curve, LRP did not become popular. The dexterity of the robot facilitated complex reconstruction deep in the pelvis, reproducing and often surpassing open surgical techniques. The feasibility and safety of the procedure has been well documented. However, techniques continue to be refined to improve functional and oncological outcomes. Today, in the US, more than 80% of RPs are performed robotically. Whether this is media and marketing hype or truly better outcomes will be decided in the future. Whatever be the technology, it is the surgeon's skill and experience that determine outcomes.</p><p>In India, the scenario is different. In the past, most patients were diagnosed in advanced stages, and treatment was bilateral orchidectomy. With an increase in the longevity of life, education, awareness and availability of prostate-specific antigen testing, the number of patients of cancer prostate is increasing every year. Cancer registries in India are reporting prostate cancer as becoming more common in men.[<xref rid="ref1" ref-type="bibr">1</xref>] There is also a stage migration, and early cases in younger men are being diagnosed. This increases the responsibility of the treating doctors vis-à-vis proper planning and management so that these patients can have prolonged survival with a good quality of life. RALP started in India in 2006. Short-term data on outcome have been published but long-term results are awaited.[<xref rid="ref2" ref-type="bibr">2</xref>]</p><p>There are several unanswered questions regarding RP. Patient selection, role in low risk, intermediate- and high-risk disease, role in salvage situations, problems of surgery in narrow pelvis and obese individuals, role of lymphadenectomy, functional outcomes like incontinence and erectile dysfunction - all need further evaluation and discussion. In this issue of the Journal, we have invited experts to answer the above questions. They have reviewed and analyzed the literature and provide current perspective in their respective fields. We are grateful to them for these endeavors. I hope that these articles will benefit the readers and help them update themselves on RP.</p> |
Newer concepts in neural anatomy and neurovascular preservation in robotic radical prostatectomy | <p>With more than 60% of radical prostatectomies being performed robotically, robotic-assisted laparoscopic prostatectomy (RALP) has largely replaced the open and laparoscopic approaches and has become the standard of care surgical treatment option for localized prostate cancer in the United States. Accomplishing negative surgical margins while preserving functional outcomes of sexual function and continence play a significant role in determining the success of surgical intervention, particularly since the advent of nerve-sparing (NS) robotic prostatectomy. Recent evidence suggests that NS surgery improves continence in addition to sexual function. In this review, we describe the neuroanatomical concepts and recent developments in the NS technique of RALP with a view to improving the “trifecta” outcomes.</p> | <contrib contrib-type="author"><name><surname>Pisipati</surname><given-names>Sailaja</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Ali</surname><given-names>Adnan</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Mandalapu</surname><given-names>Rao S.</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Haines III</surname><given-names>George K.</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib><contrib contrib-type="author"><name><surname>Singhal</surname><given-names>Paras</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Reddy</surname><given-names>Balaji N.</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Leung</surname><given-names>Robert</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Tewari</surname><given-names>Ashutosh K.</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib> | Indian Journal of Urology : IJU : Journal of the Urological Society of India | <sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>The introduction of prostate-specific antigen (PSA) screening over the last two decades has resulted in stage migration of prostate cancer.[<xref rid="ref1" ref-type="bibr">1</xref>] Radical prostatectomy (RP) for organ-confined prostate cancer is an effective treatment option but can result in erectile dysfunction (ED) and incontinence in a significant proportion of patients. The reported rates of post-operative potency vary widely from 21% to 86%.[<xref rid="ref2" ref-type="bibr">2</xref>] The prevalence of urinary incontinence following RALP ranges from 4% to 31%.[<xref rid="ref3" ref-type="bibr">3</xref>] With an increasing number of men being diagnosed at a younger age, achieving the “trifecta goals” is of utmost importance to improving the quality of life. Accomplishing negative surgical margins while preserving functional outcomes of sexual function and continence plays a significant role in determining the success of surgical intervention, particularly since the advent of nerve sparing (NS) RP. With more than 60% of RP being performed robotically, robot-assisted laparoscopic prostatectomy (RALP) has largely replaced the standard open radical prostatectomy (ORP) and laparoscopic radical prostatectomy (LRP) as a surgical treatment option for prostate cancer.[<xref rid="ref4" ref-type="bibr">4</xref>] A meta-analysis by Tewari <italic>et al</italic>. in 2012 has demonstrated that margin rates are comparable between RALP and ORP, with LRP demonstrating an increased risk for positive surgical margins (PSM). The meta-analysis also revealed that the robotic approach is the safest in terms of perioperative complications.[<xref rid="ref5" ref-type="bibr">5</xref>] Recent evidence suggests that NS not only improves sexual function but also enhances continence recovery.[<xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref7" ref-type="bibr">7</xref>] It is in the pursuit of these improved oncological outcomes along with preservation of sexual function and continence that RALP can have the greatest impact. In this review, we describe the neuroanatomical concepts and recent developments in the NS technique of RALP with a view to improving the “trifecta” outcomes.</p><sec id="sec2-1"><title/><sec id="sec3-1"><title>Prostatic neuro-anatomy re-visited</title><p>The pioneering contributions of Walsh <italic>et al</italic>. in 1982 on the anatomic dissection for preservation of the neurovascular bundles (NVBs) remains one of the most significant landmarks in urological history.[<xref rid="ref8" ref-type="bibr">8</xref>] It is well known that autonomic nerves contribute to penile erection. The inferior hypogastric plexus, comprised of the sympathetic fibers from T11-L2 ganglia and the parasympathetics from the ventral rami of S2-S4 spinal nerves, sends efferents to the pelvic viscera. The parasympathetics from this plexus situated behind the rectum travel past the seminal vesicles and along the posterolateral aspect of the prostate and descend posterior and lateral to the urethra before they penetrate the urogenital diaphragm to continue posterior to the dorsal penile artery.[<xref rid="ref8" ref-type="bibr">8</xref>] Because of the reversal of steps of RP with the robotic approach and the advantages of RALP that include seven degrees of freedom, improved ergonomics and three-dimensional vision, the anatomical foundations of the neuronal architecture were re-visited by Tewari <italic>et al</italic>.[<xref rid="ref9" ref-type="bibr">9</xref>] The course of the NVBs from its origin in the pelvic plexus down to its course along the urethra using the minimally invasive approach was studied using cadaveric models.[<xref rid="ref9" ref-type="bibr">9</xref>] The NVBs were found to be situated typically in an anterolateral location, but were found to occupy the posterolateral aspect on rare occasions.[<xref rid="ref10" ref-type="bibr">10</xref><xref rid="ref11" ref-type="bibr">11</xref>] Tewari <italic>et al</italic>. described that this network of interconnecting neural fibers around the prostate and seminal vesicles was arranged as a hammock in a trizonal distribution.[<xref rid="ref12" ref-type="bibr">12</xref>] Takenaka <italic>et al</italic>. described the fan-like distribution of the parasympathetic fibers lying about 2 cm distal to the prostato–vesical junction on the posterolateral aspect of the prostate.[<xref rid="ref13" ref-type="bibr">13</xref>] Takenaka and Tewari <italic>et al</italic>. have also demonstrated the presence and the distribution of the autonomic ganglion cells in the pelvic plexus and around the bladder and the prostate.[<xref rid="ref9" ref-type="bibr">9</xref><xref rid="ref14" ref-type="bibr">14</xref>] Additionally, the NVBs were found to be occupying a potential avascular triangular space bounded by the anterior layer of the Denonvillier's fascia posteriorly, prostatic fascia medially and the lateral pelvic fascia laterally.[<xref rid="ref9" ref-type="bibr">9</xref>] Additional erectile nerves in the “Veil of Aphrodite” along the anterolateral aspect of the prostate were identified by Menon and colleagues.[<xref rid="ref11" ref-type="bibr">11</xref>] Costello <italic>et al</italic>. identified that the array of nerve fibers coursing along the posterolateral aspect of the prostate inferior to the tip of the seminal vesicles formed a more well-defined bundle at the mid-prostatic position before re-diverging near the apex.[<xref rid="ref15" ref-type="bibr">15</xref>] According to another report, a reasonable amount of nerves were identified on the ventral aspect of the prostate in addition to the classical posterolateral location.[<xref rid="ref16" ref-type="bibr">16</xref>]</p><p>Trizonal neural architecture</p><p>The “trizonal” neural architecture is comprised of the proximal neurovascular plate (PNP), predominant neurovascular bundle (PNB) and accessory neural pathways (ANP) arranged around the prostate as a neural hammock [<xref ref-type="fig" rid="F1">Figure 1</xref>].[<xref rid="ref12" ref-type="bibr">12</xref><xref rid="ref13" ref-type="bibr">13</xref>]</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>The trizonal neural network comprising of the proximal neurovascular plate (PNP), the predominant neurovascular bundles (PNB) and accessory neural pathways (ANP) form a neural hammock around the prostate. Medical animation representing the neural hammock</p></caption><graphic xlink:href="IJU-30-399-g001"/></fig><p>The PNP is situated lateral to the bladder neck and seminal vesicles (SV) and is intermingled with branches of the inferior vesical vasculature. It is located 5 mm lateral to the SV, within 6 mm of the bladder neck, 5 mm of the endopelvic fascia and overlaps 5 mm of the prostate. It measures 3 mm thick, 7 mm wide and 9 mm long. It is the integrating center for the processing and relaying of erectogenic neural impulses and is prone to injury during incision of the endopelvic fascia, incision of the prostate-vesical junction, lateral dissection of the SV, application of a bulldog clamp and division of prostatic pedicles.[<xref rid="ref12" ref-type="bibr">12</xref>]</p><p>Enclosed within the layers of the levator fascia and/or lateral pelvic fascia and within the groove between the prostate and the rectum lies the PNB, which carries neural impulses to the erectile tissue and exhibits a variable course, shape and size. The PNB was thickest at the base, and was most variable in course and architecture near the apex. In 66% of the cases, a medial extension was noted behind the prostate, which converged medially at the apex in 33% of the cases. The ganglion cells in the PNB are attached to the prostatic capsule or embedded within the capsule; hence, the need for cautious, athermal dissection to avoid injury.[<xref rid="ref12" ref-type="bibr">12</xref>] PNBs are prone to injury during the dissection of endopelvic fascia, controlling the pedicles, during release of NVBs, apical transection and urethral anastomosis.</p><p>ANPs are putative accessory neural pathways within the layers of levator and/or lateral pelvic fascia, on the anterolateral (42%) and occasionally on the posterolateral aspect of the prostate (25%). ANPs may be damaged during dissection of the endopelvic fascia, suturing the dorsal venous complex while applying back-bleeding suture during posterior dissection, controlling the pedicle, release of NVBs, during apical transection or while performing urethral anastomosis.[<xref rid="ref12" ref-type="bibr">12</xref>]</p><p>The steps of RALP during which each of these trizonal neural structures are likely to be injured and the preventive measures to be taken to avoid such injury have been previously described by Tewari <italic>et al</italic>.[<xref rid="ref12" ref-type="bibr">12</xref>] Establishing a clear anatomical map of the neurovascular structures related to RALP has empowered surgeons with the knowledge to enhance their NS technique, which is crucial for functional recovery.</p></sec><sec id="sec3-2"><title>Techniques to preserve NVB</title><p>The majority of the fibers of the NVBs lie in between the prostatic fascia (medial layer of the lateral pelvic fascia) and the levator fascia (lateral layer of the lateral pelvic fascia). The venous/vascular layer acts as a landmark during NS surgery. Various terminologies have been coined to describe the incisions through the planes around the prostate during NS RALP.</p><p>The veil of Aphrodite technique proposed by Menon involves anterior incision of the prostatic fascia to enter the plane between the prostatic capsule and the prostatic fascia. This follows the posterior and the posterolateral dissection in the plane between the prostatic fascia and the Denonvillier's fascia. The periprostatic tissue hanging from the bladder neck down toward the urethro–apical junction is known as the “Veil of Aphrodite.”[<xref rid="ref17" ref-type="bibr">17</xref>] The technique was refined in 2009, in which the anterior prostatic fascial tissue, adherent to the capsule, dorsal venous complex and the pubovesical ligaments, was preserved. Adopting this modification known as the “superveil” technique, it was believed that the nerves interposed between the 11-o’ clock and the 1-o’ clock positions were preserved.[<xref rid="ref18" ref-type="bibr">18</xref>]</p><p>Another conventional nomenclature used for the NS approach is the intrafascial, intrafascial and extrafascial approach [<xref ref-type="table" rid="T1">Table 1</xref>]. However, this classification system is slowly being replaced by the graded NS approach.</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Intrafascial, interfascial and extrafascial approach</p></caption><graphic xlink:href="IJU-30-399-g002"/></table-wrap><p>We now routinely adopt the athermal, traction-free, risk-stratified graded NS approach described below to optimize our oncological and functional outcomes.</p><p>Athermal technique</p><p>Various modifications have been made to the technique of NS RALP over the last decade. In an attempt to minimize thermal and ischemic damage to the delicate neurovascular tissues, Tewari <italic>et al</italic>. introduced the concept of “Athermal Robotic technique” (ART) in 2005.[<xref rid="ref19" ref-type="bibr">19</xref>] Based on the understanding of the trizonal neural anatomy and by adopting the ART, 45% sexual function was achieved at 6 weeks.[<xref rid="ref20" ref-type="bibr">20</xref>] Technical feasibility of the athermal technique has been proven even in large prostates.[<xref rid="ref21" ref-type="bibr">21</xref>] In a study of 215 patients who had RALP, 87% of those who had bilateral NS, are <70 years and were pre-operatively potent, were potent at 1 year following surgery. The overall PSM rate was 6.5%.[<xref rid="ref22" ref-type="bibr">22</xref>]</p><p>In a study comparing monopolar cautery, bipolar cautery and a cautery-free technique, Ahlering <italic>et al</italic>. noticed nearly a five-fold improvement of potency recovery at 3 and 9 months with the cautery-free approach.[<xref rid="ref23" ref-type="bibr">23</xref>] A cumulative analysis of eight studies in a systematic review by Ficarra <italic>et al</italic>. revealed better potency outcomes with the athermal NS technique at 3, 6 and 12 months.[<xref rid="ref24" ref-type="bibr">24</xref>] It is hence believed that avoiding/minimizing thermal energy, particularly while dissecting the NVBs, results in better functional outcomes.</p><p>Traction-Free technique</p><p>Another modification adopted to the NS technique is a traction-free approach.[<xref rid="ref19" ref-type="bibr">19</xref>] Undue stretch on the NVB causes mechanical trauma resulting in axonotemesis and disruption of the vasa nervorum thus resulting in neural and vascular insults. In addition, tissue hypoxia can result due to injury to the accessory pudendal arteries that run along the anterolateral surface of the bladder and the prostate in 70% of the cases.[<xref rid="ref25" ref-type="bibr">25</xref>] Such vascular insults account for hypoxia, nutrient deficiency, free radical formation and accumulation of neurotoxic elements that result in ischemia and delayed recovery. It is hypothesized that reducing or avoiding traction on the NVB minimizes the chances of stretch-induced axonotemesis and tissue hypoxia thus resulting in better functional outcomes. The lack of tactile feedback in robotic surgery poses a challenge in detecting excessive traction placed on the neurovasculature during the procedure. In an attempt to overcome this drawback, Tewari <italic>et al</italic>. developed the concept of real-time intraoperative penile oxygenation monitoring as a surrogate for identifying traction. This involved the use of an auditory probe that provided feedback in the form of an alarm when the tissue oxygenation dropped below 85%. Subtle, deliberate modifications to certain steps during the procedure, based on this auditory feedback, resulted in maintenance of penile oxygenation at or above 85% during the surgery.[<xref rid="ref26" ref-type="bibr">26</xref>] Using this device, Tewari <italic>et al</italic>. reported that a significantly higher proportion of patients with bilateral NS in the study group had no ED when compared with the control group at 6 and 52 weeks post-RALP. 93.9% and 78.4% of patients in the study and control groups, respectively, had a SHIM score ≥17 at 1 year. The overall PSM rates in the study and control groups were 9.4% and 9.9%, respectively. Feedback obtained by real-time tissue oxygen monitoring has allowed subtle technical adjustments thus amounting to improved functional outcomes.</p><p>Risk-stratified graded NS</p><p>The concept of graded NS approach to improve sexual outcomes was introduced in 2008.[<xref rid="ref27" ref-type="bibr">27</xref>] In an attempt to balance the competing goals of oncological cure and sexual recovery, a “novel” risk stratification strategy has been proposed[<xref rid="ref28" ref-type="bibr">28</xref><xref rid="ref29" ref-type="bibr">29</xref>] [Figures <xref ref-type="fig" rid="F2">2</xref>–<xref ref-type="fig" rid="F4">4</xref>] [<xref ref-type="table" rid="T2">Table 2</xref>]. Based on several pre-operative parameters including PSA, clinical stage, Gleason grade on biopsy and pre-operative magnetic resonance imaging (MRI) findings, patients are categorized into one of four risk grades, where risk grade 1 patients receive NS grade 1 and so on for risk grades 2-4. By adopting this risk-stratified approach for neural hammock preservation during RALP, Tewari <italic>et al</italic>. were able to improve potency outcomes without compromising oncological outcomes in a cohort of 1263 patients. The authors reported higher rates of intercourse (90.9% and 62% for grades 1 and 4 NS, respectively) and return to baseline sexual function (81.7% and 54.4% for grades 1 and 4, respectively) in patients who had greater degrees (lower grades) of NS. The overall PSM rates for patients with NS grades 1, 2, 3 and 4 were 9.9%, 8.1%, 7.2% and 8.7%, respectively (<italic>P</italic> = 0.64). With increasing degree of NS, PSM rates were not significantly elevated; potency outcomes, however, were significantly better.[<xref rid="ref30" ref-type="bibr">30</xref>]</p><fig id="F2" position="float"><label>Figure 2</label><caption><p>Planes of dissection for nerve sparing grades 1–4. (a) Medical animation. (b) Diagrammatic representation of the layers of fascia enveloping the prostatic capsule, showing the planes of dissection. LPF = lateral pelvic fascia medial layer, i.e., prostatic fascia; LF = lateral pelvic fascia lateral layer, i.e., levator fascia; LA = levator ani. B – Reproduced with permission from [<xref rid="ref24" ref-type="bibr">24</xref>]</p></caption><graphic xlink:href="IJU-30-399-g003"/></fig><fig id="F3" position="float"><label>Figure 3</label><caption><p>Risk stratification algorithm for athermal nerve sparing robotic radical prostatectomy. (ECE = extracapsular extension; mp MRI = multiparametric magnetic resonance imaging)</p></caption><graphic xlink:href="IJU-30-399-g004"/></fig><fig id="F4" position="float"><label>Figure 4</label><caption><p>Pre-operative multiparametric magnetic resonance imaging in risk stratification and intraoperative planning for graded nerve spare, intraoperative views and corresponding histology from the edge for Grade 1 (a,b,c), Grade 2 (d, e, f), Grade 3 (g, h, i) and Grade 4 (j, k, l) nerve spare. (c – capsule; bv – blood vessel; f – periprostatic fat; n – nerve bundle). The red arrow in J points to the site of extracapsular extension</p></caption><graphic xlink:href="IJU-30-399-g005"/></fig><table-wrap id="T2" position="float"><label>Table 2</label><caption><p>Grades of nerve sparing robotic radical prostatectomy</p></caption><graphic xlink:href="IJU-30-399-g006"/></table-wrap><p>Schatloff <italic>et al</italic> described a five-point NS grading system based on intraoperative visual cues. According to their system, a non-NS procedure was assigned grade 1 and the best NS (≥95%) was graded as 5, with <50%, 50% and 75% NS being coded as grades 2, 3 and 4, respectively.[<xref rid="ref31" ref-type="bibr">31</xref>] According to a subjective model of a four-point NS score (NSS) grading system proposed by Moskovic <italic>et al</italic>., where NSS 1 meant complete preservation (i.e. full NS) and 4 was complete resection (i.e. non-NS), a lower NSS was one of the independent predictors of sexual function recovery at 24 months.[<xref rid="ref32" ref-type="bibr">32</xref>]</p><p>Available data support the fact that cavernosal preservation during RALP is no longer an “all or none” phenomenon, but is a graded entity. The grading system enables the surgeon to achieve better “trifecta” outcomes.</p><p>Intra-operative frozen sections</p><p>RP involves interplay between competing goals of cancer extirpation, NS, post-operative recovery of urinary continence and potency. This requires precise dissection in an appropriate plane to achieve effective cancer control by avoiding PSMs and performing adequate NS concurrently. At present, there is no consensus on the use of frozen sections during RP. Various studies have gauged the utility of intraoperative frozen section analysis performed at different sites of the prostate/periprostatic soft tissue, and the results vary widely. This variation is mostly due to differences in sampling methods (sites or whole margins) and sites. Neurovascular structure–adjacent frozen section examination (NeuroSAFE) technique of intraoperative frozen section (IFS) analysis allows real-time histological evaluation and helps in performing a NS procedure without compromising oncological safety. Schlomm <italic>et al</italic>. demonstrated in a cohort of 11,069 patients a feasible intraoperative technique of NeuroSAFE.[<xref rid="ref33" ref-type="bibr">33</xref>] This technique enables real-time histologic monitoring of the oncologic safety of an NS procedure. Systematic NeuroSAFE was reported to significantly increase NS frequency and reduces PSMs. Also, patients with a NeuroSAFE-detected PSM were converted to a prognostically more favorable NSM status by secondary wide resection. In this study, a false-negative IFS result was reported in 2.5% of the cases. In this context, our group is currently developing an MRI-guided intraoperative frozen section technique [<xref ref-type="fig" rid="F5">Figure 5</xref>]. The preliminary results will be available in the near future.</p><fig id="F5" position="float"><label>Figure 5</label><caption><p>Magnetic resonance imaging (MRI)-guided intraoperative frozen section. (a) T2-weighted MRI and (b) diffusion-weighted imaging (DWI) demonstrating a left-sided lesion (marked in green). (c,d) A radical prostatectomy specimen (P) with left lateral margin transected for intraoperative frozen section (left true margin marked in blue, false left margin in yellow inked as red at the apex). (e) Intraoperative frozen section from the left margin (blue and yellow represent true and false margins, respectively). (B = base; A = apex; SV = seminal vesicles)</p></caption><graphic xlink:href="IJU-30-399-g007"/></fig></sec><sec id="sec3-3"><title>Imaging and nerve mapping techniques to identify the NVB</title><p>Localization by imaging modalities</p><p>Infiltration of the neoplastic cells around the cavernosal nerve fibers and extraprostatic extension are microscopic phenomena that cannot be visualized intraoperatively even with the ×10-12 magnification of the stereoscope of the daVinci system. The inability to identify malignant cells and their association with nerves can result in incomplete removal of the cancerous tissue resulting in PSM, post-operative impotence due to damage to/excision of the cavernous nerves or a combination of both. In order to maintain the delicate balance between oncological control and preserving functional outcomes, it is crucial for the surgeon to be able to identify and better define the NVB in relation to the prostate in real-time. Optical magnification with surgical loupes, intraoperative nerve stimulation and real-time robotic transrectal ultrasound (TRUS Robot) have been attempted.</p><p>Diffusion tensor magnetic resonance imaging (DTI)</p><p>DTI is an emerging technology to facilitate treatment planning. It is based on the sensitivity of the water protons measured in the microstructural environment.[<xref rid="ref34" ref-type="bibr">34</xref>] The main quantitative measurements of DTI include average diffusivity and fractional anisotropy.[<xref rid="ref35" ref-type="bibr">35</xref>] DTI, currently used for neuroimaging applications, enables tracing of the periprostatic nerves. Its utility in human prostates was first reported by Sinha in 2004.[<xref rid="ref36" ref-type="bibr">36</xref>] In a recent study using DTI along with mp MRI, the authors demonstrated that of DTI, 2D-T2-weighted MRI and 3D-T2-weighted MRI, only DTI fiber tracking allowed assessment of the entire periprostatic nervous plexus and of all the fibers bilaterally at all levels in all the 33 patients included in the study. The authors concluded that this information could be useful for guiding proper NS surgery using an intrafascial or extrafascial robotic approach[<xref rid="ref37" ref-type="bibr">37</xref>] or even the graded NS approach, thereby ensuring recovery of erectile function after RP. <xref ref-type="fig" rid="F6">Figure 6</xref> depicts the fiber tracts from an <italic>ex vivo</italic> robotic radical prostatectomy specimen using high-resolution DTI. DTI seems to have a promising role in the future for NVB preservation during RP.</p><fig id="F6" position="float"><label>Figure 6</label><caption><p><italic>Ex vivo</italic> diffusion tensor magnetic resonance imaging obtained from robotic radical prostatectomy specimen. The various colors represent the fibers in and around the prostate</p></caption><graphic xlink:href="IJU-30-399-g008"/></fig><p>Multi-photon microscopy (MPM)</p><p>Access to high-resolution real-time imaging of the prostatic capsule, apex, sphincter and the surrounding neurovascular structures is likely to improve oncological and functional outcomes. MPM is one such novel optical imaging technology that relies on the simultaneous absorption of two or three low-energy (near-infrared) photons to cause a non-linear excitation, which reduces the potential for cellular damage.[<xref rid="ref38" ref-type="bibr">38</xref>] By adopting a stepwise approach for imaging, researchers were able to identify the cavernous nerve, major pelvic ganglion, prostatic capsule, prostatic acini, fat, vessels and pathological changes in rat models and <italic>ex vivo</italic> human prostatectomy specimens[<xref rid="ref38" ref-type="bibr">38</xref><xref rid="ref39" ref-type="bibr">39</xref>] [<xref ref-type="fig" rid="F7">Figure 7</xref>]. “Real-time tissue imaging” may help surgeons to localize the nerves in relation to the cancerous tissues and potentially identify possible perineural invasion and extraprostatic extension in real time. This would then minimize nerve damage thus enhancing sexual outcomes and reduce the incidence of PSM. Like most technological innovations, MPM imaging will have to face several potential challenges before it can be integrated into real-time applications.</p><fig id="F7" position="float"><label>Figure 7</label><caption><p>Multi-photon microscopy (MPM) images from ex vivo human prostate. (a,b) Lateral pelvic fascia showing a large artery (a), fibrocollagenous connective tissue stroma (s) and fat (c) on MPM image (A) and histology slide (B). Note the empty lumen of the artery in A and B. (c) Surgical apical margin showing a small nerve (arrow). Small arrowhead points to collagen and the large arrowhead points to elastin in the connective tissue stroma. (d) Surgical apical margin showing a small artery. Note the empty lumen (arrow) as opposed to the wavy nerve fibers in C and E. (e) Higher magnification image of a small nerve bundle at the surgical margin showing fluorescence that derives from the axoplasm or cytoplasm of the Schwann cells. (f) Prostatic capsule showing an underlying prostatic acinus (pa), capsule (c), periprostatic connective tissue (s) and fat (f). (g,h) Higher magnification of prostatic acini imaged using three detector channels. Cells emit mostly in the 420-530 nm range and thus appear green in the color-coding scheme. By contrast, the gland-associated punctate fluorescence (which could represent lipofuscin deposits) emits over a broader wavelength range and thus appears blue in the color-coding scheme (arrows point to bona fide cells with distinct nuclei). Color-coding of MPM images: Red, second harmonic generation (SHG) (355-420 nm); green, short-wavelength autofluorescence (420-530 nm); blue, long-wavelength autofluorescence (530-650 nm). Scale bars: A, C, D, H 500 μm; E 67 μm; F, G 100 μm [Reproduced and edited with permission from 31]</p></caption><graphic xlink:href="IJU-30-399-g009"/></fig><p>Optical coherence tomography (OCT)</p><p>OCT provides real-time, high-resolution, cross-sectional tissue imaging by measuring the back scatter near-infrared radiation. It is non-invasive and the energy utilized does not cause mechanical damage. Given its fiberoptic nature delivery system, portability and low cost, it can readily be integrated into endoscopic/laparoscopic surgical equipment and probes. The drawback, however, is the inadequate resolution quality for tissues >1 mm deep. Using OCT, the cavernous nerve was distinguished as an intense linear structure separate from the adjacent tissues in <italic>in vivo</italic> experiments on Sprague–Dawley rats; however, the discrimination between adjacent prostatic tissues and nerves was not adequate in <italic>ex vivo</italic> human prostatectomy specimens.[<xref rid="ref40" ref-type="bibr">40</xref>] In a feasibility study by Dangle <italic>et al</italic>., in which OCT was used on 100 <italic>ex vivo</italic> human prostatectomy specimens to identify PSM and extraprostatic extension (EPE), the results were compared against the gold standard histopathology. The reported sensitivity and specificity for PSM were 70% and 84%, respectively, with 33% and 96% positive predictive value (PPV) and negative predictive value (NPV). The sensitivity, specificity, PPV and NPV for EPE and SV invasion (SVI) were 46%, 84%, 50%, 92% and 33%, 97%, 33% and 97%, respectively. This study established the template for the visual OCT characteristics of the prostate, SV and cancerous tissue. With its high NPV, OCT could be useful to rule out PSM, EPE and SVI.[<xref rid="ref41" ref-type="bibr">41</xref>] In vivo studies are needed to prove its efficacy in real-time imaging intraoperatively. Beuvon <italic>et al</italic>. have recently tested the feasibility of OCT in prostate biopsies for diagnostic purposes and reported a 81% concordance with histopathological findings.[<xref rid="ref42" ref-type="bibr">42</xref>] OCT might have a potential role in the future both in the diagnostic and the therapeutic pathways.</p><p>Localization by physiological stimulation</p><p>A number of nerve mapping technologies have been investigated to aid in localization of periprostatic nerves for augmenting NS during radical prostatectomy. Mapping is usually performed by stimulating a nerve either by optical or by electrical means and then detecting a physiologic response, such as penile tumescence/detumescence, intracavernosal pressure, intraurethral pressure, impedance or an action potential. Examples of such devices include CaverMap,[<xref rid="ref43" ref-type="bibr">43</xref>] ProPep, NIMEclipse and optical nerve monitoring. <xref ref-type="table" rid="T3">Table 3</xref> describes the currently available nerve stimulation devices.</p><table-wrap id="T3" position="float"><label>Table 3</label><caption><p>Characteristics of the nerve stimulation devices</p></caption><graphic xlink:href="IJU-30-399-g010"/></table-wrap><p>Dye-based visualization</p><p>Nerves can be stained using fluorescent dyes and can be identified based on specific characteristics such as the method of delivery, nerve specificity, time for staining and resolution. When applied directly by local infiltration into the base of the penis, these dyes travel via the retrograde transport mechanism along the erectile nerves. Systemic administration of the dye results in labeling of all or most of the nerves, and hence the labeled nerves may not be responsible for erectile function. Local injections have a limited utility as well, as they label only one nerve fiber tract at a time. In addition, axonal transport is a slow process and can take a long time, sometimes up to several months. Currently, indocyanine green and fluorescein are the only FDA-approved dyes that have been studied in RP. Other examples of nerve dyes include compounds from Avelas and General Electric, fluorescent cholera toxin subunit B, indocyanine green, fluorescent-inactivated herpes simplex 2 and Fluoro-Gold.[<xref rid="ref48" ref-type="bibr">48</xref>]</p><p>Neurovascular bundle reconstruction</p><p>Wide excision of the NVBs is a prudent approach followed by most surgeons when there is a high index of suspicion of ECE and NVB invasion by tumor based on pre-operative parameters. However, there is no consensus on the ideal method for NVB reconstruction. While there are a few existing options to choose from, namely sural nerve grafting,[<xref rid="ref49" ref-type="bibr">49</xref>] use of embryonic stem cells or growth factors to enhance neural regeneration,[<xref rid="ref50" ref-type="bibr">50</xref>] entubulization model of cavernosal nerve[<xref rid="ref51" ref-type="bibr">51</xref>] or nerve advancement with end-to-end reconstruction,[<xref rid="ref52" ref-type="bibr">52</xref>] none of them are employed routinely.</p><p>Nerve advancement technique (NAT) is one such technique based on neuroscientific concepts of peripheral nerve repair that attempts to establish continuity between the proximal and distal nerve stumps by end-to-end anastomosis of the partially resected NVB [<xref ref-type="fig" rid="F8">Figure 8</xref>]. Geuna <italic>et al</italic>. and Terzis <italic>et al</italic>. reported that if continuity is restored by end-to-end suturing, bands of Bungner arise from axons upstream of the point of transection and grow along the glial columns in the distal nerve stump to eventually re-innervate the denervated structures.[<xref rid="ref53" ref-type="bibr">53</xref><xref rid="ref54" ref-type="bibr">54</xref>] Tewari <italic>et al</italic>. performed nerve advancement and end-to-end, tension-free anastomosis of the proximal and distal neural stumps following partial resection of NVBs in a pilot study of seven pre-operatively potent, high-risk patients. One patient had a PSM and five of the seven men regained potency. They reported that NAT is technically feasible, oncologically safe and is associated with promising sexual outcomes.[<xref rid="ref52" ref-type="bibr">52</xref>]</p><fig id="F8" position="float"><label>Figure 8</label><caption><p>Nerve advancement technique (NAT). (a) Black arrow heads mark the location of partially resected neurovascular bundle. (b) Black arrow heads mark the approximated ends of neurovascular bundles using the nerve advancement technique (NAT)</p></caption><graphic xlink:href="IJU-30-399-g011"/></fig></sec></sec></sec><sec sec-type="conclusion" id="sec1-2"><title>CONCLUSION</title><p>Much of the success achieved in the last decade in terms of improved trifecta outcomes following robotic radical prostatectomy relates to the adoption of an athermal, traction-free, risk-stratified, graded nerve spare approach to preserve the neural hammock. Innovative techniques to incorporate real-time intraoperative imaging and nerve mapping methodologies to identify and preserve the cavernosal nerves seem to have a challenging but promising role in the future.</p></sec> |
Role of robot-assisted radical prostatectomy in the management of high-risk prostate cancer | <p>We aimed to evaluate the role of robot-assisted radical prostatectomy (RARP) in the management of high-risk prostate cancer (PCa), with a focus on oncological, functional and perioperative outcomes. Further, we also aimed to briefly describe our novel modification to conventional RARP that allows immediate organ retrieval and examination for intra-operative surgical margin assessment. A literature search of PubMed was performed for articles on the management of high-risk PCa. Papers written in English and concerning clinical outcomes following RARP for locally advanced and high-risk PCa were selected. Outcomes data from our own center were also included. A total of 10 contemporary series were evaluated. Biopsy Gleason score ≥ 8 was the most common cause for classification of patients into the high-risk PCa group. Biochemical failure rate, in the few series that looked at long-term follow-up, varied from 9% to 26% at 1 year. The positive surgical margin rate varied from 12% to 53.3%. Urinary continence rates varied from 78% to 92% at 1 year. The overall complication rates varied from 2.4% to 30%, with anastomotic leak and lymphocele being the most common complications. Long-term data on oncological control following RARP in high-risk patients is lacking. Short-term oncological outcomes and functional outcomes are equivalent to open radical prostatectomy (RP). Safety outcomes are better in patients undergoing RARP when compared with open RP. Improved tools for predicting the presence of organ-confined disease (OCD) are available. High-risk patients with OCD would be ideal candidates for RARP and would benefit most from surgery alone.</p> | <contrib contrib-type="author"><name><surname>Sood</surname><given-names>Akshay</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Jeong</surname><given-names>Wooju</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Dalela</surname><given-names>Deepansh</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Klett</surname><given-names>Dane E.</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Abdollah</surname><given-names>Firas</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Sammon</surname><given-names>Jesse D.</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Menon</surname><given-names>Mani</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Bhandari</surname><given-names>Mahendra</given-names></name><xref ref-type="aff" rid="aff1"/></contrib> | Indian Journal of Urology : IJU : Journal of the Urological Society of India | <sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Prostate-specific antigen (PSA) screening was introduced in the US in the late 1980s for early detection of prostate cancer (PCa), and has resulted in both an increase in PCa diagnoses and an earlier identification of these tumors. Despite the downward stage migration associated with PSA testing and the growing number of low-risk and organ-confined (OC) tumors, roughly 14.8% and 3.5% of men with newly diagnosed PCa are currently found to have high-risk and locally advanced disease (cT3NX/+M0), respectively.[<xref rid="ref1" ref-type="bibr">1</xref><xref rid="ref2" ref-type="bibr">2</xref>] Although these proportions have declined when compared with the pre-PSA screening era, they remain significant and have remained relatively stable over the past decade. Those men with high-risk PCa at the time of presentation have increased rates of secondary therapy and metastasis and contribute disproportionately to PCa mortality.[<xref rid="ref3" ref-type="bibr">3</xref><xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref5" ref-type="bibr">5</xref>] Improved treatments for such men would have a significant positive impact on overall morbidity and mortality due to this disease.</p><p>There is no consensus on the ideal management of these high-risk PCa patients. Multiple challenges exist in optimally treating men with high-risk disease, including, but not limited to, the biologic behavior of the cancer and the lack of staging accuracy of current diagnostic tools; furthermore, breakdown of various nomograms at these extremes make prognostic and outcome assessment difficult.[<xref rid="ref6" ref-type="bibr">6</xref>] Significant differences may exist within the high-risk group because patients may have anywhere from one to three high-risk features and yet be similarly classified.[<xref rid="ref7" ref-type="bibr">7</xref>]</p><p>With robot-assisted radical prostatectomy (RARP) being increasingly utilized for PCa treatment,[<xref rid="ref8" ref-type="bibr">8</xref><xref rid="ref9" ref-type="bibr">9</xref>] in this review, we discuss oncological, functional and perioperative outcomes following RARP, the general role of surgery for the management of high-risk disease and technical modifications that may help improve patient outcomes.</p></sec><sec sec-type="materials|methods" id="sec1-2"><title>MATERIALS AND METHODS</title><p>A literature search of PubMed was performed to retrieve all published articles on the management of high-risk PCa. We used the search terms “high-risk prostate cancer” AND “radical prostatectomy” OR “robotic radical prostatectomy.” Papers written in English and concerning clinical outcomes following RARP for high-risk PCa were selected. Reference lists of retrieved papers were scrutinized for additional relevant articles. Outcomes data following RARP from our own center were also included.</p><p><xref ref-type="table" rid="T1">Table 1</xref> summarizes the studies evaluating outcomes of RARP in high-risk PCa. We found 10 studies evaluating the role of RARP in high-risk PCa. All studies represent contemporary experiences and were published between 2008 and 2013. In 90% of the studies, data were collected prospectively and the mean cohort size was 104 patients (range 30-160 patients).</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Summary of series evaluating outcomes after robot-assisted radical prostatectomy in patients with high-risk PCa</p></caption><graphic xlink:href="IJU-30-410-g001"/></table-wrap></sec><sec id="sec1-3"><title>RESULTS AND DISCUSSION</title><sec id="sec2-1"><title/><sec id="sec3-1"><title>Definition of high-risk PCa and epidemiology</title><p>High-risk PCa is characterized by the following criteria [D’Amico], either</p><p>
<list list-type="bullet"><list-item><p>PSA value >20 ng/mL or</p></list-item><list-item><p>Biopsy Gleason score (GS) of ≥8 or</p></list-item><list-item><p>Clinical stage (cT) of T2c or above</p></list-item><list-item><p>A combination of either of these.[<xref rid="ref3" ref-type="bibr">3</xref>]</p></list-item></list>
</p><p>The National Comprehensive Cancer Network (NCCN) and UCSF Cancer of the Prostate Risk Assessment (UCSF–CAPRA) classifications are other widely used criteria for defining high-risk PCa [<xref ref-type="fig" rid="F1">Figure 1</xref>]. NCCN differs from the D’Amico criteria with regard to the clinical stage assignment for high risk,[<xref rid="ref20" ref-type="bibr">20</xref>] where clinical stage T3a or higher is considered high risk. In the UCSF–CAPRA classification,[<xref rid="ref21" ref-type="bibr">21</xref>] points (0-4) are assigned to individual risk factors and a score of ≥6 is considered high-risk disease. Most studies included in the current review utilized D’Amico risk stratification (70.0%) for defining high-risk PCa. Nguyen <italic>et al</italic>. compared the predictive value of six different definitions of high-risk PCa (consisting of varying combinations of clinical stage, serum PSA and GS) and found 5-year and 10-year BCRFS to range from 36% to 58% and 25% to 43% respectively, concluding that BCRFS for high-risk PCa did not differ significantly regardless of the definition used.[<xref rid="ref22" ref-type="bibr">22</xref>]</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Different classification schemes of high-risk prostate cancer</p></caption><graphic xlink:href="IJU-30-410-g002"/></fig><p>With respect to epidemiology, as mentioned previously, the overall incidence of high-risk PCa has decreased since the advent of PSA screening, and currently stands at 14.8% as compared with 40.9% in the late 1980s. At present, approximately 9.8%, 8.1% and 3.5% of men newly diagnosed with PCa present with GS ≥ 8, PSA > 20 ng/dL and cT ≥ T2c, respectively.[<xref rid="ref1" ref-type="bibr">1</xref>] Accordingly, GS is currently the major factor determining patient assignment to the high-risk category, followed by PSA and then by clinical stage with 61.5%, 50.8% and 21.9% of high-risk patients having GS ≥ 8, PSA > 20 ng/dL and cT ≥ T2c, respectively.[<xref rid="ref1" ref-type="bibr">1</xref>] This trend was evident in the current review as well, as in all the series reviewed that used > 1 risk factor to define high-risk PCa, 91.6-29.7% of patients had GS ≥ 8 and 30.0-15.9% had a PSA > 20 ng/dL [<xref ref-type="table" rid="T1">Table 1</xref>].</p></sec><sec id="sec3-2"><title>Literature review of outcomes following RARP in high-risk PCa patients</title><p>Oncological outcomes</p><p><xref ref-type="table" rid="T2">Table 2</xref> summarizes the oncological outcomes in patients with high-risk PCa. Long-term follow-up data were not available in all the series. In the studies that did assess long-term oncological control, the rate of biochemical recurrence (BCR) varied from 9% to 26% at 1 year.[<xref rid="ref11" ref-type="bibr">11</xref><xref rid="ref13" ref-type="bibr">13</xref>] At 3 years, it was between 14%[<xref rid="ref11" ref-type="bibr">11</xref>] and 55%.[<xref rid="ref13" ref-type="bibr">13</xref>] Sukumar <italic>et al</italic>. looked at the oncological outcomes of 4803 patients with PCa undergoing RARP, including 1556 patients with non-organ confined disease (OCD).[<xref rid="ref23" ref-type="bibr">23</xref>] They demonstrated a BCR of 18.3% in this group of patients over a period of mean follow-up of 34.6 months (range 1-116.7 months). All studies used a PSA ≥ 0.2 ng/mL as the definition of BCR, except for Shikanov <italic>et al</italic>.,[<xref rid="ref19" ref-type="bibr">19</xref>] who defined BCR as a PSA > 0.1 ng/mL and reported an 18% BCR rate at 1 year.</p><table-wrap id="T2" position="float"><label>Table 2</label><caption><p>Summary of oncological outcomes in series looking at robot-assisted radical prostatectomy outcomes in patients with high-risk PCa</p></caption><graphic xlink:href="IJU-30-410-g003"/></table-wrap><p>The positive surgical margin (PSM) rate varied from 12.0%[<xref rid="ref14" ref-type="bibr">14</xref>] to 53.3%.[<xref rid="ref10" ref-type="bibr">10</xref>] However, in most studies, the PSM rate was between 20% and 30%.[<xref rid="ref12" ref-type="bibr">12</xref><xref rid="ref13" ref-type="bibr">13</xref><xref rid="ref15" ref-type="bibr">15</xref><xref rid="ref17" ref-type="bibr">17</xref><xref rid="ref18" ref-type="bibr">18</xref><xref rid="ref19" ref-type="bibr">19</xref>] The lymph node positivity rate was also widely variable, from 1.4% to 33.3% overall. A 33.3% positivity rate was reported by Yuh <italic>et al</italic>.[<xref rid="ref15" ref-type="bibr">15</xref>] in their study specifically assessing the role of robotic-extended lymph node dissection in intermediate and high-risk PCa, and might reflect the most accurate lymph node involvement rate in this subset of patients. That being said, the PSM and the lymph node positivity results from these reports should be interpreted cautiously as many of these studies did not provide details on the protocol used for sampling radical prostatectomy (RP) specimens and the extent of lymph node dissection performed, both of which are well-know factors affecting the PSM rate[<xref rid="ref24" ref-type="bibr">24</xref>] and lymph node positivity rate.[<xref rid="ref15" ref-type="bibr">15</xref><xref rid="ref25" ref-type="bibr">25</xref>] respectively.</p><p>These BCR and PSM rates are comparable to the results reported by Briganti <italic>et al</italic>. in their review of open RP (ORP) outcomes, where they found a mean BCR rate of 31% at 5 years and a mean PSM rate of 45% following ORP in patients with high-risk disease.[<xref rid="ref26" ref-type="bibr">26</xref>] Harty <italic>et al</italic>. compared the PSM rates for open versus minimally invasive RP in 445 high-risk PCa patients and found that they were not significantly different: 52.9% in ORP, 50% in RARP and 41.4% in laparoscopic RP (LRP) (<italic>P</italic> = 0.13).[<xref rid="ref27" ref-type="bibr">27</xref>] The PSM rate did not differ when comparing ORP with a combined group of RARP and LRP (<italic>P</italic> = 0.16). Similar results were seen by Pierorazio <italic>et al</italic>. when they compared 913 men undergoing open versus minimally invasive RARP and LRP for high-risk PCa at a single center over a 10-year period and found no significant difference in the PSM or BCR rates across the groups.[<xref rid="ref28" ref-type="bibr">28</xref>]</p><p>Sukumar <italic>et al</italic>., in the study mentioned previously, looked at 99 patients with node-positive disease (N1), which, even though not strictly classified under high-risk PCa, represents an aggressive cohort with a relatively high risk of BCR.[<xref rid="ref23" ref-type="bibr">23</xref>] In their series, this group had a mean PSA of 12.3 ng/mL; 46.5% of them had a biopsy GS of 8-10 and 15.2% had cT2c-cT3 disease. BCR for this cohort was 58.6% over a mean follow-up of 34.6 months (range 1-116.7 months). Actuarial 5-year BCR-free survival, metastasis-free survival and cancer-specific survival (CSS) were 26.3% (SE: 7.3), 77.7% (SE: 11.7) and 96.1% (SE: 2.7), respectively, for these patients with N1 disease.</p><p>Functional outcomes</p><p><xref ref-type="table" rid="T3">Table 3</xref> summarizes the functional outcomes. The 12-month continence rates using a 0-1 safety pad definition varied from 78% to 92%.[<xref rid="ref13" ref-type="bibr">13</xref><xref rid="ref18" ref-type="bibr">18</xref>] Ou <italic>et al</italic>., using a 0 pad definition for continence, reported a 95% continence rate in men undergoing RARP for high-risk disease.[<xref rid="ref10" ref-type="bibr">10</xref>] Potency rates varied from 52% to 60% at 12 months.[<xref rid="ref10" ref-type="bibr">10</xref><xref rid="ref17" ref-type="bibr">17</xref>] Rogers <italic>et al</italic>. reported a 33.3% potency rate at 26 months, but their study population was aged ≥ 70 years.[<xref rid="ref11" ref-type="bibr">11</xref>] In select patients with high-risk disease who underwent bilateral nerve sparing, Ou <italic>et al</italic>. found potency rates of 71%.[<xref rid="ref10" ref-type="bibr">10</xref>]</p><table-wrap id="T3" position="float"><label>Table 3</label><caption><p>Summary of functional outcomes in series looking at robot-assisted radical prostatectomy outcomes in patients with high-risk PCa</p></caption><graphic xlink:href="IJU-30-410-g004"/></table-wrap><p>Perioperative outcomes</p><p><xref ref-type="table" rid="T4">Table 4</xref> provides details on the perioperative outcomes of RARP. The mean operative time was 168 min, with operative times ranging from 111 to 186 min and mean estimated blood loss was 189 mL (range 84-200 mL). Length of hospital stay (LOS) varied from 1 to 3.4 days. The overall complication rates ranged from 2.4% to 30%, although many series did not fulfill the Martin criteria requirement for reporting of complications,[<xref rid="ref29" ref-type="bibr">29</xref>] and this could lead to underreporting of adverse events. The most common complications reported were urethrovesical anastomotic leaks in approximately 10% of the patients,[<xref rid="ref15" ref-type="bibr">15</xref>] lymphocele (range 6.6-2.4%)[<xref rid="ref14" ref-type="bibr">14</xref><xref rid="ref15" ref-type="bibr">15</xref>] and deep venous thrombosis in approximately 3.3% of patients.[<xref rid="ref15" ref-type="bibr">15</xref>] Rectal injuries were extremely rare.[<xref rid="ref15" ref-type="bibr">15</xref>]</p><table-wrap id="T4" position="float"><label>Table 4</label><caption><p>Summary of perioperative outcomes in a series looking at robot-assisted radical prostatectomy outcomes in patients with high-risk PCa</p></caption><graphic xlink:href="IJU-30-410-g005"/></table-wrap><p>In a retrospective, propensity score-matched analysis comparing the perioperative outcomes of 1512 patients with high-risk PCa undergoing RARP versus ORP, Gandaglia <italic>et al</italic>. found no significant differences in complications (<italic>P</italic> = 0.6), PSM (<italic>P</italic> = 0.4) or additional therapy needed after surgery (<italic>P</italic> = 0.2) between patients treated with RARP and ORP.[<xref rid="ref30" ref-type="bibr">30</xref>] In multivariable analyses, however, patients undergoing RARP were less likely to receive a blood transfusion (<italic>P</italic> = 0.002) or to experience a prolonged LOS (<italic>P</italic> < 0.001) compared with men treated with ORP.</p><p>Similar results were obtained by Punnen <italic>et al</italic>., who retrospectively analyzed oncological and perioperative outcomes of 410 patients undergoing ORP versus RARP at a single center and found that, beside a longer LOS, more blood loss and higher transfusion rate (all <italic>P</italic> < 0.01), more men undergoing RARP had a complete nerve-sparing procedure compared with men undergoing ORP (54% vs. 34%, <italic>P</italic> < 0.01).[<xref rid="ref31" ref-type="bibr">31</xref>] Oncological outcomes measured, in terms of PSM and BCR-free survival, did not differ significantly between the two groups.</p></sec><sec id="sec3-3"><title>Role of surgery in high-risk PCa</title><p>Randomized studies specifically looking at the comparative effectiveness of different treatment modalities for high-risk PCa are lacking, but significant information may be gleaned from high-risk subsets of randomized controlled trials (RCTs) evaluating various treatment modalities for all-risk PCa. In the supplement to the landmark paper published by Wilt <italic>et al</italic>. in 2012 comparing RP versus observation for treatment of localized PCa, they performed a subset analysis of patients with high-risk PCa.[<xref rid="ref32" ref-type="bibr">32</xref>] The subset analysis [<xref ref-type="fig" rid="F2">Figure 2</xref>] showed that cancer-specific mortality (CSM) was greatly reduced in high-risk PCa patients who underwent RP versus those who underwent observation (11.5% vs. 20.0%, respectively, <italic>P</italic> = 0.05). The overall mortality was also lower in patients undergoing RP as compared with observation, although it was not statistically significant (55% vs. 59%, respectively, <italic>P</italic> = 0.25). Their subset analysis showed survival benefit after RP was greater in patients with high-risk PCa in comparison with patients with low-risk PCa. Similarly, Bill-Axelson <italic>et al</italic>. in their Scandinavian Prostate Cancer Group-4 RCT showed that patients who have high-risk PCa are benefited by surgery as compared with watchful waiting.[<xref rid="ref33" ref-type="bibr">33</xref>]</p><fig id="F2" position="float"><label>Figure 2</label><caption><p>(a) Overall survival trend in patients with high-risk prostate cancer. (b) Cancer-specific survival trend in patients with high-risk prostate cancer (Adapted with permission from Supplementary Data to Wilt <italic>et al.</italic>, 2012 New England Journal of Medicine. All rights belong to the Massachusetts Medical Society.)</p></caption><graphic xlink:href="IJU-30-410-g006"/></fig><p>In addition to the aforementioned RCTs, multiple other studies[<xref rid="ref34" ref-type="bibr">34</xref><xref rid="ref35" ref-type="bibr">35</xref>] substantiate the favorable outcomes of surgery for high-risk disease patients. A recent study by Briganti <italic>et al</italic>., which evaluated outcomes in 1366 patients after RP with 15 years of follow-up, found that the CSS was as high as 91.0% at 10 years.[<xref rid="ref26" ref-type="bibr">26</xref>] Boorjian <italic>et al</italic>. in their study looking at the comparative effectiveness of RP vs. external-beam radiotherapy (EBRT) in high-risk PCa patients found that overall survival (OS) was significantly better in patients who underwent RP compared with patients who underwent EBRT with or without androgen-deprivation therapy (ADT) (RP OS: 77% compared with the patients who received EBRT + ADT OS: 67% or EBRT alone OS: 52%; <italic>P</italic> < 0.001).[<xref rid="ref36" ref-type="bibr">36</xref>] Similarly, Abdollah <italic>et al</italic>.[<xref rid="ref37" ref-type="bibr">37</xref>] showed that the 10-year CSM rates were significantly better in patients who underwent RP as compared with radiotherapy or observation (3.6%, 6.5% and 10.8%, respectively; <italic>P</italic> < 0.001).</p><p>RP offers several distinct advantages. First, and most importantly, it provides pathologic specimens for more accurate staging. Recent studies have shown that up to 35% of patients are staged inaccurately,[<xref rid="ref38" ref-type="bibr">38</xref>] and up to 50% of high-risk, post-RP patients have more favorable disease on final pathological review.[<xref rid="ref39" ref-type="bibr">39</xref>] Thus, RP allows clinicians to offer targeted therapy based on more accurate staging and to avoid morbidity associated with unnecessary adjuvant treatment. Second, RP provides excellent local cancer control. Inman <italic>et al</italic>. reported a recurrence rate of just 13% in men who underwent RP as compared with a recurrence rate of 41-61% in men who underwent EBRT.[<xref rid="ref40" ref-type="bibr">40</xref>] Third, EBRT, which is considered the main alternative to RP, is associated with significant morbidity including late-onset erectile dysfunction, chronic bladder irritation, hemorrhagic cystitis, infertility, strictures, bowel/bladder incontinence and secondary risk of cancer [Ischia <italic>et al</italic>.,]. In addition, men undergoing RP are 3.5 times less likely to require ADT, and they also have significantly longer intervals of ADT-free survival compared with men undergoing EBRT.[<xref rid="ref41" ref-type="bibr">41</xref>] ADT is associated with significant morbidity including hyperlipidemia, metabolic syndrome, diabetes, cardiovascular disease, anemia, osteoporosis, periodontal disease, infertility, sexual dysfunction, fatigue, hot flashes and/or cognitive deficits.[<xref rid="ref42" ref-type="bibr">42</xref>] Finally, primary tumors play a significant role in tumor shedding and cytokine/growth factor production. Therefore, RP provides definitive tumor debulking (i.e. removal of the primary tumor) and may improve overall outcomes.[<xref rid="ref43" ref-type="bibr">43</xref>]</p><p>These RCTs and cohort studies constitute level-1b and 2b evidence, respectively, and clearly demonstrate the beneficial role of surgery in high-risk PCa. Having said that, we should recognize that the high-risk PCa group is a heterogeneous group, as shown by Yossepowitch <italic>et al</italic>., Yossepowitch <italic>et al</italic>. compared eight high-risk subsets in nearly 6000 men undergoing RP and found varying rates of BCRFS, need for secondary therapy and metastatic progression, with 10-year PCa-specific mortality ranging from 3% to 11%.[<xref rid="ref5" ref-type="bibr">5</xref>] Thus, patients falling under the umbrella of high-risk PCa should ideally be treated on a case-to-case basis, as a patient with high PSA and high GS but with an OCD would benefit most from surgery alone while a patient with non-OCD might benefit more from a non-surgical option.</p><p>This review demonstrates many points. First, outcomes following RARP for the management of high-risk PCa are equivalent to ORP in terms of functional and short-term oncological outcomes, but better in terms of safety outcomes. Second, RARP is an effective and safe option for select high-risk patients. Third, functional outcomes for patients with high-risk PCa are highly variable, and in general are inferior when compared with all-risk or low-risk PCa.[<xref rid="ref44" ref-type="bibr">44</xref>] Fourth, surgery as part of a multimodal management strategy seems to offer the best survival rates as compared with other modalities alone or in combination, but there is a need for better stratification of these high-risk patients for their optimal management, which can only be achieved by devising better modalities of stage assessment and prediction of biological behavior of the disease utilizing various imaging and translational tumor markers, which are still in discovery and testing phase. At this time, it can be said that RARP is as effective as other available modalities, but may be the best choice for select patients with localized, high-risk PCa.</p></sec><sec id="sec3-4"><title>RARP for high-risk PCa - Our MORE technique</title><p>We recently developed a Modification to the Vattikuti Institute Prostatectomy technique[<xref rid="ref45" ref-type="bibr">45</xref><xref rid="ref46" ref-type="bibr">46</xref><xref rid="ref47" ref-type="bibr">47</xref><xref rid="ref48" ref-type="bibr">48</xref><xref rid="ref49" ref-type="bibr">49</xref>] of RP, which allows immediate Organ Retrieval after excision for intra-operative Examination and frozen-section analysis (the MORE technique).</p><p>Briefly, with the help of a GelPOINT™ platform (Applied Medical, Rancho Santa Margarita, CA, USA), a hand-access platform, the excised RP specimen, is easily extracted without needing to undock the robot or any loss of pneumoperitoneum. The specimen is then examined on-table by the surgeon. Frozen-section biopsies may be taken from areas that appear suspicious for PSM on inspection and/or bimanual examination, and after carefully marking the samples for anatomical orientation they are sent for analysis. Biopsies that are positive or suspicious for cancer result in more tissue being removed. While waiting for the results of the frozen section analysis, the surgeon continues with lymph node dissection and hence there is no increase in the overall operative time.</p><p>With this technical modification, we showed an absolute risk reduction of 26.6% (<italic>P</italic> = 0.04) in the PSM rate in patients with pT3a disease. MORE is a promising technique and a prospective trial is currently underway in our institution.</p></sec></sec></sec><sec sec-type="conclusions" id="sec1-4"><title>CONCLUSIONS</title><p>There is substantial evidence to support that RP with or without robotic assistance achieves extremely favorable oncological outcomes in patients with high-risk PCa compared with other treatment modalities. In addition, patients who are considered to have high-risk disease are an internally heterogeneous group, and all patients should not be treated alike. The key is to predict which patients, despite having high-risk characteristics, may harbor OCD. The current literature suggests that at least 40%[<xref rid="ref26" ref-type="bibr">26</xref>] of patients classified as high risk fall into this category, but the proportion may be as high as 80%.[<xref rid="ref1" ref-type="bibr">1</xref>] With new nomograms specifically tailored for predicting OCD in high-risk disease patients,[<xref rid="ref26" ref-type="bibr">26</xref>] and the improvement in the accuracy of magnetic resonance imaging technology in assessing nodal disease/non-OCD, patients with a high likelihood of having OCD, despite being high risk, would be ideal candidates for RP and would gain the most benefit from surgery alone.</p></sec> |
Handling difficult anastomosis. Tips and tricks in obese patients and narrow pelvis | <p>Vesico-urethral anastomosis (VUA) is a technically challenging step in robotic-assisted laparoscopic prostatectomy (RALP) in obese individuals. We describe technical modifications to facilitate VUA encountered in obese individuals and in patients with a narrow pelvis. A Pubmed literature search was performed between 2000 and 2012 to review all articles related to RALP, obesity and VUA for evaluation of technique, complications and outcomes of VUA in obese individuals. In addition to the technical modifications described in the literature, we describe our own experience to encounter the technical challenges induced by obesity and narrow pelvis. In obese patients, technical modifications like use of air seal trocar technology, steep Trendlenburg positioning, bariatric trocars, alterations in trocar placement, barbed suture and use of modified posterior reconstruction facilitate VUA in robotic-assisted radical prostatectomy. The dexterity of the robot and the technical modifications help to perform the VUA in challenging patients with lesser difficulty. The experience of the surgeon is a critical factor in outcomes in these technically challenging patients, and obese individuals are best avoided during the initial phase of the learning curve.</p> | <contrib contrib-type="author"><name><surname>Samavedi</surname><given-names>Srinivas</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Abdul-Muhsin</surname><given-names>Haidar</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Pigilam</surname><given-names>Suneel</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Sivaraman</surname><given-names>Ananth</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib><contrib contrib-type="author"><name><surname>Patel</surname><given-names>Vipul R.</given-names></name><xref ref-type="aff" rid="aff1"/></contrib> | Indian Journal of Urology : IJU : Journal of the Urological Society of India | <sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Prostate cancer is the most common non-cutaneous solid malignancy among men in the United States and the second leading cause of cancer death.[<xref rid="ref1" ref-type="bibr">1</xref>] With the advent of new diagnostic advances and early screening, more than 90% of the cases identified have organ-confined disease and are potentially curable.[<xref rid="ref2" ref-type="bibr">2</xref>] In general, radical prostatectomy (RP) is the treatment of choice for patients with clinically localized prostate cancer and life expectancy >10 years.[<xref rid="ref3" ref-type="bibr">3</xref>] By 2008, around 80% of RPs in the United States were performed with robotic assistance.[<xref rid="ref2" ref-type="bibr">2</xref>] The feasibility and safety of the procedure has been well documented; however, techniques continue to be refined to improve functional and oncological outcomes.[<xref rid="ref3" ref-type="bibr">3</xref><xref rid="ref4" ref-type="bibr">4</xref>] Robotic-assisted laparoscopic prostatectomy (RALP) offers several advantages including decreased blood loss, shorter hospital stay and less peri-operative morbidity compared with traditional open methods.[<xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref7" ref-type="bibr">7</xref>]</p><p>Obesity represents a major healthcare problem that is significantly affecting people of all ages in developed countries. A body mass index >30 kg/m<sup>2</sup> is increasing in incidence and is a major health issue in the USA and elsewhere. In 2009 and 2010, the National Health and Nutrition Examination Survey (NHANES) reported the overall prevalence of obesity as 35.7% among adults in the USA.[<xref rid="ref8" ref-type="bibr">8</xref><xref rid="ref9" ref-type="bibr">9</xref>] Several previous studies have reported a correlation of obesity with increased odds of prostate cancer risk[<xref rid="ref10" ref-type="bibr">10</xref>] higher grade prostate cancer and progression that increases death from prostate cancer.[<xref rid="ref11" ref-type="bibr">11</xref><xref rid="ref12" ref-type="bibr">12</xref>] Given the increasing incidence of obesity in men, several authors have reported the impact of body mass index (BMI) on surgical outcomes for patients with prostate cancer.[<xref rid="ref13" ref-type="bibr">13</xref>] A Pubmed literature search was between 2000 and 2012 to review all articles related to RALP, obesity and vesico-urethral anastomosis (VUA) for evaluation of technique, complications and outcomes of VUA in obese individuals. Obesity increased the operative time and blood loss, elevated the transfusion rate and made radical prostatectomy challenging.[<xref rid="ref13" ref-type="bibr">13</xref><xref rid="ref14" ref-type="bibr">14</xref><xref rid="ref15" ref-type="bibr">15</xref><xref rid="ref16" ref-type="bibr">16</xref>]</p><p>The final step of VUA is a very challenging and critical step of RALP, particularly in obese patients. The goals that are achieved by an ideal VUA are absence of peri-operative urine leakage and earlier return of continence. Persistent efforts have been made to reduce the urinary extravasation and achieve urinary continence both in open surgery and in minimally invasive prostatectomy.[<xref rid="ref4" ref-type="bibr">4</xref>] The dexterity of the robot had made it possible to facilitate complex reconstruction in the deep pelvis reproducing and surpassing the open techniques. Failure to achieve a watertight anastomosis is associated with post-operative urinary leak and its consequences such as paralytic ileus, prolonged catheterization, urinary peritonitis, possibly re-intervention, bladder neck contracture and delayed continence.[<xref rid="ref3" ref-type="bibr">3</xref><xref rid="ref4" ref-type="bibr">4</xref>] Thus, it is very important and critical to have a watertight anastomosis. Several techniques of urethra-vesical anastomosis have been tested in the past aiming to reduce the post-operative urine leak and its consequences. These include the use of interrupted sutures, two independent running sutures[<xref rid="ref17" ref-type="bibr">17</xref>] and pre-tied running sutures, the van Velthoven method[<xref rid="ref18" ref-type="bibr">18</xref>] and the use of unidirectional[<xref rid="ref19" ref-type="bibr">19</xref><xref rid="ref20" ref-type="bibr">20</xref>] and bidirectional barbed[<xref rid="ref21" ref-type="bibr">21</xref><xref rid="ref22" ref-type="bibr">22</xref>] sutures. Despite these variations, the rates of urine leak have been reported to range between 4.5% and 7.5% at high-volume centers.[<xref rid="ref23" ref-type="bibr">23</xref><xref rid="ref24" ref-type="bibr">24</xref>] Use of barbed sutures has reduced the anastomotic time in general.[<xref rid="ref22" ref-type="bibr">22</xref>]</p><p>Numerous technical modifications have been used to improve early return and overall continence following surgery (both open and minimally invasive), including the anterior urethropexy[<xref rid="ref25" ref-type="bibr">25</xref>] bladder neck preservation,[<xref rid="ref26" ref-type="bibr">26</xref>] intussusception of the bladder neck,[<xref rid="ref27" ref-type="bibr">27</xref>] puboprostatic ligament sparing and sling construction.[<xref rid="ref28" ref-type="bibr">28</xref>] The actual benefits of these modifications are controversial. Many of the surgical reconstruction techniques for RALP have been based on the posterior reconstruction described by Rocco and his colleagues in 2001.[<xref rid="ref29" ref-type="bibr">29</xref>] In posterior reconstruction, the posterior rhabdo-sphincter is joined to the posterior Denonvilliers’ fascia and fixed to the bladder wall 1-2 cm cranial to the new bladder neck to avoid caudal retraction of the urethra-sphincteric complex, prior to completing the standard VUA. The modified posterior reconstruction, described by Rocco <italic>et al</italic>. is especially a very good adjunct technique for reconstruction in obese individuals.</p><sec id="sec2-1"><title/><sec id="sec3-1"><title>Difficulties in obese individuals</title><p>Obese patients can present a technical challenge for RRP (13-16, 41-42) because of excess abdominal fat, which makes access to the prostate and pelvic organs difficulty. In men who elect to undergo robotic prostatectomy, the body habitus can present a challenge to even the most experienced surgeon. Other challenges usually induced by obesity include a deeper and narrowed true pelvis combined with occasional exostosis of the pubic symphysis. During the apical dissection and preservation of the intra-pelvic urethra, the surgeon encounters peri-prostatic fat that reduces the visibility and requires effort to clear. The urethral anastomosis in obese patients can be more challenging due to intra-abdominal fat obscuring the visual field. Abdominal contour resulting in acute angulation of arms, huge omental and mesenteric fat pushing the bladder and reducing the working space, large prostates with limited working space in the true pelvis, overriding pubic bone obscuring the apical dissection and VUA, inability to reach for visualization due to increased distance between the ports and depth of the pelvis, difficulty in getting the bladder caudad for anastomosis because of fat and big prostates, fragile bladder neck secondary to large prostate and tension on bladder and a difficult to visualize retractile urethra are factors responsible for difficulty in obese individuals [<xref ref-type="table" rid="T1">Table 1</xref>].</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Problems and solutions for difficult anastomosis in obese patients</p></caption><graphic xlink:href="IJU-30-418-g001"/></table-wrap></sec><sec id="sec3-2"><title>Steps to facilitate vesico-urethral anastomosis and posterior reconstruction in obese patients-</title><p>To maximize the vision in the operative field, the following maneuvers need be performed: Trendlenburg position is increased and this modification mandates caution during positioning to prevent sliding of the patient by usage of the gel pads and the bean bag. The usual angle of the table is around 25 degrees, and it may be extended to 30 degrees. If difficulty is encountered with pubic bone interposition, the scope is changed from 30 to 0 degrees for dissection of the apex and anastomosis. If it is difficult to visualize the bladder neck and posterior sphincter complex, the scope is switched from 30 to 0 degree. Two instruments are used to retract the fat and the bladder and to prevent fat from falling into the operative field.</p><p>Based on our continuing experience, we have modified out technique to facilitate RALP in the obese patients. Port placement should be adjusted to the body habitus with trocars shifted farther away from the pelvis and more laterally, enabling a deeper reach [<xref ref-type="fig" rid="F1">Figure 1</xref>]. Air-seal trocars<sup>®</sup> technology[<xref rid="ref30" ref-type="bibr">30</xref>] has been a useful new addition to the armamentarium for laparoscopic procedures, reducing the number of episodes of pressure loss <8 mmHg helping in maintaining already compromised working space in these populations. After establishing a pneumo-peritoneum in overweight patients, the instrument's path may be obstructed by the pubic symphysis and the pelvic brim due to a more vertical angle. Depressing the robotic arms to prevent the instruments from hitting the pelvic brim can help avoid this. In the patients with protuberant abdomen, ports must be placed at a greater distance from the pubic symphysis as measured on the body surface after insufflation, typically translating a distance of 15 cm to 17-18 cm from the pubic symphysis. Additionally, robotic trocars may have to be inserted deeper into the abdominal cavity and the arms deflected laterally to flatten the working angle of the robotic arm as they reach deep into the pelvis under the pubic bone. The use of extra-long Da-vinci trocars (Intuitive surgical, Sunnyvale, CA, USA) is helpful for the purpose. Optimally, the trocars should be inserted into the peritoneal cavity perpendicular to the abdominal wall. Placing the 12 mm Air seal<sup>®</sup> bariatric assistant lateral port pointing towards the umbilicus is helpful in preventing the port being buried under pre-peritoneal fat and avoiding bowel injury. The midline camera port is a bariatric 12 mm port. A larger beanbag is necessary to accommodate larger patients and it is imperative to pad all bony points to avoid pressure trauma, and the anesthesiologist should monitor the respiratory status in a steep Trendlenburg position.</p><p>Before performing VUA, modified reconstruction of the pelvic floor, reattaching the Denonvilliers fascia to the rhabdo-sphincter as described by Francesco Rocco <italic>et al</italic>.[<xref rid="ref29" ref-type="bibr">29</xref><xref rid="ref31" ref-type="bibr">31</xref><xref rid="ref32" ref-type="bibr">32</xref>] is performed. For this step, a 12-cm double-armed 2/0 quill suture<sup>®</sup>[<xref rid="ref21" ref-type="bibr">21</xref>] on a RB1 needle is used. The free edge of the Denonvilliers fascia is approximated to the posterior aspect of the rhabdo-sphincter and the posterior median raphe running one of the arms. A second layer is then run with the second arm of the suture, approximating the posterior bladder neck to the posterior lip of the urethra. A continuous modified van Velthoven VUA[<xref rid="ref18" ref-type="bibr">18</xref>] is then performed. A 12-inch double-armed quill<sup>®</sup> 2/0 on RB1is used. The posterior urethral anastomosis is performed first with one arm of the suture starting at the 5 o clock position and reaching the 10 o clock position in the clock-wise position. This is followed by the completion of the anterior urethral anastomosis with the second arm of the suture in a counter-clock fashion and then tying the sutures on the urethral stump.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Picture showing cephalad placement of the camera and the 8-mm robotic ports compared with the non-obese individuals</p></caption><graphic xlink:href="IJU-30-418-g002"/></fig></sec><sec id="sec3-3"><title>Difficulties in individuals with narrow pelvis</title><p>Several authors have reported various degrees of difficulties both in extraperitoneal and trans-peritoneal robotic prostatectomy in patients with narrow pelvis. In a study by Mason <italic>et al</italic>. and his colleagues,[<xref rid="ref33" ref-type="bibr">33</xref>] the PCI (the pelvic cavity index is the pelvic inlet multiplied by the inter-spinous distance and divided by the pelvic depth) in patients undergoing prostatectomy was calculated and used to estimate the probable robotic working space. Patients with larger prostates and with narrow, deep pelvises were predicted to have a more difficult RALP. The PV (prostate volume) to PCI ratio statistically predicted lengthier and bloodier procedures but did not predict positive surgical margins[<xref rid="ref34" ref-type="bibr">34</xref>] or transfusion risk on regression analysis. Most of the studies[<xref rid="ref34" ref-type="bibr">34</xref><xref rid="ref35" ref-type="bibr">35</xref><xref rid="ref36" ref-type="bibr">36</xref>] did not show a positive correlation between smaller pelvic volume and negative prostatectomy outcomes. Two technical issues in patients with narrow pelvis are decreased intra-pelvic working space and clashing of robotic instruments externally. Clashing between the third and fourth arm is common in patients with smaller BMI and narrow pelvis. A minimum distance of 8 cm will negate the instrument clashing externally, with additional maneuvers of depressing the 4<sup>th</sup> arm, elevating the third arm and medially rotating the 3<sup>rd</sup> arm helping to prevent clashing. Another maneuver to prevent clashing is using a three-arm robot with additional assistant ports at the 4<sup>th</sup> arm. Intra-operative clashing can be avoided with experience; hence, smaller BMI and narrow pelvis can be avoided in the initial learning curve.</p></sec><sec id="sec3-4"><title>Studies and outcomes of VUA in obese individuals</title><p>Several studies have reported the functional and oncological outcomes in obese individuals [<xref ref-type="table" rid="T2">Table 2</xref>].[<xref rid="ref16" ref-type="bibr">16</xref><xref rid="ref37" ref-type="bibr">37</xref><xref rid="ref38" ref-type="bibr">38</xref>] Operative times ranged between 84 to 353 min, length of hospital stay ranged between 1.1 to 4.3 days and blood loss ranged between 100 to 457 ml.[<xref rid="ref13" ref-type="bibr">13</xref><xref rid="ref14" ref-type="bibr">14</xref><xref rid="ref15" ref-type="bibr">15</xref><xref rid="ref16" ref-type="bibr">16</xref>] Technical modifications that have shown to improve better operating time were use of Air seal technology and use of barbed suture, either unidirectional[<xref rid="ref19" ref-type="bibr">19</xref><xref rid="ref20" ref-type="bibr">20</xref>] or bidirectional.[<xref rid="ref21" ref-type="bibr">21</xref><xref rid="ref22" ref-type="bibr">22</xref>] AirSeal<sup>®</sup> trocar[<xref rid="ref30" ref-type="bibr">30</xref>] allows reducing the number of ingress incisions and facilitates simultaneous as well as consecutive use of multiple instruments. Barbed suture has shown to decrease the anastomotic time but did not affect the urinary extravasation or long term continence rates.</p><table-wrap id="T2" position="float"><label>Table 2</label><caption><p>Review of the literature</p></caption><graphic xlink:href="IJU-30-418-g003"/></table-wrap></sec></sec></sec><sec sec-type="conclusions" id="sec1-2"><title>CONCLUSIONS</title><p>Obesity is a significant health problem in the world. As more and more patients seek surgical management for localized cancer prostate, it is logical the surgeons will need to operate on increasing numbers of obese individuals. In obese patients, this novel technique of air seal technology, bariatric trocars, alterations in trocar placement, barbed suture and use of modified posterior reconstruction facilitate urethra-vesical anastomosis in robotic assisted radical prostatectomy. The experience of the surgeon is a critical factor in outcomes in these technically challenging patients, and obese individuals are best avoided during the initial phase of the learning curve.</p></sec> |
Significance and management of positive surgical margins at the time of radical prostatectomy | <p>Positive surgical margins (PSM) at the time of radical prostatectomy (RP) result in an increased risk of biochemical recurrence (BCR) and secondary treatment. We review current literature with a focus on stratifying the characteristics of the PSM that may define its significance, the impact of modern imaging and surgical approaches in avoidance of PSM, and management strategies when PSM do occur. We performed a review of the available literature to identify factors associated with PSM and their management. PSM have been repeatedly demonstrated to be associated with an increased risk of BCR following RP. The specific characteristics (size, number, location, Gleason score at the margin) of the PSM may influence the risk of recurrence. Novel imaging and surgical approaches are being investigated and may allow for reductions of PSM in the future. The use of adjuvant treatment for a PSM remains controversial and should be decided on an individual basis after a discussion about the risks and benefits. The goal of RP is complete resection of the tumor. PSM are associated with increased risk of BCR and secondary treatments. Of the risk factors associated with BCR after RP, a PSM is directly influenced by surgical technique.</p> | <contrib contrib-type="author"><name><surname>Silberstein</surname><given-names>Jonathan L.</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Eastham</surname><given-names>James A.</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib> | Indian Journal of Urology : IJU : Journal of the Urological Society of India | <sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Wide variations in the incidence of positive surgical margins (11-48%) have been reported at the time of radical prostatectomy (RP).[<xref rid="ref1" ref-type="bibr">1</xref><xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref3" ref-type="bibr">3</xref><xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref8" ref-type="bibr">8</xref>] Centers of excellence tend to report PSM in the lower end of this range[<xref rid="ref2" ref-type="bibr">2</xref>] while population-based studies demonstrate results on the upper end which may be more reflective of most clinicians experience.[<xref rid="ref1" ref-type="bibr">1</xref>] Cancer registries such as Surveillance, Epidemiology and End Results Program (SEER) have been shown to grossly underreport PSM and may be inaccurate.[<xref rid="ref5" ref-type="bibr">5</xref>] Regardless, the incidence of PSM depends on both the characteristics of the cancer and the technique of the surgeon. Achieving a negative surgical margin may be the most significant opportunity the surgeon has to influence the natural history of the disease, as patients with PSM have been repeatedly demonstrated to have greater rates of biochemical recurrence (BCR).[<xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref8" ref-type="bibr">8</xref>] Additionally, some clinicians view PSM as a trigger for adjuvant radiation therapy.</p><p>Attaining a negative surgical margin at the time of RP is the primary goal of the surgeon, but it is not an isolated goal. Preserving the neurovascular tissue and maintaining maximal urethral length are crucial for erectile functional and continence outcomes. Balancing oncologic and functional goals which are at odds with one another is fundamental to successfully performing RP regardless of surgical approach.</p><p>When a PSM is encountered, the specific characteristics of the PSM may influence the risk of BCR and subsequent disease progression. The specific pathologic characteristics of the PSM (length, number, location and Gleason score at the PSM) may all influence the risk of BCR. Because of the increased risk of BCR with PSM, some advocate immediate adjuvant treatment; however this may result in deterioration in quality of life and over treatment for many patients. In this review we will consider the definition and significance of a PSM, the pathologic characteristics that influence the significance of the margin, recent surgical and imaging techniques that may reduce the rates of PSM and management of PSM when they are encountered.</p></sec><sec id="sec1-2"><title>DEFINITION OF A SURGICAL MARGIN</title><p>In theory, the definition of a PSM is clear; “tumor that extends to the surface of the prostate wherein the surgeon has cut across the tissue plane.”[<xref rid="ref9" ref-type="bibr">9</xref>] However, because the prostate lacks a true histologic capsule, in practice the definition can become confusing. In order to facilitate defining surgical margins (SM) status upon receipt by the pathologist, the entire surgical specimen should be inked and fixed. A positive margin is simply identified as “cancer cells extending to the inked surface of the specimen”. Margin status is negative if tumor cells are microscopically close to (<0.1 mm), but not actually in contact with the inked surface or when they are at the surface of the tissue lacking any ink.[<xref rid="ref10" ref-type="bibr">10</xref>] Even with proper handling of the specimen by the pathologist, SM assessment may be complicated by crush, thermal, or electrocautery artifact, partial tearing of the extraprostatic soft tissue during processing or tissue banking and incomplete or irregular tracking of ink.[<xref rid="ref11" ref-type="bibr">11</xref>] Such findings may contribute to interobserver variation with reported kappa values of 0.45 (moderate agreement) between local pathologists and expert pathologists and values of 0.74 between expert pathologists.[<xref rid="ref11" ref-type="bibr">11</xref><xref rid="ref12" ref-type="bibr">12</xref>]</p><p>Surgical margins in the presence of extra-prostatic extension (EPE) may represent an over enthusiastic effort on the part of the surgeon to preserve the neurovascular bundle (NVB) or tumor that invades into vital structures and could not be completely resected [<xref ref-type="fig" rid="F1">Figure 1a</xref>]. PSM in the absence of EPE, usually represent a capsular incision into tumor, an iatrogenic positive margin, due to an improper dissection plane with incision into the prostate and into the tumor [<xref ref-type="fig" rid="F1">Figure 1b</xref>].[<xref rid="ref13" ref-type="bibr">13</xref>] Importantly this scenario, pT2+, has prognostic significance. Such patients have greater rates of biochemical recurrence than patients with either pT2 cancers with negative SM or cancers with EPE and negative SM (pT3a, SM negative).[<xref rid="ref14" ref-type="bibr">14</xref>]</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>(a) Prostatectomy specimen demonstrating extraprostatic extension with tumor extending beyond the capsule of the prostate and a positive surgical margin (pT3a+). (b) Prostatectomy specimen demonstrating an organ confined tumor with tumor extending to the inked margin due to capsular incision (pT2+) (Figure adapted from Meeks and Eastham[<xref rid="ref13" ref-type="bibr">13</xref>])</p></caption><graphic xlink:href="IJU-30-423-g001"/></fig></sec><sec id="sec1-3"><title>PATHOLOGIC CHARACTERISTICS OF SURGICAL MARGINS</title><p>Patients with PSM have increased risk of BCR.[<xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref4" ref-type="bibr">4</xref><xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref8" ref-type="bibr">8</xref><xref rid="ref12" ref-type="bibr">12</xref>] For example in a multi-institutional study of more than 7000 patients the 5 and 10 year BCR rate for PSM were 0.53 (95% CI 0.494, 0.566) and 0.36 (95% CI 0.28, 0.45), [<xref ref-type="fig" rid="F2">Figure 2</xref>], a Kaplan-Meier curve adapted from this publication demonstrating BCR stratified according to margin status.[<xref rid="ref4" ref-type="bibr">4</xref>] This figure also demonstrates that many patients, despite the presence of PSM will not develop BCR even with 10 years of follow. Many investigators have attempted to better define the pathologic characteristics of PSM in order to better risk stratify patients and potentially offer adjuvant intervention for those at high risk of progression while sparing over treatment for others.</p><fig id="F2" position="float"><label>Figure 2</label><caption><p>Kaplan-Meier curve demonstrating BCR according to SM status based on the collected data from 7816 consecutively treated patients from eight institutions. PSM are associated with greater rates of BCR over time, adapted from Karakiewicz <italic>et al</italic>.[<xref rid="ref4" ref-type="bibr">4</xref>]</p></caption><graphic xlink:href="IJU-30-423-g002"/></fig><sec id="sec2-1"><title/><sec id="sec3-1"><title>“Amount” of positive margin</title><p>Multiple investigators have sought to quantify the “amount” of PSM either by counting the number of positive margins in a given specimen, or the extent of the positive margin quantified as binary variable such as focal versus extensive often seen in older studies or as a more reproducible linear extent. The rationale behind these attempts assumes that a greater amount of PSM is associated with greater quantity of tumor left behind and a greater potential for growth, biochemical recurrence, and metastases. Multiple analyses from our institution and others have demonstrated that multiple PSM confer increased risk of BCR when compared with a solitary margin.[<xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref8" ref-type="bibr">8</xref>] Although the number of positive margins may be an independent predictor in multivariable analysis for BCR, the number of positive margins may not significantly impact the predictive accuracy of nomogram predictions compared to a PSM modeled more simply as positive or negative.[<xref rid="ref7" ref-type="bibr">7</xref>]</p><p>Multiple investigators have attempted to determine if the extent of the positive margin has prognostic significance. Examining our data as focal compared to extensive as part of a multicenter analysis and then more rigorously examining linear length at our institution alone, both analyses demonstrated that longer PSM were associated with greater rates of BCR. However, both analyses demonstrated that the concordance index in a model incorporating length of positive margin was not meaningfully enhanced compared to a model that just included the status of the margin (positive versus negative).[<xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref15" ref-type="bibr">15</xref>] Other groups have independently corroborated these findings and repeatedly demonstrated that the length of PSM is associated with BCR.[<xref rid="ref16" ref-type="bibr">16</xref><xref rid="ref17" ref-type="bibr">17</xref>] The International Society of Urological Pathologists (ISUP) reviewed the available data and reached a consensus in 2008 that PSM should be recorded as millimeters of involvement.[<xref rid="ref9" ref-type="bibr">9</xref>]</p></sec><sec id="sec3-2"><title>Anatomic location of positive surgical margin</title><p>Efforts to reduce PSM have lead surgeons and pathologists to define their anatomic location and determine if the site-specific location impacts rates of recurrence. Repeatedly studies have demonstrated that the two most likely locations for PSM are the apex of the prostate and the posterolateral margins. Together these sites make up the majority of PSM accounting for 60-75% of PSM in most reported series of either open retropubic or robotic approaches.[<xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref18" ref-type="bibr">18</xref>] The apex of the prostate has less supporting tissue than the rest of the gland, it contains the least amount of capsule, and even benign glands can become admixed with skeletal muscle at this location.[<xref rid="ref19" ref-type="bibr">19</xref>] This coupled with the increased traction placed on the apex during various parts of the procedure and efforts to maintain urethral length may explain the increased rates of PSM in this location. The posterolateral margin of the prostate is the second most common location of PSMs and this is likely due to attempts to preserve as much of the neurovascular bundle as possible which run in this location.</p></sec><sec id="sec3-3"><title>Gleason score at positive surgical margin</title><p>Recently, investigators have begun to investigate the importance of the Gleason score at the PSM. Theoretically when a higher Gleason score is found at the PSM, a more aggressive tumor remains in the patient with potentially higher rates of BCR. Several studies have demonstrated that grade of cancer at the PSM is associated with greater rates of BCR.[<xref rid="ref15" ref-type="bibr">15</xref><xref rid="ref16" ref-type="bibr">16</xref><xref rid="ref20" ref-type="bibr">20</xref>] Gleason score in the primary tumor is highly correlated with Gleason score at the margin for Gleason 6 tumors but this concordance rate diminishes rapidly as the primary tumor Gleason score increases.[<xref rid="ref21" ref-type="bibr">21</xref>] At MSKCC, Udo and colleagues noted that Gleason grade was associated with increased risk of BCR in a univariate analysis but it did not significantly enhance the concordance index of a model incorporating specimen Gleason Score and overall SM status.[<xref rid="ref15" ref-type="bibr">15</xref>] While ISUP currently recommends that reporting Gleason score at PSM remain at the discretion of the reporting pathologist, it may remain an important discriminator for determining the importance of a positive margin, particularly for those with Gleason 7 or greater disease, further study will be needed to verify the importance of these findings.</p><p>All together these data suggest that length of the PSM, the number of PSMs, the Gleason score at the PSM, and potentially even the location of the PSM may each play important roles in defining the risk of BCR following RP. Inter-institution variability in reporting makes comparisons or collaborations difficult. Potentially one could envision a scenario in which the characteristics (length, location, number, Gleason score) of a margin would have value in determining who should receive adjuvant therapy; however, this has yet to be demonstrated convincingly. Furthermore, while each of these factors may have significance on their own they have not demonstrated benefit in predicting outcomes when added to existing models.</p></sec></sec></sec><sec id="sec1-4"><title>BLADDER NECK MARGIN</title><p>Extraprostatic extension with microscopic invasion of the bladder neck-previously designated as T4 according to the American joint commission on cancer (AJCC)-has recently been revised to be included in T3a category. This reclassification is based on the work of several retrospective series which have demonstrated that patients with isolated positive bladder neck margins have outcomes that more closely approximate T3 lesions.[<xref rid="ref22" ref-type="bibr">22</xref><xref rid="ref23" ref-type="bibr">23</xref><xref rid="ref24" ref-type="bibr">24</xref>] Typically, direct extension of cancer from the organ of origin into surrounding structures is designated as T4; however, at the bladder neck, the interwoven nature of the outer layers of the bladder with the smooth muscle of the prostate obscure distinct tissue boundaries. It is unclear if a PSM at the bladder neck is associated with a worse prognosis than PSM in other locations, as isolated bladder neck margins are rare and often associated with multiple high-risk features.[<xref rid="ref22" ref-type="bibr">22</xref>] Further investigation is needed to more clearly define whether isolated bladder neck margin truly does have a worse prognosis than margins in other locations, if confirmed, clarification of the AJCC might be to define bladder neck invasion as T3b and seminal vesicle invasion as T3c as suggested by some groups.[<xref rid="ref23" ref-type="bibr">23</xref>]</p></sec><sec id="sec1-5"><title>SURGICAL APPROACH</title><p>With the huge shift towards robotics and away from open RP that has occurred over the last decade, invariable the question is asked does the surgical approach influence SM status.[<xref rid="ref25" ref-type="bibr">25</xref>] Currently, there are no large prospective randomized surgical trials designed to answer this question and even if there were, a larger problem might still remain. Large heterogeneity in PSM exists between individual surgeons performing RP through the same approach even after adjusting for case mix, surgery date, and surgery volume. In a review of the SMS of 44 different surgeons at two large urban centers PSM ranged, for each surgeon, from 10% to 48%.[<xref rid="ref3" ref-type="bibr">3</xref>] Substantial variation in outcomes remained even when analyses are limited to high or very high volume surgeons and are likely to dwarf differences between surgical approaches limiting the utility of comparative studies.</p><p>Despite these limitations various investigators have compared margin rates, location, and length for one surgical approach with another. Because of the increased use of robotic-assisted laparoscopic prostatectomy investigators have been interested in the impact that this novel surgical approach has on SM status. In a recently published meta-analysis with propensity adjustment for patient, surgeon, and hospital factors, the authors found no difference in PSM for open and robotic surgery.[<xref rid="ref26" ref-type="bibr">26</xref>] A prior meta-analysis that limited its analysis to comparative studies only demonstrated that PSM rates were similar between approaches.[<xref rid="ref27" ref-type="bibr">27</xref>] Administrative care datasets have not been able to directly compare PSM for differing surgical approaches but have demonstrated similar rates in the use of secondary therapies between different surgical approaches as a surrogate.[<xref rid="ref28" ref-type="bibr">28</xref>] At our institution we have found no significant difference in PSM or BCR for one surgical approach compared with another, but again individual surgeon variation is likely to be of greater importance than surgical approach.[<xref rid="ref29" ref-type="bibr">29</xref><xref rid="ref30" ref-type="bibr">30</xref>]</p></sec><sec id="sec1-6"><title>IMAGING TOOLS PREDICT OR PREVENT POSITIVE MARGINS</title><p>Partial preservation of the neurovascular bundle (NVB) is likely the best compromise between oncologic and erectile functional outcomes in men at risk for EPE in the area of the NVB. Key to this approach is identification of the specific location of the cancer in relation to the NVB. Pre-operative MRI has been demonstrated to alter surgical plan prior to RP in approximately 40% of patients; however, MRI is reader dependent with significant interobserver variability.[<xref rid="ref31" ref-type="bibr">31</xref><xref rid="ref32" ref-type="bibr">32</xref>]</p><p>Another strategy has been the use of a real-time transrectal ultrasound during RP to help outline the suspected area of EPE. Using this technology, one group demonstrated a reduction in their PSM rates from 29% to 9%.[<xref rid="ref33" ref-type="bibr">33</xref>] More recently urologists have begun to incorporate the use of a transrectal ultrasound probe with concurrent use of the TilePro to display the ultrasound images on the da Vinci surgical system console.[<xref rid="ref34" ref-type="bibr">34</xref>] Mounting interest in MR-US fusion technology is likely to result in utilizing this technology in a similar fashion to attempt to minimize PSM and maximize preservation of the NVB.</p><p>Near-infrared fluorescence imaging has been used for the identification of renal tumors[<xref rid="ref35" ref-type="bibr">35</xref>] and sentinel lymph nodes for prostate cancer.[<xref rid="ref36" ref-type="bibr">36</xref>] In the future similar types of technology may assist in the identification of the NVB or the location of the prostate tumor in order to reduce the rates of PSM.</p></sec><sec id="sec1-7"><title>MANAGEMENT OF POSITIVE MARGINS</title><p>Large multi-institutional studies have demonstrated that patients with PSM are more than twice as likely to experience BCR as patients without, even after adjusting for age, PSA, pathologic Gleason score, pathologic stage, and year of surgery.[<xref rid="ref7" ref-type="bibr">7</xref>] This leaves clinicians and patients in the challenging position of considering the role for additional treatment in the absence of any detectable disease. Unfortunately, adjuvant radiotherapy comes at the cost of increased risk of urinary incontinence, urinary stricture disease, proctitis, and rectal bleeding. Furthermore, although patients with PSM are at an increased risk of developing BCR many never do and are exposed to the potential harms of adjuvant radiotherapy without benefit.</p><p>Three randomized trials have examined the role of adjuvant radiotherapy in men with ‘adverse’ pathologic features in the RP specimen.[<xref rid="ref37" ref-type="bibr">37</xref><xref rid="ref38" ref-type="bibr">38</xref><xref rid="ref39" ref-type="bibr">39</xref>] Eligible patients were randomized to either adjuvant radiotherapy or “wait and see”. All three trials documented improvement in BCR free-survival with adjuvant radiotherapy compared to a “wait and see’ approach. Two of these trials also demonstrated a reduction in clinical locoregional failure with adjuvant radiotherapy.[<xref rid="ref37" ref-type="bibr">37</xref><xref rid="ref38" ref-type="bibr">38</xref>] In all three studies the group gaining the most benefit from adjuvant radiotherapy was men with PSM. Based largely on the results of these three trials the American Urological Association (AUA) and the American Society for Therapeutic Radiology Organization (ASTRO) released joint guidelines stating that patients with adverse pathologic features (including but not limited to a PSM) should be offered ART.[<xref rid="ref40" ref-type="bibr">40</xref>] The guidelines continue on to state that the decision of whether to receive adjuvant radiotherapy should be based on a shared decision making process by a multidisciplinary team and the patient with consideration of the “patient's history, functional status, values, preferences, and tolerance for potential toxicities and QoL effects of radiotherapy.”</p><p>A remaining and important limitation of the existing data is that none of these randomized trials have compared adjuvant radiotherapy to early salvage radiotherapy. It has been demonstrated that salvage therapy administered at lower PSA levels is associated with greatest effectiveness. The ability to detect PSA at very low levels has led many to conclude that a preferable strategy would be to offer early salvage treatment when patients have low but detectable PSA rather than adjuvant radiotherapy. Such a strategy may reduce the over treatment of patients who are never destined to develop BCR while maintaining the potential advantage of radiotherapy. Two ongoing randomized clinical trials, RADICALS (Radiotherapy and Androgen Deprivation In Combination After Local Surgery, NCT # 00541047) and RAVES (Radiotherapy Adjuvant vs. Early Salvage, NCT # 00860652), are evaluating whether progression-free and/or prostate cancer specific and overall survival are significantly prolonged by the use adjuvant radiotherapy compared to early salvage radiotherapy at the time of PSA failure.</p><p>Lastly, some practitioners use androgen deprivation therapy (ADT) alone for patients with adverse pathologic characteristics including a PSM. In one small randomized trial, whose results have not been confirmed, ADT following prostatectomy for patients with lymph node positive disease was demonstrated to result in overall survival benefit[<xref rid="ref41" ref-type="bibr">41</xref>] but for node negative patients ADT has never been demonstrated to have similar benefit. ADT has the potential for significant harm, reduces QoL, and should only be considered for patients with a positive lymph node or those undergoing adjuvant or salvage radiotherapy.</p></sec><sec sec-type="conclusions" id="sec1-8"><title>CONCLUSIONS</title><p>PSM are associated with an increased risk of BCR. The presence of a PSM may be more influenced by the individual surgeon than the surgical approach used to perform RP. Longer PSM (>3 mm), multiple PSM, and higher Gleason score at the PSM are associated with an increased likelihood of BCR, while isolated apical PSM have a lower risk of BCR. MRI and intra-operative imaging modalities may help to reduce the incidence of PSM although further study is needed to ascertain the role of pre-or intra-operative imaging in improving outcomes. Finally when a PSM is encountered consistent with the AUA/ASTRO recommendations a thoughtful discussion should be had about the risks and benefits of immediate ART.[<xref rid="ref37" ref-type="bibr">37</xref>] Until the results of two ongoing randomized trials evaluating the impact of ART versus early salvage radiotherapy report results, a nuanced strategy which considers the specific characteristics of the individuals oncologic factors as well as those of the PSM-with a tendency toward ART for multiple high risk features and toward early salvage for those with few-may be the most rational approach.</p></sec> |
Salvage robotic radical prostatectomy | <p>Failure of non-surgical primary treatment for localized prostate cancer is a common occurrence, with rates of disease recurrence ranging from 20% to 60%. In a large proportion of patients, disease recurrence is clinically localized and therefore potentially curable. Unfortunately, due to the complex and potentially morbid nature of salvage treatment, radical salvage surgery is uncommonly performed. In an attempt to decrease the morbidity of salvage therapy without sacrificing oncologic efficacy, a number of experienced centers have utilized robotic assistance to perform minimally invasive salvage radical prostatectomy. Herein, we critically evaluate the existing literature on salvage robotic radical prostatectomy with a focus on patient selection, perioperative complications and functional and early oncologic outcomes. These results are compared with contemporary and historical open salvage radical prostatectomy series and supplemented with insights we have gained from our experience with salvage robotic radical prostatectomy. The body of evidence by which conclusions regarding the efficacy and safety of robotic salvage radical prostatectomy can be drawn comprises fewer than 200 patients with limited follow-up. Preliminary results are promising and some outcomes have been favorable when compared with contemporary open salvage prostatectomy series. Advantages of the robotic platform in the performance of salvage radical prostatectomy include decreased blood loss, short length of stay and improved visualization. Greater experience is required to confirm the long-term oncologic efficacy and functional outcomes as well as the generalizability of results achieved at experienced centers.</p> | <contrib contrib-type="author"><name><surname>Kaffenberger</surname><given-names>Samuel D.</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Smith</surname><given-names>Joseph A.</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib> | Indian Journal of Urology : IJU : Journal of the Urological Society of India | <sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Radical prostatectomy and radiation therapy remain the mainstays of primary treatment for clinically localized prostate cancer. Recurrence after primary therapy failure represents a significant and common clinical dilemma, with rates of prostate cancer recurrence reported to range from 15% to 60% depending on the choice of primary treatment and length of follow-up.[<xref rid="ref1" ref-type="bibr">1</xref><xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref3" ref-type="bibr">3</xref><xref rid="ref4" ref-type="bibr">4</xref>] While the overall utilization of potentially curative local salvage therapies after failure of primary therapy has increased in recent years, this trend has been exclusive of salvage radical prostatectomy (SRP), which is still rarely performed despite evidence of improved safety and oncologic efficacy in modern series.[<xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref7" ref-type="bibr">7</xref>] Furthermore, it has been shown that a high proportion of patients with radiation failure have clinically localized disease recurrence, which may be amenable to cure by local salvage therapy, although the burden of co-morbid conditions in this patient population combined with the difficulty and morbidity of local salvage therapies like SRP limit the applicability of these findings.[<xref rid="ref5" ref-type="bibr">5</xref><xref rid="ref8" ref-type="bibr">8</xref>]</p><p>Patients who are not acceptable surgical candidates or who have limited life expectancy may be more appropriately treated with either salvage cryotherapy or androgen deprivation therapy. However, in appropriately selected patients, it has been shown that SRP can result in excellent cancer-specific survival as well as durable biochemical recurrence (BCR)-free survival.[<xref rid="ref6" ref-type="bibr">6</xref>] SRP has also been suggested to provide superior cancer-free survival when compared with salvage cryotherapy, and, although salvage cryotherapy results have improved with newer cryoablation systems, further follow-up is required to confirm the oncologic efficacy of salvage cryotherapy.[<xref rid="ref9" ref-type="bibr">9</xref><xref rid="ref10" ref-type="bibr">10</xref>]</p><p>Despite the potential for cure and avoidance of androgen deprivation therapy afforded by SRP, it is rarely performed.[<xref rid="ref3" ref-type="bibr">3</xref><xref rid="ref5" ref-type="bibr">5</xref>] While the often-elderly and co-morbid status of patients with radio-recurrent disease compounded with concerns for the presence of advanced disease certainly impacts the low rates of SRP, the challenging technical aspects of the procedure, poor functional outcomes and risk of major complications likely contribute as well. In historical series, rates of rectal injury approached 15% and rates of anastomotic stricture have been as high as 32%.[<xref rid="ref7" ref-type="bibr">7</xref>] While these rates are daunting, improvements in technique and experience have vastly increased the safety of SRP, with rectal injury rates falling to 2-5%.[<xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref10" ref-type="bibr">10</xref><xref rid="ref11" ref-type="bibr">11</xref>]</p><p>The rapid increase in utilization of the robotic platform in the performance of radical prostatectomy in the United States has led to the use of robotic assistance in the salvage setting.[<xref rid="ref12" ref-type="bibr">12</xref><xref rid="ref13" ref-type="bibr">13</xref><xref rid="ref14" ref-type="bibr">14</xref><xref rid="ref15" ref-type="bibr">15</xref><xref rid="ref16" ref-type="bibr">16</xref><xref rid="ref17" ref-type="bibr">17</xref><xref rid="ref18" ref-type="bibr">18</xref><xref rid="ref19" ref-type="bibr">19</xref><xref rid="ref20" ref-type="bibr">20</xref>] Salvage robotic radical prostatectomy (sRRP) has shown promising early results and appears to be an excellent alternative to open SRP. As for any salvage procedure, prudent patient selection and surgical experience remain crucial for optimal perioperative and oncologic outcomes.</p><p>Since 2008, at least six series of sRRP have been published, encompassing approximately 134 patients.[<xref rid="ref13" ref-type="bibr">13</xref><xref rid="ref14" ref-type="bibr">14</xref><xref rid="ref15" ref-type="bibr">15</xref><xref rid="ref16" ref-type="bibr">16</xref><xref rid="ref17" ref-type="bibr">17</xref><xref rid="ref19" ref-type="bibr">19</xref><xref rid="ref20" ref-type="bibr">20</xref>] In our experience to date, sRRP has been safe and efficacious, with excellent early oncologic results, and has become our preferred method of performing SRP. The precision and dexterity of the robotic instrumentation combined with the superior visualization offered by the three-dimensional magnification of the operative field all facilitate the challenging aspects of the operation-namely, scarring and loss of tissue planes.</p><sec id="sec2-1"><title/><sec id="sec3-1"><title>Patient selection</title><p>Several groups have sought to define disease characteristics associated with poor outcomes after SRP in order to assist in proper patient selection.[<xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref19" ref-type="bibr">19</xref>] Patient selection is particularly important in the salvage setting given the potentially substantial impact on quality of life and narrow risk–benefit ratio when compared with prostatectomy in the primary setting. In general, patients selected for sRRP should have a life expectancy of at least 10-15 years and should have clinical stage ≤ T3, post-radiation failure prostate biopsy-proven localized disease with no evidence of metastases on pre-operative imaging. Computerized tomography (CT) scanning can be useful to identify nodal metastases and magnetic resonance imaging (MRI) can be useful to identify the local extent of disease. Other pre-operative clinical characteristics reflective of possible systemic disease are elevated pre-sRRP prostate-specific antigen (PSA) and high pre-operative biopsy Gleason score, short PSA doubling time and short time from primary therapy to recurrence. Pre-operative PSA and biopsy Gleason score have been shown to be predictive of BCR following SRP as well as for the development of metastatic disease.[<xref rid="ref6" ref-type="bibr">6</xref>]</p><p>A thorough discussion should be undertaken with patients giving consideration to sRRP encompassing the substantial risk of impotence and incontinence as well as the increased risk of perioperative complications including the small but increased risk of rectal laceration. We recommend pre-operative mechanical bowel preparation with one bottle of magnesium citrate the day prior to surgery and a clear liquid diet until midnight the night before surgery. We do not recommend the neoadjuvant administration of androgen deprivation therapy as an adjunct to surgery as it has not been shown to be useful.</p></sec><sec id="sec3-2"><title>Operative technique</title><p>The surgical technique for sRRP does not significantly depart from that of standard robotic-assisted laparoscopic prostatectomy (RALP). We utilize the six-port transperitoneal approach for both standard RALP and sRRP. The patient is placed in the dorsal lithotomy position and the arms are tucked and padded. After confirming stability on the bed, a Foley catheter is placed on the field and the bladder is emptied. We do not administer pharmaceutical deep vein thrombosis prophylaxis prior to RALP or sRRP. After receiving a single intravenous dose of a first-generation cephalosporin, the patient is positioned in the steep Trendelenburg position after insufflation and the ports are placed in the standard configuration, with a 12 mm supra-umbilical trocar, three robotic trocars, a right lower quadrant assistant 12 mm trocar and, if necessary, an additional 5 mm right-sided assistant trocar.</p><p>While others have utilized a posterior approach to first dissect free the seminal vesicles and the plane between the prostate and the rectum before taking down the bladder, we generally perform an anterior approach for both standard RALP and sRRP.[<xref rid="ref20" ref-type="bibr">20</xref>] Many surgeons-especially those with less experience-may find it easier to identify the posterior plane with the posterior approach, although in our experience the bladder neck (anterior) approach is preferable. For any given patient, either approach is acceptable and it is to the discretion of the individual surgeon based upon training and experience as to which approach is utilized. The initial steps of sRRP are identical to a standard RALP-the peritoneum is incised lateral to each medial umbilical ligament and the space of Retzius is developed after division of the urachus and medial umbilical ligaments. The periprostatic fatty tissue is dissected free and the superficial dorsal venous branches are cauterized and divided, exposing the endopelvic fascia. The effects of prior radiation therapy are often readily apparent at the level of the endopelvic fascia, which is frequently thickened, fibrotic and adherent to the underlying tissues [<xref ref-type="fig" rid="F1">Figure 1</xref>]. The endopelvic fascia is carefully incised with judicious cautery as required to cut through the thick tissue [<xref ref-type="fig" rid="F2">Figure 2</xref>]. During salvage procedures, we generally omit ligation of the deep dorsal venous complex at this point to allow for improved mobility during dissection of the prostatic apex. Furthermore, it has been our experience that dorsal venous complex bleeding is minimal in the salvage setting due to radiation effects.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Fibrotic and thickened endopelvic fascia in a patient undergoing salvage robotic radical prostatectomy after external beam radiation therapy</p></caption><graphic xlink:href="IJU-30-429-g001"/></fig><fig id="F2" position="float"><label>Figure 2</label><caption><p>The thickened right endopelvic fascia is incised</p></caption><graphic xlink:href="IJU-30-429-g002"/></fig><p>The bladder neck is then identified and incised as per a standard RALP and the bilateral vasa deferentia are divided and the seminal vesicles dissected free. Complete excision of the seminal vesicles is prudent during sRRP as 30–35% of patients will have seminal vesicle invasion.[<xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref19" ref-type="bibr">19</xref>] The posterior plane is then developed. Although the inter-fascial plane above the Denonvillier's fascia can be quite adherent to the prostate, Denonvillier's fascia is relatively inviolate and the plane posterior to Denonvillier's fascia is generally well preserved. Furthermore, developing this plane anterior to the perirectal fat allows for wide excision of the prostate in case of extracapsular extension [<xref ref-type="fig" rid="F3">Figure 3</xref>]. The improved visualization of this plane with the robotic platform greatly facilitates this dissection, although caution is still required. Because of the high incidence of locally advanced disease in this population, we generally perform a wide excision of the lateral prostatic fascia and neurovascular bundle to optimize oncologic outcomes.</p><fig id="F3" position="float"><label>Figure 3</label><caption><p>Dissection of the posterior plane deep to Denonvillier's fascia is almost completed. The rectum is tented up as the last remaining attachments between posterior prostatic apex and rectum are divided</p></caption><graphic xlink:href="IJU-30-429-g003"/></fig><p>The apex of the prostate is often quite immobile in the post-radiation setting and, in the case of primary brachytherapy, the posterior prostatic apex is often particularly fibrotic and adherent [<xref ref-type="fig" rid="F4">Figure 4</xref>]. Therefore, the lateral margins of the prostatic are completely freed in order to allow full mobilization of the prostate prior to division of the dorsal venous complex. The prostate is retracted posteriorly and cranially with the fourth arm as the dorsal venous complex is divided and then laterally as needed to improve visualization of the urethra and posterior apex. The urethra is sharply divided, the catheter is removed and any remaining apical tissue is dissected free. The improved visualization provided by robotic approach is remarkably useful at this point as periapical fibrosis may obscure the boundaries of the prostate. Additional margins can be taken as needed. Further hemostatic sutures can be placed in the dorsal venous complex at this time if required.</p><fig id="F4" position="float"><label>Figure 4</label><caption><p>The dorsal venous complex has been divided. Note the marked periapical fibrosis</p></caption><graphic xlink:href="IJU-30-429-g004"/></fig><p>A standard bilateral pelvic lymphadenectomy is generally performed and, although we have become more aggressive with node dissections in patients with advanced disease in recent years, the improved staging and possibility of trivial survival benefit with lymphadenectomy must be weighed against the increased morbidity of a surgical node dissection. This is particularly relevant in patients with favorable disease characteristics. The vesicourethral anastomosis is then performed in the standard fashion and a surgical drain is placed through the left lateral robotic trocar site only if a lymphadenectomy was performed.</p></sec><sec id="sec3-3"><title>Post-operative care</title><p>Post-operative management after sRRP follows our standard RALP pathway, which has been previously published and includes early ambulation, a full liquid diet on the first post-operative morning, intravenous ketorolac to reduce the utilization of narcotics and an aggressive bowel regimen including milk of magnesia and bisacodyl suppositories.[<xref rid="ref21" ref-type="bibr">21</xref>] Surgical drains are generally removed on post-operative day one, unless the output is greater than 200 cc over 24 hours. It has been our experience that salvage status should not prolong length of hospital stay, and greater than 94% of patients undergoing sRRP have been discharged on the first post-operative day.[<xref rid="ref19" ref-type="bibr">19</xref>] Post-operative cystography is rarely performed and Foley catheters are left in place for 10-14 days.</p></sec><sec id="sec3-4"><title>Complications</title><p>Complication rates in published sRRP series are generally comparable to or lower than those of contemporary open SRP series. Major complications are uncommon and a distinct advantage of performing SRP with robotic assistance is decreased blood loss. Median estimated blood loss has ranged from 75 cc to 175 cc in published series, with no blood transfusions or conversions from laparoscopic to open in all 134 patients.[<xref rid="ref13" ref-type="bibr">13</xref><xref rid="ref14" ref-type="bibr">14</xref><xref rid="ref15" ref-type="bibr">15</xref><xref rid="ref16" ref-type="bibr">16</xref><xref rid="ref17" ref-type="bibr">17</xref><xref rid="ref19" ref-type="bibr">19</xref><xref rid="ref20" ref-type="bibr">20</xref>] Another distinct advantage of the robotic approach is the low rate of anastomotic stricture, ranging from 0% to 17% in the six sRRP series.[<xref rid="ref14" ref-type="bibr">14</xref><xref rid="ref15" ref-type="bibr">15</xref><xref rid="ref16" ref-type="bibr">16</xref><xref rid="ref17" ref-type="bibr">17</xref><xref rid="ref19" ref-type="bibr">19</xref><xref rid="ref20" ref-type="bibr">20</xref>] Even in contemporary open SRP series, anastomotic stricture rates remain troublesome, ranging from 11% to 30%.[<xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref10" ref-type="bibr">10</xref><xref rid="ref11" ref-type="bibr">11</xref>] A small percentage (0–33%) of patients develop anastomotic leaks following sRRP requiring prolonged catheterization.[<xref rid="ref14" ref-type="bibr">14</xref><xref rid="ref15" ref-type="bibr">15</xref><xref rid="ref16" ref-type="bibr">16</xref><xref rid="ref17" ref-type="bibr">17</xref><xref rid="ref19" ref-type="bibr">19</xref><xref rid="ref20" ref-type="bibr">20</xref>] Four patients with thrombotic complications (deep vein thrombosis or pulmonary emboli) have been reported.[<xref rid="ref17" ref-type="bibr">17</xref><xref rid="ref19" ref-type="bibr">19</xref><xref rid="ref20" ref-type="bibr">20</xref>] Two enterotomies during lysis of adhesions have also been reported.[<xref rid="ref16" ref-type="bibr">16</xref><xref rid="ref20" ref-type="bibr">20</xref>] Of the 134 patients undergoing sRRP in published series, only two (1.5%) rectal injuries have been described-a rate lower than most contemporary open SRP series, which have ranged from 2% to 4%.[<xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref10" ref-type="bibr">10</xref><xref rid="ref11" ref-type="bibr">11</xref><xref rid="ref14" ref-type="bibr">14</xref><xref rid="ref15" ref-type="bibr">15</xref><xref rid="ref16" ref-type="bibr">16</xref><xref rid="ref17" ref-type="bibr">17</xref><xref rid="ref19" ref-type="bibr">19</xref><xref rid="ref20" ref-type="bibr">20</xref>] If a rectal laceration occurs, we recommend primary, multilayer closure, tissue interposition and strong consideration for fecal diversion given the increased propensity for rectourethral fistula in the salvage setting.</p></sec><sec id="sec3-5"><title>Oncologic outcomes</title><p>The assessment of the oncologic efficacy of sRRP is limited at this point with a median follow-up ranging from 4 to 36 months in published series.[<xref rid="ref14" ref-type="bibr">14</xref><xref rid="ref15" ref-type="bibr">15</xref><xref rid="ref16" ref-type="bibr">16</xref><xref rid="ref17" ref-type="bibr">17</xref><xref rid="ref19" ref-type="bibr">19</xref><xref rid="ref20" ref-type="bibr">20</xref>] Despite this, preliminary results are encouraging. Of the 134 sRRP patients described in the literature, 34 (25%) had positive margins, comparable to contemporary open SRP margin rates, which have ranged from 11% to 33%.[<xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref13" ref-type="bibr">13</xref><xref rid="ref14" ref-type="bibr">14</xref><xref rid="ref15" ref-type="bibr">15</xref><xref rid="ref16" ref-type="bibr">16</xref><xref rid="ref17" ref-type="bibr">17</xref><xref rid="ref19" ref-type="bibr">19</xref><xref rid="ref20" ref-type="bibr">20</xref><xref rid="ref22" ref-type="bibr">22</xref>] Positive margins have been shown to be associated with BCR following radical prostatectomy in the primary and salvage settings.[<xref rid="ref22" ref-type="bibr">22</xref><xref rid="ref23" ref-type="bibr">23</xref>]</p><p>Of the 117 patients in published series undergoing sRRP who received a bilateral pelvic lymphadenectomy, seven (5.2%) were detected to have positive nodes.[<xref rid="ref14" ref-type="bibr">14</xref><xref rid="ref15" ref-type="bibr">15</xref><xref rid="ref16" ref-type="bibr">16</xref><xref rid="ref17" ref-type="bibr">17</xref><xref rid="ref19" ref-type="bibr">19</xref><xref rid="ref20" ref-type="bibr">20</xref>] In our experience, six patients (18%) developed BCR with 16 months of median follow-up.[<xref rid="ref19" ref-type="bibr">19</xref>] In another large series of 55 patients undergoing sRRP, 10 (18%) developed BCR with a median follow-up of 36 months.[<xref rid="ref20" ref-type="bibr">20</xref>] These results are similar to the 25% BCR rate at a median follow-up of 16 months reported by a large multi-institutional series of more than 400 patients receiving open SRP.[<xref rid="ref6" ref-type="bibr">6</xref>]</p></sec><sec id="sec3-6"><title>Functional outcomes</title><p>Functional outcomes following SRP are generally worse than radical prostatectomy performed in the primary setting. While follow-up is limited in many sRRP series, continence rates (0-1 pad per day) have ranged from 33% to 80%, with the two largest series reporting rates of 39-45%.[<xref rid="ref14" ref-type="bibr">14</xref><xref rid="ref15" ref-type="bibr">15</xref><xref rid="ref16" ref-type="bibr">16</xref><xref rid="ref17" ref-type="bibr">17</xref><xref rid="ref19" ref-type="bibr">19</xref><xref rid="ref20" ref-type="bibr">20</xref>] Reported continence rates will likely improve as sRRP series mature and follow-up increases.</p><p>Pre-operative and post-operative erectile function in the post-radiation setting is universally poor. The majority of patients have impaired pre-operative erectile function-only 21-23% of patients were considered potent pre-operatively in the two largest sRRP series.[<xref rid="ref19" ref-type="bibr">19</xref><xref rid="ref20" ref-type="bibr">20</xref>] High rates of post-operative erectile dysfunction have been a consistent finding in other sRRP series and, although limited follow-up may underestimate improvements in erectile function, potency rates in contemporary, mature open SRP series are invariably poor.[<xref rid="ref6" ref-type="bibr">6</xref><xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref11" ref-type="bibr">11</xref><xref rid="ref14" ref-type="bibr">14</xref><xref rid="ref15" ref-type="bibr">15</xref><xref rid="ref16" ref-type="bibr">16</xref><xref rid="ref17" ref-type="bibr">17</xref>]</p></sec></sec></sec><sec sec-type="conclusions" id="sec1-2"><title>CONCLUSIONS</title><p>Performance of radical prostatectomy in the salvage setting, whether open or robotic, is a technically formidable procedure. While this is in part related to the radiation changes to the tissue and subsequent increased risk of serious complication, it is also related to the increased incidence of locally advanced disease. Therefore, patient selection is crucial and sRRP is not recommended for the novice robotic surgeon. Utilization of robotic assistance is particularly well suited for the performance of radical prostatectomy in the post-radiation setting, and is almost universally our modality of choice in this patient population. Improved visualization and precise tissue handling greatly facilitate performance of the procedure. The literature in support of sRRP to date has demonstrated excellent safety with low rates of perioperative complications. Some outcomes may compare favorably to open SRP series, including low blood loss, short length of stay and decreased anastomotic stricture rates. Furthermore, early oncologic results are encouraging, although improvements in functional outcomes are still required. As follow-up increases and experience is gained, it is hoped that wider and safer delivery of a potentially curative therapeutic option to a difficult patient population may be facilitated by the robotic platform.</p></sec> |
Erectile dysfunction in robotic radical prostatectomy: Outcomes and management | <p>Robot-assisted laparoscopic prostatectomy (RALP) has emerged as the most common treatment for localized prostate cancer. With improved surgical precision, RALP has produced hope of improved potency rates, especially with the advent of nerve-sparing and other modified techniques. However, erectile dysfunction (ED) remains a significant problem for many men regardless of surgical technique. To identify the functional outcomes of robotic versus open and laparoscopic techniques, new robotic surgical techniques and current treatment options of ED following RALP. A Medline search was performed in March 2014 to identify studies comparing RALP with open retropubic radical prostatectomy (RRP) and laparoscopic radical prostatectomy, modified RALP techniques and treatment options and management for ED following radical prostatectomy. RALP demonstrates adequate potency rates without compromising oncologic benefit, with observed benefit for potency rates compared with RRP. Additionally, specific surgical technical modifications appear to provide benefit over traditional RALP. Phosphodiesterase-5 inhibitors (PDE5I) demonstrate benefit for ED treatment compared with placebo. However, long-term benefit is often lost after use. Other therapies have been less extensively studied. Additionally, correct patient identification is important for greatest clinical benefit. RALP appears to provide beneficial potency rates compared with RRP; however, these effects are most pronounced at high-volume centers with experienced surgeons. No optimal rehabilitation program with PDE5Is has been identified based on current data. Additionally, vacuum erection devices, intracavernosal injections and other techniques have not been well validated for post RALP ED treatment.</p> | <contrib contrib-type="author"><name><surname>Whelan</surname><given-names>Patrick</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Ekbal</surname><given-names>Shahid</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Nehra</surname><given-names>Ajay</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib> | Indian Journal of Urology : IJU : Journal of the Urological Society of India | <sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Prostate cancer (CaP) remains the most common malignancy in men in the United States.[<xref rid="ref1" ref-type="bibr">1</xref>] Retropubic radical prostatectomy (RRP) remains the gold standard for locally invasive disease;[<xref rid="ref2" ref-type="bibr">2</xref>] however, robot-assisted laparoscopic prostatectomy (RALP) has emerged as the most common technique.[<xref rid="ref3" ref-type="bibr">3</xref>] Although RALP has reduced complications and length of stay,[<xref rid="ref4" ref-type="bibr">4</xref>] there is still unclear evidence whether RALP reduces erectile dysfunction (ED) post-operatively. Data show that 60% of men report ED 18 months post-operatively,[<xref rid="ref5" ref-type="bibr">5</xref>] 20% report erections strong enough for intercourse at —5 years of follow-up[<xref rid="ref5" ref-type="bibr">5</xref>] and only 20% of men return to pre-operative erectile function at —1 year post-operatively.[<xref rid="ref6" ref-type="bibr">6</xref>] These findings have led to the development of penile rehabilitation programs to improve long-term erectile function. The functional outcomes of robotic versus open and laparoscopic techniques, new robotic surgical techniques and current treatment options and management of ED following RALP are examined in this paper.</p></sec><sec sec-type="materials|methods" id="sec1-2"><title>MATERIALS AND METHODS</title><p>A systematic review using the Medline database was performed in March 2014. The search terms included “radical prostatectomy,” “erectile dysfunction” and “robot” and “radical prostatectomy,” “erectile dysfunction” and “robotic” with the following limits used: Humans, English and gender (male). Articles were screened using abstracts and those selected underwent full review. Two hundred and thirty-three articles were obtained. All articles’ abstracts were reviewed. Those that compared RALP with RRP or laparoscopic radical prostatectomy (LRP) and articles comparing modified RALP techniques with traditional RALP were included. All articles’ abstracts were reviewed. To identify treatment options for ED post-RALP, a similar Medline database search was performed. The search terms included “radical prostatectomy,” “erectile dysfunction” and “penile rehabilitation.” One hundred and twenty articles were obtained. All abstracts were reviewed. Given the numerous articles available for such a broad subject matter, those that are included in this article were determined to be most critical to the subject matter.</p></sec><sec sec-type="results" id="sec1-3"><title>RESULTS</title><sec id="sec2-1"><title/><sec id="sec3-1"><title>Erectile function outcomes following RRP</title><p>After a thorough review of the articles obtained, 23 articles addressing ED following RALP were included in this review as outlined in <xref ref-type="table" rid="T1">Table 1</xref>. Improved surgical precision and reduced complication rates have provided hope that RALP provides greater potency rates with preservation of oncologic outcomes. A meta-analysis by Ficarra <italic>et al</italic>. identified 31 studies that assessed potency following RALP.[<xref rid="ref7" ref-type="bibr">7</xref>] At 1 and —2 years, the potency rates were 70% (54-90%) and 79% (63-94%), respectively. When including only studies that fulfilled six or more of the Mulhall criteria, the 1- and 2-year potency rates were 76% (62-90%) and 82% (69-94%), respectively. The Mulhall criteria were developed to better assess the validity of reported ED rates following radical prostatectomy (RP).[<xref rid="ref8" ref-type="bibr">8</xref>] Criteria from the analysis determined to be most important were study population factors, population demographics, means of data acquisition, variability in questionnaire use, duration post-operatively at evaluation, baseline erectile function status, the definition of adequate erectile function and the definition of quality and consistency of erection. When comparing multiple studies, it is easier to compare the results if a greater number of these criteria are met.</p><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Studies included in the analysis for ED outcomes following RALP</p></caption><graphic xlink:href="IJU-30-434-g001"/></table-wrap><p>In the same study, seven studies compared RALP with RRP.[<xref rid="ref7" ref-type="bibr">7</xref>] The 1-year potency rates were superior for RALP (OR 2.84, <italic>P</italic> = 0.002). A recent meta-analysis from Moran <italic>et al</italic>.[<xref rid="ref9" ref-type="bibr">9</xref>] identified nine studies comparing potency following RALP and RRP. Men undergoing RALP were more likely to regain sexual function at —1 year post-operatively than RRP (RR 1.60, <italic>P</italic> < 0.001). One randomized control study produced 1-year potency rates of 77% and 32% (<italic>P</italic> < 0.0001) in 52 and 64 men who underwent RALP and LRP, respectively.[<xref rid="ref10" ref-type="bibr">10</xref>] However, meta-analyses of RALP versus LRP have only shown a trend in favor of RALP in potency recovery (OR 1.89, <italic>P</italic> = 0.21;[<xref rid="ref7" ref-type="bibr">7</xref>] RR 1.49, <italic>P</italic> = 0.392[<xref rid="ref9" ref-type="bibr">9</xref>]).</p><p>Novara <italic>et al</italic>. analyzed the potency rates (International Index of Erectile Function-Erectile Function domain [IIEF-EF] >18) of 208 men who underwent bilateral nerve sparing (BNS) RALP.[<xref rid="ref11" ref-type="bibr">11</xref>] Potency was 62% at 12 months post-operatively. Independent predictors of potency were age (HR 2.828, <italic>P</italic> < 0.001), Charlson comorbidity index (CCI) (HR 2.992, <italic>P</italic> = 0.007) and baseline IIEF-EF score (HR 0.843, <italic>P</italic> < 0.001). The potency rates were 81.9%, 56.7% and 28.6% (<italic>P</italic> < 0.001) for the low-, intermediate- and high-risk groups, respectively, as proposed by Briganti <italic>et al</italic>.[<xref rid="ref12" ref-type="bibr">12</xref>] Further, substratification of the intermediate-risk group to pre-operative IIEF-EF scores of 18-21 and 11-17 produced potency rates of 68.8% and 27% (<italic>P</italic> < 0.001), respectively. A prospective comparative study of 609 patients treated with BNS RALP or RRP[<xref rid="ref13" ref-type="bibr">13</xref>] stratified the patients similarly.[<xref rid="ref12" ref-type="bibr">12</xref>] The 2-year potency rates (IIEF-EF > 22) were higher in the overall, low- and intermediate-risk populations for RALP versus RRP (67.8% vs. 52.1%, <italic>P</italic> < 0.001; 87.6% vs. 77.5%, <italic>P</italic> < 0.001; 67.2% vs. 55.7%, <italic>P</italic> < 0.001). Further studies have shown that age (OR 0.92, <italic>P</italic> < 0.0001;[<xref rid="ref14" ref-type="bibr">14</xref>] OR 0.95, <italic>P</italic> = 0.004[<xref rid="ref15" ref-type="bibr">15</xref>]), baseline Sexual Health Inventory of Men (SHIM) score (OR 1.1, <italic>P</italic> < 0.0001),[<xref rid="ref14" ref-type="bibr">14</xref>] erection suitable for intercourse (ESI) at baseline (OR 0.95, <italic>P</italic> = 0.019)[<xref rid="ref15" ref-type="bibr">15</xref>] and BNS (OR 2.92, <italic>P</italic> < 0.001)[<xref rid="ref14" ref-type="bibr">14</xref>] were independently associated with recovering erectile function. However, this is in contrast to data reporting 87.5% and 89% of Medicare-aged men having “moderate or big problems with sexual function” for RALP and RRP, respectively, at an average of 14 months of follow-up.[<xref rid="ref16" ref-type="bibr">16</xref>]</p><p>Several attempts at modified RALP techniques have been performed and the results are shown in <xref ref-type="table" rid="T2">Table 2</xref>.</p><table-wrap id="T2" position="float"><label>Table 2</label><caption><p>Potency rates observed during various modified RALP techniques</p></caption><graphic xlink:href="IJU-30-434-g002"/></table-wrap><p>Data comparing extraperitoneal versus transperitoneal BNS RALP did not identify a difference in the 12-month potency rates.[<xref rid="ref17" ref-type="bibr">17</xref>] Comparing cautery and non-cautery techniques has produced conflicting results, with Ahlering <italic>et al</italic>. showing a benefit with the cautery-free technique[<xref rid="ref18" ref-type="bibr">18</xref>] but Samadi <italic>et al</italic>. not demonstrating any benefit with the athermal technique in a larger study.[<xref rid="ref19" ref-type="bibr">19</xref>] Traction-free techniques have also been conflicting. In a technique where O<sub>2</sub> tissue monitoring allowed intraoperative surgical modification for reduced traction, benefit was noted.[<xref rid="ref20" ref-type="bibr">20</xref>] However, a study from Kowalczyk <italic>et al</italic>. did not demonstrate benefit in the traction-free technique at 12 months.[<xref rid="ref15" ref-type="bibr">15</xref>] Thus far, only retrograde[<xref rid="ref21" ref-type="bibr">21</xref>] and intraoperative cooling[<xref rid="ref22" ref-type="bibr">22</xref>] dissection have proven beneficial in potency rates.</p><p>The Veil of Aphrodite technique was developed to provide the greatest nerve sparing (NS) possible. Now known as the Vattikuti Institute prostatectomy (VIP) technique, the technique has been further modified and now involves “superveiling,” bladder drainage through a suprapubic tube and limited obturator and internal iliac node dissection with preference to the external iliac nodes for low- and intermediate-risk patients.[<xref rid="ref23" ref-type="bibr">23</xref>] In 85 men with these modifications, 94% of men were able to complete successful intercourse at 6-18 months post-operatively with an average SHIM score = 18.[<xref rid="ref23" ref-type="bibr">23</xref>] These modifications are in addition to previously described early transection of the bladder neck, preservation of the prostatic fascia and control of the dorsal vein complex after dissection of the prostatic apex.[<xref rid="ref24" ref-type="bibr">24</xref>] Analysis of the original VIP technique of 1142 men with a pre-operative SHIM score >21 showed that 93% of men reported ESI with or without phosphodiesterase-5 inhibitors (PDE5Is) at >1 year follow-up.[<xref rid="ref24" ref-type="bibr">24</xref>] However, only 51% returned to baseline. A prospective, non-randomized analysis of pre-operatively potent (SHIM ≥ 18) men undergoing VIP (<italic>n</italic> = 200) compared with RRP (<italic>n</italic> = 100) showed that return to erection and intercourse were 180 versus 440 days (<italic>P</italic> < 0.05).[<xref rid="ref25" ref-type="bibr">25</xref>]</p><p>Although most studies broadly classify patients who have undergone nerve sparing radical prostatectomy (NSRP), the NS technique is not an all-or-none technique. Correlation between degree of NS was shown, where potency rates for 1335 men undergoing RALP with >1 year follow-up and pre-operative SHIM score > 21 were 90.6, 76.2, 60.5 and 57.1% for NS grades 1, 2, 3 and 4, respectively (<italic>P</italic> < 0.001).[<xref rid="ref26" ref-type="bibr">26</xref>] Additionally, comparison of interfascial and extrafascial NS technique produced 12-month potency rates of 64% and 40% (<italic>P</italic> = 0.02), respectively.[<xref rid="ref27" ref-type="bibr">27</xref>] Additionally, men with larger prostates (>100 vs. <50 g) have decreased post-operative potency rates (61.9% vs. 72.9%, <italic>P</italic> < 0.05) at 12 months post-operatively.[<xref rid="ref28" ref-type="bibr">28</xref>] When examining extended pelvic lymph node dissection (PLND) in a single-center study of 561 men (SHIM ≥ 17) who underwent RALP, men with a lymph node yield ≥20 and <20 reported potency rates of 55.2% and 70%, respectively (<italic>P</italic> = 0.020<sup>)</sup>.[<xref rid="ref29" ref-type="bibr">29</xref>]</p></sec><sec id="sec3-2"><title>Timing and patient selection</title><p>After a thorough review, 17 articles were determined to be most relevant for clinical application of treatment of ED post-RALP. Those addressing PDE5Is are shown in <xref ref-type="table" rid="T3">Table 3</xref>. All other forms of post-RALP ED treatment are shown in <xref ref-type="table" rid="T4">Table 4</xref>. The purpose of penile rehabilitation has been proposed to prevent alterations of the smooth muscle of the corpora cavernosa, limit venous leak development and maximize the chances of returning to pre-operative erectile function.[<xref rid="ref30" ref-type="bibr">30</xref>] Iacono <italic>et al</italic>. demonstrated increased evidence of fibrosis at increasing time points post-operatively.[<xref rid="ref31" ref-type="bibr">31</xref>] In a study of bilateral NSRP patients, 84 subjects were divided into a penile rehabilitation program starting either less than 6 months or >6 months after surgery.[<xref rid="ref30" ref-type="bibr">30</xref>] Two years post-operatively, the early rehabilation group reported superior IIEF-EF scores (22 vs. 16, <italic>P</italic> < 0.001), higher Sildenafil-assisted erection rates (86% vs. 45%, <italic>P</italic> < 0.01), and higher Sildenafil-unassisted erection rates (58% vs. 30%, <italic>P</italic> < 0.01) than the delayed rehabilitation group.</p><table-wrap id="T3" position="float"><label>Table 3</label><caption><p>Study design and primary outcomes of long-term, randomized control trials evaluating PDE5Is for penile rehabilitation after RP</p></caption><graphic xlink:href="IJU-30-434-g003"/></table-wrap><table-wrap id="T4" position="float"><label>Table 4</label><caption><p>Studies included in analysis for ED management following RALP</p></caption><graphic xlink:href="IJU-30-434-g004"/></table-wrap><p>Compliance with therapy is important as potency recovery may require 1 year or more. Seventy-seven men were prospectively followed after NS RALP and enrolled in a penile rehabilitation program with sildenafil or tadalafil three times weekly.[<xref rid="ref32" ref-type="bibr">32</xref>] At <2 months after RALP, 32% of men stopped therapy and 39% or more stopped therapy by 6 months, with high cost (65%) being the primary reason. Pre-operative ED and long-term compliance were independent predictors of potency.</p><p>Active surveillance (AS) is frequently employed for the management of CaP. A group of 367 low-risk patients post-RALP, who were well matched and divided into single and multiple biopsy groups, reported 6-month potency rates of 80% and 57% (<italic>P</italic> = 0.03), respectively.[<xref rid="ref33" ref-type="bibr">33</xref>]</p></sec><sec id="sec3-3"><title>PDE5Is</title><p>PDE5Is have emerged as the gold standard of treatment for ED. Several animal models have demonstrated histological and functional benefit with PDE5Is in animal studies.[<xref rid="ref34" ref-type="bibr">34</xref><xref rid="ref35" ref-type="bibr">35</xref><xref rid="ref36" ref-type="bibr">36</xref><xref rid="ref37" ref-type="bibr">37</xref>] Although the pathways behind these results are not clear, it has been theorized that PDE5Is can provide a protective role to preventing post-operative ED following RP.</p><p>Currently, three large, randomized, double-blind, placebo-controlled studies evaluating PDE5Is as penile rehabilitation following RP have been performed. Additionally, a randomized control trial without placebo evaluating sildenafil was also performed. Trial designs and primary outcomes are outlined in <xref ref-type="table" rid="T3">Table 3</xref>.</p><p>Padma-Nathan <italic>et al</italic>. were the first to demonstrate benefit from PDE5I use versus placebo.[<xref rid="ref38" ref-type="bibr">38</xref>] Additionally, the IIEF-EF score was higher in the sildenafil group compared with placebo (13.1 vs. 8.8). However, the study was terminated prematurely after the interim review showed response rates less than expected compared with the rates of spontaneous recovery in the literature.</p><p>Mortosi <italic>et al</italic>. demonstrated that although on-demand vardenafil was associated with higher IIEF-EF scores after 9 months (<italic>P</italic> < 0.0001), benefit was lost after drug-free washout (DFW).[<xref rid="ref39" ref-type="bibr">39</xref>] Similarly, although benefit was noted with SEP-3 success rates after 9 months with nightly and on-demand use (34.5% vs. 25.0%, <italic>P</italic> = 0.0344; 45.9% vs. 25.0%, <italic>P</italic> < 0.0001), benefit was lost after DFW. Vardenafil on-demand was also superior to vardenafil nightly for SEP-3 success rates (45.9% vs. 34.5%, <italic>P</italic> = 0.0114).</p><p>Bannowsky demonstrated superior IIEF-EF scores with sildenafil compared with no treatment.[<xref rid="ref40" ref-type="bibr">40</xref>] Additionally, spontaneous erection rates were 47% and 28% for men receiving sildenafil and no treatment, which improved to 86% and 66%, respectively, with the use of sildenafil on-demand.</p><p>Montorsi <italic>et al</italic>. demonstrated that although nightly tadalafil was associated with a greater percentage of men with IIEF-EF ≥22 after 9 months versus placebo (25.2% vs. 14.2%, <italic>P</italic> = 0.016), benefit was lost after DFW.[<xref rid="ref41" ref-type="bibr">41</xref>] Similarly, although nightly tadalafil demonstrated superior SEP-1-5 response rates at 9 months, benefit was lost after DFW. However, superior SEP-1 and -2 success rates were observed after open-label therapy for both formulations of tadalafil. Further statistical analysis demonstrated positive response for tadalafil nightly versus placebo (<italic>P</italic> = 0.019) at 9 months and tadalafil nightly at 9 months (<italic>P</italic> = 0.007) and 13.5 months (<italic>P</italic> = 0.010), respectively. Significantly less penile shrinkage was noted in the tadalafil nightly group versus placebo at 9 months (2.2 mm vs. 6.3 mm, <italic>P</italic> = 0.003).</p><p>Briganti <italic>et al</italic>. proposed that the correct selection of patients is critical to those who will benefit from a penile rehabilitation program.[<xref rid="ref12" ref-type="bibr">12</xref>] A retrospective study divided 435 men post-bilateral nerve sparing radical prostatectomy (BNSRP) into three groups based on risk of ED post-operatively: Low (age < 65 years or IIEF-EF = 26 or CCI < 1 [<italic>n</italic> = 184]), intermediate (age 66-69 years or IIEF-EF = 11-25 or CCI > 1 [<italic>n</italic> = 115]) and high (age > 70 years or IIEF-EF <10 or CCI > 2 [<italic>n</italic> = 136]). The population included 193 men untreated after surgery; 147 using on-demand PDE5Is and 95 using daily/every other day PDE5Is. Potency (IIEF-EF > 22) was greater in those receiving any PDE5Is compared with no treatment. Only in the intermediate-risk group was potency superior with daily versus on-demand PDE5I use (74% vs. 52%, <italic>P</italic> = 0.02).</p></sec><sec id="sec3-4"><title>Vacuum erection device</title><p>VED benefit was first demonstrated in several markers of inflammation, fibrosis and erectile function in animal models.[<xref rid="ref42" ref-type="bibr">42</xref><xref rid="ref43" ref-type="bibr">43</xref>] Recently, a study randomized 20 men post-BNSRP to tadalafil 20 mg three times weekly with or without VED.[<xref rid="ref44" ref-type="bibr">44</xref>] The combination group had higher SHIM scores at 6, 9 and 12 months and higher penile hardness scores at 6 and 9 months. In the combination and tadalafil groups, 92% and 57% of patients, respectively, were able to achieve erection satisfactory for vaginal penetration.</p><p>Other results with VED use have been less conclusive. A study of 28 men post-unilateral nerve sparing or BNSRP randomized patients to receive penile rehabilitation with VED starting at 1 or 6 months post-operatively.[<xref rid="ref45" ref-type="bibr">45</xref>] No spontaneous erections adequate for intercourse or difference of IIEF scores were observed at 12 months. Stretched penile length was greater in the early intervention group (<italic>P</italic> < 0.044); however, there was no difference in either group relative to their own pre-operative measurements.</p><p>A study randomized 109 men to daily VED for 9 months after RP or no treatment.[<xref rid="ref46" ref-type="bibr">46</xref>] After 9 months, the SHIM score was higher in the treatment group (16 vs. 11.1, <italic>P</italic> < 0.05). However, there was no difference in the spontaneous erection rates or ESI rate. In those who quit and completed VED use, 85% versus 23% of patients reported decreased penile length and girth.</p></sec><sec id="sec3-5"><title>Intraurethral injections/intracavernosal injections</title><p>Prostaglandin E1 (PGE1) causes erection via vasodilation and smooth muscle relaxation to expand the corpora. Montorsi <italic>et al</italic>. first demonstrated penile rehabilitation benefit using ICI of PGE1 three times weekly or no treatment for 12 weeks in men post-NSRP.[<xref rid="ref47" ref-type="bibr">47</xref>] The ICI and no treatment groups reported potency rates (needing ICI < 50% of attempts at intercourse) of 67% and 20% (<italic>P</italic> < 0.01), respectively.</p><p>A non-randomized study of 132 men post-RP examined 58 men receiving penile rehabilitation (23% used PDE5Is, 77% required ICI).[<xref rid="ref48" ref-type="bibr">48</xref>] The non-rehab group was composed of 74 patients who did not desire rehabilitation. The rehab group and non-rehab group reported spontaneous ESI rates of 52% and 19% (<italic>P</italic> < 0.01), respectively, at 18 months post-operatively. Additionally, those who underwent rehab had improved responses to sildenafil (64% vs. 24%, <italic>P</italic> < 0.001) and ICI (95% vs. 76%, <italic>P</italic> < 0.01).</p><p>A study of medicated urethral system for erections (MUSE) therapy randomized 139 men following BNSRP to intraurethral alprostadil or nightly sildenafil 50 mg for 1 months, 1 month DFW and open-label sildenafil 100 mg for 1 month.[<xref rid="ref49" ref-type="bibr">49</xref>] There were no differences in IIEF-EF scores after completion of therapy. A non-randomized study of 91 men following BNSRP investigated 56 men with MUSE therapy (three times per week for 6 months) and 35 men with no treatment.[<xref rid="ref50" ref-type="bibr">50</xref>] ESI rates of 74% and 37% and SHIM scores of 18.9 and 15.8 were reported in each group.</p></sec><sec id="sec3-6"><title>Psychosocial and alternative interventions</title><p>Psychological factors play an important role for sexual function after RALP.[<xref rid="ref51" ref-type="bibr">51</xref><xref rid="ref52" ref-type="bibr">52</xref>] A randomized study of 101 men post-RP showed that cognitive-behavioral stress management for 10 weeks resulted in improved sexual function 2-3 weeks after the completion of counseling.[<xref rid="ref53" ref-type="bibr">53</xref>] A study of 84 men who had undergone curative treatment for CaP with ED underwent four sessions of counseling.[<xref rid="ref54" ref-type="bibr">54</xref>] Although short-term benefits were observed in several IIEF subscales, most were lost at —6 months of follow-up.</p><p>A study randomized 52 men to receive early post-operative pelvic-floor biofeedback weekly for 3 months or a control group with verbal instructions to contract the pelvic floor.[<xref rid="ref55" ref-type="bibr">55</xref>] The 12-month potency rates (SHIM > 20) were 47.1% and 12.5% in the treatment and control groups, respectively.</p></sec><sec id="sec3-7"><title>Inflatable penile prosthesis</title><p>IPP remains the most definitive surgical treatment for ED refractory to oral or other therapies. However, it should only be used as a last resort, as, once installed, IPP is the only means by which erection can be achieved and natural erections are no longer attainable.</p></sec></sec></sec><sec sec-type="discussion" id="sec1-4"><title>DISCUSSION</title><p>CaP will continue to remain a serious and prevalent disease that requires RP for treatment. Although current robotic techniques are improving, there is still clear evidence that ED will be encountered for men choosing to undergo RALP. All men should be counseled that potency return could take up to 1 year or more. The wide range of reported potency rates (54-94%)[<xref rid="ref7" ref-type="bibr">7</xref>] is likely due to the different inclusions of pre-operative potency, definitions of potency, penile rehabilitation used and surgical technique. The risk stratification proposed by Briganti <italic>et al</italic>.[<xref rid="ref12" ref-type="bibr">12</xref>] has been the best-validated predictor of recovery, which has now been externally validated by Novara <italic>et al</italic>.[<xref rid="ref11" ref-type="bibr">11</xref>] and Gandaglia <italic>et al</italic>.[<xref rid="ref13" ref-type="bibr">13</xref>] Other studies have confirmed that age and baseline erectile function are independent predictors of potency recovery.[<xref rid="ref14" ref-type="bibr">14</xref><xref rid="ref15" ref-type="bibr">15</xref>] This provides the greatest information for educating patients pre-operatively to potency recovery.</p><p>There is evidence that certain surgical techniques performed at high-volume centers lead to more promising results. Nerve sparing techniques have been confirmed to be superior to non-nerve sparing techniques. Unilateral nerve sparing should be considered when bilateral nerve sparing is not feasible for oncologic control. Additionally, although NS techniques are superior, it is evident that the degree of NS leads to different post-operative potency rates.[<xref rid="ref26" ref-type="bibr">26</xref>] Various dissection techniques of fascial planes have been developed[<xref rid="ref27" ref-type="bibr">27</xref>] in addition to the VIP.[<xref rid="ref23" ref-type="bibr">23</xref><xref rid="ref24" ref-type="bibr">24</xref><xref rid="ref25" ref-type="bibr">25</xref>] More accurate description of the NS technique would allow for better comparison of operative techniques. Although retrograde[<xref rid="ref21" ref-type="bibr">21</xref>] and intraoperative cooling[<xref rid="ref22" ref-type="bibr">22</xref>] have demonstrated benefit in potency recovery, these are single-center studies. Although traction-free techniques have been proposed to reduce neuropraxia, results between studies have been conflicting.[<xref rid="ref15" ref-type="bibr">15</xref><xref rid="ref26" ref-type="bibr">26</xref>] Additionally, athermal dissection theoretically results in less damage to the cavernosal nerves. However, similar to traction-free techniques, athermal studies have produced conflicting results.[<xref rid="ref18" ref-type="bibr">18</xref><xref rid="ref19" ref-type="bibr">19</xref>] Nonetheless, there is early benefit from the athermal technique, and it is likely that any potential harm is minimized.</p><p>Two recent meta-analyses have demonstrated that RALP is superior to RRP for potency recovery without oncologic compromise.[<xref rid="ref7" ref-type="bibr">7</xref><xref rid="ref9" ref-type="bibr">9</xref>] This is in addition to the recent prospective control series from Gandaglia <italic>et al</italic>., where RALP demonstrated superior potency rates versus RRP.[<xref rid="ref13" ref-type="bibr">13</xref>] However, no randomized prospective control studies have been performed. Additionally, the use of validated questionnaires has been inconsistent. ESI or various cut-offs of SHIM or IIEF-EF scores have been used. A firmer definition of potency would allow for greater comparison in future studies. The retrospective population study from Barry[<xref rid="ref16" ref-type="bibr">16</xref>] should be interpreted with caution as a non-validated questionnaire was used to assess potency. Patients were not matched for pre-operative erectile function, age, comorbidities or other factors. Additionally, 58% of men were >70 years old, a population which is not ideal population for radical prostatectomy, nerve sparing or with a strong interest in sexual activity.[<xref rid="ref56" ref-type="bibr">56</xref>]</p><p>Recovery from this ED is very important to improving patients’ quality of life. Although the evidence from animal models suggests that PDE5Is can help prevent and provide recovery from ED,[<xref rid="ref34" ref-type="bibr">34</xref><xref rid="ref35" ref-type="bibr">35</xref><xref rid="ref36" ref-type="bibr">36</xref><xref rid="ref37" ref-type="bibr">37</xref>] this has not always translated to humans. PDE5Is provide benefit compared with placebo during active treatment and are an effective treatment for post-RALP ED.[<xref rid="ref38" ref-type="bibr">38</xref><xref rid="ref39" ref-type="bibr">39</xref><xref rid="ref40" ref-type="bibr">40</xref><xref rid="ref41" ref-type="bibr">41</xref>] However, the long-term benefit of PDE5Is in preventing ED has not been demonstrated.[<xref rid="ref38" ref-type="bibr">38</xref><xref rid="ref39" ref-type="bibr">39</xref><xref rid="ref40" ref-type="bibr">40</xref><xref rid="ref41" ref-type="bibr">41</xref>] Given that potency can take over 1 year to return, it is possible that current studies and treatment duration were not long enough to demonstrate benefit in the current clinical studies. Additionally, the psychological effect of not receiving treatment during DFW might result in a regression of erectile function. Furthermore, there have been no studies comparing the effect of different PDE5Is with each other. Although, theoretically, they have the same mechanism of action, different half-lives may contribute to the different results observed in on-demand or daily PDE5I use. There is likely benefit to compare different PDE5Is in future trials. Additionally, there is a lack of consistency in use of placebo, DFW, trial length, inclusion criteria, degree of NS technique, use of robotic or open technique and, maybe most notably, potency definition.[<xref rid="ref38" ref-type="bibr">38</xref><xref rid="ref39" ref-type="bibr">39</xref><xref rid="ref40" ref-type="bibr">40</xref><xref rid="ref41" ref-type="bibr">41</xref>] To date, no trials have evaluated PDE5I penile rehabilitation specifically in patients undergoing RALP. Current evidence suggests that PDE5Is can play a role in penile rehabilitation, although the dosing, frequency and PDE5I used cannot be recommended based on current data.</p><p>VEDs have increasingly been used in penile rehabilitation programs.[<xref rid="ref44" ref-type="bibr">44</xref><xref rid="ref45" ref-type="bibr">45</xref><xref rid="ref46" ref-type="bibr">46</xref>] Although there is evidence that VED can prevent shortening of penile length,[<xref rid="ref45" ref-type="bibr">45</xref>] long-term potency benefits have not been demonstrated. ICI and MUSE appear to have some role in penile rehabilitation; however, long-term data are lacking.</p><p>When counseling a patient on RALP, it is necessary to explain that recovery of erectile function can take up to 1 year, if not greater. Therefore, it is important to identify the patients that will be willing to complete a rehabilitation program to provide the greatest clinical benefit.</p></sec><sec sec-type="conclusion" id="sec1-5"><title>CONCLUSION</title><p>RALP demonstrates beneficial potency rates without compromising oncologic benefit compared with RRP. However, these data lack prospective randomized control studies. Retrograde dissection, intraoperative cooling and VIP have demonstrated benefit for potency recovery compared with traditional RALP. Although benefit is demonstrated during use of PDE5Is compared with placebo, it is often lost after use. Subsequently, no optimal rehabilitation program with PDE5Is has been developed. Although VED, ICI and MUSE have shown promise in penile rehabilitation, long-term, randomized control studies have not been performed. It is important to identify patients who will most likely recover erectile function for patient education. Although the future is promising with improved robotic techniques, an optimal robotic technique has not been identified. Additionally, no definitive recommendations regarding a penile rehabilitation program can be made without further evidence.</p></sec> |
Ureteritis cystica: A rare entity in children | <p>Ureteritis cystica (UC) is a benign condition. Although it can often be diagnosed with imaging techniques, we report a case of a child for whom we planned nephrectomy and ureteral augmentation cystoplasty, but abandoned the cystoplasty due to extensive UC in the ureter.</p> | <contrib contrib-type="author"><name><surname>Dönmez</surname><given-names>M. İrfan</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Beksaç</surname><given-names>A. Tuna</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Doğan</surname><given-names>H. Serkan</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Koni</surname><given-names>Artan</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Baydar</surname><given-names>Dilek Ertoy</given-names></name><xref ref-type="aff" rid="aff2">1</xref></contrib><contrib contrib-type="author"><name><surname>Tekgül</surname><given-names>Serdar</given-names></name><xref ref-type="aff" rid="aff1"/></contrib> | Indian Journal of Urology : IJU : Journal of the Urological Society of India | <sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Ureteritis cystica (UC) is a benign pathology, which develops following chronic inflammation. A variety of diseases with obstruction and chronic inflammation may lead to UC. Although a diagnosis is often made with imaging techniques, we report a case of a child in whom was discovered during a nephrectomy. To the best of our knowledge, this is the first pediatric case of UC.</p></sec><sec id="sec1-2"><title>CASE REPORT</title><p>The patient was a 12 year old child who, as a newborn, had undergone surgery for anal atresia with rectourethral fistula repair and colostomy. His postnatal ultrasound (US) and Dimercaptosuccinic acid (DMSA) scan had shown a hypoplasic left kidney and bilateral minimal hydronephrosis. Voiding cystourethrography (VCU) showed bilateral grade 5 vesicoureteral reflux. At 1 year of age, the colostomy was closed and DMSA was repeated and differential function of the left kidney was found 22%, whereas the right kidney was 78%. In the 3<sup>rd</sup> year of life, a second VCU revealed clinically significant residual urine, which required clean intermittent catheterization (CIC). He underwent bilateral Cohen ureteroneocystostomy for recurrent urinary tract infections (UTIs) under antibiotic prophylaxis. Ultrasound prior to surgery showed bilateral high grade hydronephrosis. On the follow-up, DMSA scan showed 63% differential function on the right kidney. At the age of 7, US showed persistent bilateral high grade hydronephrosis. When he became a 10-year-old, hemodialysis was initiated for the first time and he had been placed on a renal transplant waiting list. The patient was on CIC 6 times a day when another urodynamic evaluation was obtained at 12 years of age. The study revealed hypocompliant bladder with decreased anatomic capacity accompanying detrusor overactivity with no demonstrable vesicoureteric reflux. Due to the low functional bladder capacity, nephrectomy with ureteral augmentation cystoplasty was planned.</p><p>During surgery, after left simple nephrectomy, left ureter was dissected and spatulated, but ureteral mucosa was found to be filled with polyps [<xref ref-type="fig" rid="F1">Figure 1</xref>]. It was decided not to use the ureter and the augmentation cystoplasty was postponed.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Innumerable 2-4 mm pearly raised vesicular/polipoid lesions covering entire mucosa of the ureter</p></caption><graphic xlink:href="IJU-30-443-g001"/></fig><p>Histopathological examination of the ureter showed polypoid ureteritis and exuberant UC producing exophytic mucosal lesions in a background of chronically inflamed lamina propria [<xref ref-type="fig" rid="F2">Figure 2</xref>]. UC typically consisted of expanded cystic lesions containing colloid like eosinophilic fluid. Cysts were lined by flattened urothelial cells without atypia.</p><fig id="F2" position="float"><label>Figure 2</label><caption><p>Florid ureteritis cystica with chronic inflammation in lamina propria. Large and small cysts lined by flattened urothelial cells leading to polipoid mucosal protrusions into the lumen; some filled with eosinophilic secretion ((a) H and E, ×40; (b) H and E, ×400)</p></caption><graphic xlink:href="IJU-30-443-g002"/></fig></sec><sec sec-type="discussion" id="sec1-3"><title>DISCUSSION</title><p>Ureteritis cystica is a rare and benign pathology of inflammatory origin first reported by Morgagni in 1761 and described by Richmond and Robb in 1967.[<xref rid="ref1" ref-type="bibr">1</xref>] It is the cystic transformation of von Brunn's nests.[<xref rid="ref2" ref-type="bibr">2</xref>] A PubMed search with keywords of “ureteritis, cystica” revealed 72 papers those mostly presenting case reports without any pediatric age patients. Although many reports state female dominancy, Menendez <italic>et al</italic>. have reported this may not be true after evaluating 34 cases and showing only 44% of them being female patients.[<xref rid="ref3" ref-type="bibr">3</xref>]</p><p>Ureteritis cystica is associated with chronic irritation. Some studies have shown nephrolithiasis and recurrent UTIs to be the cause. It is most frequently diagnosed incidentally.[<xref rid="ref1" ref-type="bibr">1</xref>] UC is generally seen in patients during the evaluation of UTI (82%), urolithiasis (53%), and hematuria (52%).[<xref rid="ref4" ref-type="bibr">4</xref>] Furthermore, it can present as renal colic. The differential diagnosis includes ureteral tumors, pseudodiverticula, urolithiasis, polyps, vascular indentations, tuberculosis, UTIs, and submucosal hemorrhages.[<xref rid="ref1" ref-type="bibr">1</xref>] In the long-term, UC may result in an atrophic kidney due to obstruction.</p><p>In this particular case, the etiology was thought to be persisting recurrent UTIs initially due to the bilateral high grade reflux and then due to the obstruction that developed following antireflux surgery. Mahboubi <italic>et al</italic>. have reported UC in a patient who received formalin treatment for cyclophosphamide induced hemorrhagic cystitis.[<xref rid="ref5" ref-type="bibr">5</xref>] Although, intravenous pyelography and retrograde urography were the preferred diagnostic tools earlier, in the last decade magnetic resonance and computed tomography urography have become more available and accurate. Despite advanced technology, imaging studies may be inadequate in some cases. Ureteroscopy is a definitive diagnostic approach in these cases[<xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref3" ref-type="bibr">3</xref>] In the present case, we did not have any imaging studies other than US since such pathology was not expected. Moreover, the patient's chronic renal failure precluded the use of imaging studies with contrast agents. Therefore, in such patients with history of chronic UTI, inflammation or obstruction, the possibility of this pathology should be taken into consideration and appropriate imaging modalities such as retrograde ureteropyelography or direct examination by ureterorenoscopy may be needed before reconstructive surgeries using the ureter. Limited data are present in the literature regarding follow-up and malignant transformation, however follow-up is advised annually. Duffin <italic>et al</italic>. have reported a case after follow-up of 17 years without malignant transformation, whereas there is a report of a case complicated with adenocarcinoma of the ureter.[<xref rid="ref4" ref-type="bibr">4</xref>]</p><p>During the surgery we abstained from using this ureter as we did not have the facility of frozen section pathology and also had no experience of using such a ureter for augmentation. No bladder biopsies were taken during or after the surgery. The treatment of this pathology, which is believed to be reversible, depends on the elimination of the underlying causes.</p></sec><sec sec-type="conclusion" id="sec1-4"><title>CONCLUSION</title><p>Ureteritis cystica is a rare pathology that should be considered in the presence of chronic inflammation and obstruction of the upper urinary tract. Presence of such pathology needs to be considered in cases undergoing ureterocystoplasty.</p></sec> |
Small bowel intussusception due to metastatic bladder carcinoma | <p>The common sites of metastasis from a urinary bladder carcinoma include lymph nodes, bones, lung, liver, and peritoneum. Only a few cases of small bowel metastasis from urinary bladder malignancy have been reported in the English literature. Only one of these cases presented with bowel intussusception. We report a case of an adult small bowel intussusception due to metastasis from a urinary bladder carcinoma which is extremely rare as small bowel metastasis occur mostly from breast, lungs or melanomas.</p> | <contrib contrib-type="author"><name><surname>Singh</surname><given-names>Seema</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Ranjan</surname><given-names>Rakesh</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Sharma</surname><given-names>Naveen</given-names></name><xref ref-type="aff" rid="aff1"/></contrib> | Indian Journal of Urology : IJU : Journal of the Urological Society of India | <sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Small bowel intussusception is seen more commonly in pediatric population as compared to adults. Adult small bowel intussusceptions occur almost always due to a demonstrable lead point, mostly benign, at times malignant.[<xref rid="ref1" ref-type="bibr">1</xref>] These malignant lesions can be either a primary small bowel tumor or a metastatic tumor from a distant primary. Metastatic tumors arise mostly from breast, lungs or melanomas. On the other hand, urinary bladder malignancies spread usually to lymph nodes, bones, lung, liver, and peritoneum. We report a rare case of small bowel intussusception due to metastasis from urinary bladder carcinoma.</p></sec><sec id="sec1-2"><title>CASE REPORT</title><p>A 40-year-old male patient was admitted to the hospital for work up of hematuria. Ultrasonography of the abdomen revealed a lesion with mixed echogenicity in the urinary bladder suggestive of carcinoma urinary bladder. Cystoscopy showed a growth over the anterior wall of the urinary bladder. The patient was provisionally diagnosed as a case of carcinoma urinary bladder and planned for further imaging. During the course of hospital stay, however, he developed features of acute intestinal obstruction. Repeat ultrasonography of the abdomen at this point showed dilated and thickened small bowel loops with bowel within the bowel appearance, suggestive of intussusception.</p><p>The patient underwent an exploratory laparotomy and was found to have an ileoileal intussusception two feet proximal to ileocecal junction [<xref ref-type="fig" rid="F1">Figure 1</xref>]. On reduction of the intussusception, a growth of size 3 cm × 3 cm was found to be the lead point [<xref ref-type="fig" rid="F2">Figure 2</xref>]. There were no other metastatic deposits in the abdomen. Resection and anastomosis of the ileal segment was done.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Operative picture showing small bowel intussusception</p></caption><graphic xlink:href="IJU-30-445-g001"/></fig><fig id="F2" position="float"><label>Figure 2</label><caption><p>Tumor marked with a hemostat</p></caption><graphic xlink:href="IJU-30-445-g002"/></fig><p>Histopathological examination of the resected bowel showed a 2.5 cm × 2 cm × 1.5 cm growth, microscopic examination of which revealed metastatic urothelial carcinoma involving serosa and reaching up to the mucosa with lymphovascular invasion.</p></sec><sec sec-type="discussion" id="sec1-3"><title>DISCUSSION</title><p>Intussusception refers to a condition whereby a segment of intestine becomes drawn into the lumen of the adjacent distal bowel. The most common locations of intussusception are at the junctions between the freely moving segments of bowel and segments fixed due to adhesions or retroperitoneal attachments.</p><p>Intussusception is the leading cause of intestinal obstruction in children. Adult intussusception on the other hand is a rare disease and accounts for only 5% of all cases of intussusception. It is also a rare cause of intestinal obstruction in adults accounting for less than 1% of all cases of bowel obstruction.[<xref rid="ref1" ref-type="bibr">1</xref>]</p><p>The presentation of pediatric intussusception often is acute with sudden onset of intermittent colicky pain, vomiting, and bloody mucoid stools, and the presence of a palpable mass. In contrast, the adult entity may present with acute, subacute, or chronic non-specific symptoms.[<xref rid="ref1" ref-type="bibr">1</xref>] The usual clinical presentation of adult intussusception is with features of intestinal obstruction and in many of these cases a preoperative diagnosis of intussusception cannot be made. In a study of 44 cases of adult intussusception by Barussaud <italic>et al</italic>., a preoperative diagnosis of intussusception was made in only 52% of the cases. The sensitivities of the different radiological methods in diagnosing intussusception preoperatively were abdominal ultrasounds (35%), upper gastrointestinal barium study (33%), abdominal computed tomography (CT) (58%) and barium enema (73%).[<xref rid="ref2" ref-type="bibr">2</xref>]</p><p>Adult intussusceptions have been found to have an organic lead point in 70-90% of the cases in various studies.[<xref rid="ref1" ref-type="bibr">1</xref>] Small bowel intussusceptions may occur due to adhesions, Meckel's diverticulum, inflammatory bowel disease, lymphoma, primary malignancy or metastatic disease, as opposed to large bowel intussusceptions, which are due to an underlying malignancy in the majority of the cases. Malignant lesions as the cause of small bowel intussusception have been reported in 17-30% of the cases in various studies as opposed to 66% of large bowel intussusceptions.[<xref rid="ref1" ref-type="bibr">1</xref>]</p><p>Metastatic spread to small intestine can occur by direct invasion, hematogenous spread, or intraperitoneal seeding. Colon and pancreatic cancers are the most common primary sites for direct invasion. Hematogenous metastases occur most frequently from lung and breast carcinoma or melanoma. Peritoneal seeding may arise from any intra-abdominal malignancy including gastric, hepatic, ovarian, appendiceal, and colonic primary tumors.[<xref rid="ref3" ref-type="bibr">3</xref>]</p><p>In a study of 392 cases of bladder cancer by Shinagare <italic>et al</italic>., lymph nodes, bones, lung, liver, and peritoneum were the most common sites of metastasis. Two patients with peritoneal carcinomatosis in this study had intestinal obstruction.[<xref rid="ref4" ref-type="bibr">4</xref>] Although there have been other case reports of carcinoma bladder metastasizing to intestines and causing obstruction,[<xref rid="ref5" ref-type="bibr">5</xref>] we could identify only one reported case of bowel intussusception due to metastasis from a bladder carcinoma to large bowel.[<xref rid="ref6" ref-type="bibr">6</xref>]</p><p>The management of adult intussusception is essentially surgical in view of high probability of the presence of an organic lead point. However, there remains a controversy regarding the extent of surgery in these cases. Some authors recommend an en bloc resection without initial reduction in view of high incidence of underlying malignancy as reduction in these cases carries a risk of intraluminal seeding and venous embolization. Others recommend en bloc resection in all cases of colonic intussusception and a selective approach in small bowel in view of lower rate of malignancy. Recently, laparoscopic approach has also been advocated in these cases especially in view of often doubtful preoperative diagnosis.[<xref rid="ref1" ref-type="bibr">1</xref>]</p></sec> |
Arteriovenous malformation causing obstructive uropathy: A different dimension to ovarian vein syndrome | <p>Ovarian vein syndrome is defined as obstructive uropathy caused by dilated ovarian vein with or without thrombosis. This is seen in the puerperal period as an acute condition with abdominal pain and fever and in multipara women with chronic recurrent abdominal pain. We report an ovarian vein syndrome caused by a true vascular anomaly in an 8-year-old child. Laparoscopic ureterolysis was performed with ligation of the arteriovenous malformation during the first operation. As ureterolysis was not effective, the patient was reoperated and ureteroureterostomy was performed after 3 months, which emphasizes the importance of removing the diseased segment even if it looks normal.</p> | <contrib contrib-type="author"><name><surname>Kone</surname><given-names>Kalyana Ram</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib> | Indian Journal of Urology : IJU : Journal of the Urological Society of India | <sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Ovarian vein syndrome is defined as ureteric obstruction caused by dilated ovarian vein with or without thrombosis and is a controversial and rare diagnosis. All cases reported so far are in adults where an abnormal gonadal vein compressed the ureter and caused obstructive uropathy mostly on the right side and lower ureter. Here we report the ovarian vein syndrome caused by an arteriovenous malformation affecting the left upper ureter in an 8-year-old child.</p></sec><sec id="sec1-2"><title>CASE REPORT</title><p>An 8-year-old girl presented with recurrent attacks of left flank pain of 6 months duration. She had no episodes of urinary tract infection. Ultrasound revealed left hydronephrosis with dilatation of the upper ureter. Intravenous pyelography (IVP) showed hydronephrosis with dilated left upper ureter with sudden tapering and normal lower ureter below the area of tapering [<xref ref-type="fig" rid="F1">Figure 1</xref>]. She was diagnosed to have left upper ureteric obstruction and was taken up for laparoscopic exploration.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Intravenous urography showing a ureter - sudden narrowing and tapering of the upper ureter with normal distal ureter</p></caption><graphic xlink:href="IJU-30-448-g001"/></fig><p>During surgery, we found that the upper ureter near the site of obstruction was surrounded by a sheet of vessels [<xref ref-type="fig" rid="F2">Figure 2</xref>]. The ureter was sandwiched between a dilated periureteric vein and the gonadal vein. The gonadal vein instead of crossing from the lateral to medial side of the ureter, made a U shaped loop laterally, attached to the ureter at the apex of U and was seen draining into an abnormally placed lumbar vein with tributaries coming from above and below the confluence. The part of gonadal vein attached to the ureter was thrombosed with turbulent flow seen near the junction of gonadal vein with the lumbar vein. A small artery was seen entering the vein above the site of clot and causing turbulent flow in the vein. All the vessels were ligated proximally and distally laparoscopically and the parts of vessels attached to ureter were removed. The dilated gonadal vein attached to the left ureter was found to contain clotted blood. Complete ureterolysis was done at the site of obstruction. As ureter seemed normal, ureteroureterostomy was not done A line diagram is produced here for understanding the anomaly [<xref ref-type="fig" rid="F3">Figure 3</xref>].</p><fig id="F2" position="float"><label>Figure 2</label><caption><p>Intraoperative pictures showing vascular anomaly surrounding the upper ureter and its ligation (a) Ureter. (b) Dilated periureteric vein. (c) Thrombosed gonadal vein surrounding ureter. (d) Turbulent flow in gonadal vein. (e) Psoas major muscle. (f) Reflected colon. (g) Ligation of arteriovenous malformation above and below. (h) Arterial communication ligated</p></caption><graphic xlink:href="IJU-30-448-g002"/></fig><fig id="F3" position="float"><label>Figure 3</label><caption><p>Line diagram showing the abnormal course of the gonadal vein and its communication with the lumbar vein (a) inferior vena cava. (b) Aorta. (c) Kidney. (d) Gonadal vein. (e) Gonadal Artery. (f) Communication of gonadal vein with a periureteric vein. (g) Dilated periureteric vein. (h) Thrombosed gonadal vein. (i) Arterial communication with the dilated gonadal vein. (j) Lumbar vein. (k) Ureter</p></caption><graphic xlink:href="IJU-30-448-g003"/></fig><p>Postoperatively, Doppler study was done to uncover major vascular anomalies and was found to be normal. Double J (DJ) stent was removed after 15 days. Subsequently the patient continued to have abdominal pain again and a repeat IVP at 2 months showed similar obstruction as in the past. The patient was operated again and the obstructing segment was excised and ureteroureterostomy was performed. Ultrasound done after 3 months showed complete resolution of hydronephrosis and the patient remained asymptomatic.</p></sec><sec sec-type="discussion" id="sec1-3"><title>DISCUSSION</title><p>Clarke described ovarian vein syndrome for the first time in 1964.[<xref rid="ref1" ref-type="bibr">1</xref>] Following initial controversy, it was accepted as a clinical entity after further cases were reported.[<xref rid="ref2" ref-type="bibr">2</xref>]</p><p>Two forms of the ovarian vein syndrome are now recognized. The first is a form of pelvic congestion syndrome analogous to varicocele in males. The right ovarian vein is most commonly involved. All patients are multipara, leading to the hypothesis that the dilatation of the ovarian vein is due to recurrent obstruction by the gravid uterus. However, as this condition is rare, it is always a diagnosis of exclusion. The patients usually present with recurrent abdominal, flank or pelvic pain, which increases in the lying down posture, mainly in the pre-menstrual period. The diagnosis is confirmed by imaging studies - ultrasound, Doppler, computed tomography and magnetic resonance imaging. The treatment consists of either percutaneous embolization or surgical ligation of the dilated vein.[<xref rid="ref3" ref-type="bibr">3</xref>]</p><p>The second one is due to puerperal ovarian vein thrombophlebitis, which causes perivenous phlegmon formation and resultant periureteritis. The patients present with nonspecific abdominal pain and fever 2-3 days after delivery. The majority of patients respond to conservative measures with antibiotics, although some patients may require temporary DJ stenting or surgical ureterolysis with ligation of the ovarian vein.[<xref rid="ref4" ref-type="bibr">4</xref>]</p><p>The present case adds a new dimension to the two types already described. This is the first case in a child, and is a true vascular anomaly. The abnormally placed gonadal vein was thrombosed and the resultant collaterals communicated with a branch of gonadal artery and caused this anomaly. The case also highlights the need for excising the obstructed segment of ureter along with ureterolysis in cases of complex vascular obstruction of upper ureter.</p></sec> |
Massive left hemothorax following laparoscopic pyeloplasty | <p>Laparoscopic pyeloplasty is viable standard minimally invasive alternative to open pyeloplasty for the treatment of ureteropelvic junction obstruction. Intrathoracic bleeding is an extremely rare complication after laparoscopic urological surgery, but it should be suspected and promptly diagnosed in case of worsening hemodynamic status and respiratory parameters during the intra or post-operative course. We report a case of hemothorax complicating an otherwise uneventful LP in an 18-year-old girl.</p> | <contrib contrib-type="author"><name><surname>Rao</surname><given-names>Manjula</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>D’Souza</surname><given-names>Nischith</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Khan</surname><given-names>Altaf</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Rahiman</surname><given-names>Mujeebu</given-names></name><xref ref-type="aff" rid="aff1"/></contrib> | Indian Journal of Urology : IJU : Journal of the Urological Society of India | <sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Laparoscopic pyeloplasty (LP) is a minimally invasive alternative to open pyeloplasty for the treatment of ureteropelvic junction obstruction. It has been shown to provide lower patient morbidity, shorter hospitalization and faster convalescence. However, there is an inherent risk of surgical (blind trocar insertion, colonic injury, hemorrhage, ileus, urinoma formation) and anesthetic complications (gas embolism, extraperitoneal insufflation and surgical emphysema, pneumothorax and pneumomediastinum).[<xref rid="ref1" ref-type="bibr">1</xref>] We report an isolated case of hemothorax, complicating an otherwise uneventful LP.</p></sec><sec id="sec1-2"><title>CASE REPORT</title><p>An 18-year-old girl was diagnosed to have left pelviureteral junction obstruction during evaluation of left flank pain. A transperitoneal LP was planned. The Veress<sup>®</sup> needle insertion and insufflation was uneventful. High resolution camera and monitor systems were used. Surgical cautery was used for dissection. Three trocars, including a 10 mm umbilical trocar, a 10 mm trocar midway between the umbilicus and the symphysis, and a 5 mm trocar midway between the umbilicus and xiphoid were used. Pneumo-peritoneum was kept constant at 12 cm H<sub>2</sub>O. No impairment of respiratory parameters was observed by the anesthesiologist during the procedure. The entire procedure was completed in 90 minutes, and a drain was placed after confirming hemostasis.</p><p>However, at the end of the procedure, before evacuation of pneumoperitoneum, the patient developed hypotension. This was initially responsive to fluid administration, but quickly deteriorated after weaning, resulting in severe hypovolemic shock with worsening of respiratory parameters (tidal volumes, peak pressures and blood gases).</p><p>An on-table clinical examination suggested decreased air entry in the left hemithorax. A pneumothorax was suspected, and a needle was inserted into the left fifth intercostal space. However, it drained blood and a chest X-ray revealed a hazy left hemithorax. Screening ultrasound and contrast enhanced chest computed tomography (CT) scan showed a massive hemothorax with no hemoperitoneum (ultrasound and CT installations are within the operation theatre complex).</p><p>A 28 French intercostal tube was inserted in the left fifth intercostal space, and 2 L of blood was drained. The patient continued to remain hypotensive despite resuscitation and blood transfusion and more than 500 ml blood drained through the intercostal tube during the subsequent 2 h. An emergency surgical exploration was planned. In any case of hemothorax following an abdominal procedure, the first suspicion is towards an abdominal cause and a laparotomy was performed but it did not reveal any blood in the peritoneal cavity.</p><p>A subsequent left mini-thoracotomy revealed fresh and clotted blood in the pleural cavity, which was evacuated. An active bleeding vessel became evident in the thoracic aspect of the left diaphragm near the anterior costophrenic angle secondary to a small diaphragmatic cautery burn. Hemostasis was achieved by under-running the bleeding vessel, and an intercostal drain was placed. Careful inspection of the pleural cavity did not reveal any other injuries. The post-operative course was uneventful, and complete lung re-expansion was achieved. The chest drainage tube was subsequently removed, and the patient discharged.</p></sec><sec sec-type="discussion" id="sec1-3"><title>DISCUSSION</title><p>Thoracic bleeding is a rare complication of elective abdominal surgery, mainly resulting from unrecognized, iatrogenic diaphragmatic tears. This has been described during gastrointestinal surgical procedures. Cristian <italic>et al</italic>.[<xref rid="ref1" ref-type="bibr">1</xref>], have reported a case of massive right hemothorax causing hemorrhagic shock after laparoscopic cholecystectomy. The bleed was from a laceration of the parieto-diaphragmatic adherence, which was attributed to change in diaphragmatic position during pneumoperitoneum, as it has been demonstrated in animal models.[<xref rid="ref2" ref-type="bibr">2</xref>]</p><p>Kyo <italic>et al</italic>.[<xref rid="ref3" ref-type="bibr">3</xref>] have reported severe hemothorax after laparoscopic surgery for endometriosis, which was attributed to pulsatile active bleeding from scattered small endometriotic lesions present on the pleural surface of the right diaphragm.</p><p>Documentation of thoracic complications during urological laparoscopic surgery is rare. Abreu <italic>et al</italic>. have reported that thoracic surgical complications occurred in only 0.5% of the patients. Of the 1,129 patients operated laparoscopically, only one patient demonstrated a hemothorax (0.08%), which was attributed to accidental supracostal placement of the posterior port.[<xref rid="ref4" ref-type="bibr">4</xref>] Most complications occur during creation of pneumoperitoneum and blind insertion of the first trocar, not because of the laparoscopic surgery.</p><p>In this case, the hemothorax was probably caused due to injury to the diaphragm by an unrecognized cautery burn, following which the bleeding vessel retracted into the thorax. The colon is routinely mobilized during LP. The cautery burn probably occurred during the mobilization of the splenic flexure with the monopolar cautery. This was not evident during the entire procedure due to the intact pneumoperitoneum. Hypotension due to blood loss was evident only after evacuation of the intra-abdominal gas and the natural suspicion was of an intra-abdominal source of bleeding. There needs to be a high index of suspicion of possible thoracic complications during laparoscopic urological procedures.</p></sec> |
Spontaneous perinephric hemorrhage (Wunderlich syndrome) secondary to polyarteritis nodosa: Computed tomography and angiographic findings | <p>We report the case of a young man who presented with spontaneous left perinephric hematoma and per-rectal bleeding. Evaluation revealed renal and superior mesenteric arterial aneurysms secondary to polyarteritis nodosa (PAN). Computed tomography and angiographic findings are presented. The aetiology of spontaneous perinephric hemorrhage along with relevant features of PAN are discussed.</p> | <contrib contrib-type="author"><name><surname>Venkatramani</surname><given-names>Vivek</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Banerji</surname><given-names>John S.</given-names></name><xref ref-type="aff" rid="aff1"/></contrib> | Indian Journal of Urology : IJU : Journal of the Urological Society of India | <p>Spontaneous perinephric hematoma is a rare entity with malignancies being the most common cause.[<xref rid="ref1" ref-type="bibr">1</xref>] Vasculites are the next most common cause, and of these polyarteritis nodosa (PAN) accounts for most cases.[<xref rid="ref1" ref-type="bibr">1</xref>] We report the case of a young man who presented with spontaneous left perinephric hematoma and per-rectal bleeding. Evaluation revealed renal and superior mesenteric arterial aneurysms.</p><sec id="sec1-1"><title>CASE REPORT</title><p>A 20-year-old boy presented to the emergency department with sudden onset severe left flank pain for a week and no associated vomiting or urinary symptoms. He had a history of per-rectal bleeding 6 months earlier for which he was evaluated elsewhere. He had noticed a weight loss of 4 kg over these past 6 months. Examination was unremarkable. Blood investigations were unremarkable except for hemoglobin of 9.3 g%. Urinalysis was normal. An ultrasound done elsewhere the previous day was suggestive of a left perinephric mass, hence a contrast enhanced computed tomography (CT) of the abdomen was performed. It revealed an 8 × 7 cm hyperdense lesion in the perinephric space displacing the kidney anteriorly, with an intensely enhancing lesion adjacent to the hemorrhage, indicating an arterial aneurysm with surrounding hemorrhage [<xref ref-type="fig" rid="F1">Figure 1</xref>]. Subsequently, he underwent renal angiography. It revealed multiple small left renal arterial aneurysms with no active bleeding [<xref ref-type="fig" rid="F2">Figure 2a</xref>]. It also revealed similar aneurysms in the superior mesenteric and lumbar arteries [<xref ref-type="fig" rid="F2">Figure 2b</xref>]. Further evaluation revealed a raised erythrocyte sedimentation rate and C-reactive protein. Anti-nuclear antibodies, hepatitis B and C serology were negative, and complement levels were normal. A diagnosis of PAN was made and he was started on steroids and mycophenolate mofetil, as well as anti-hypertensive medication. He was well at 9 months follow-up and CT angiogram showed no beading of the renal vasculature [<xref ref-type="fig" rid="F3">Figure 3</xref>].</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>Computed tomography of the abdomen showing perinephric hematoma (arrow) with adjacent intensely enhancing lesion suggestive of an aneurysm (arrowhead)</p></caption><graphic xlink:href="IJU-30-452-g001"/></fig><fig id="F2" position="float"><label>Figure 2</label><caption><p>Abdominal arteriography (a) left renal arteriogram showing multiple small aneurysms involving segmental and interlobar branches of renal artery (arrows) (b) aortogram showing involvement of superior mesenteric and lumbar vasculature (arrows)</p></caption><graphic xlink:href="IJU-30-452-g002"/></fig><fig id="F3" position="float"><label>Figure 3</label><caption><p>Follow-up computed tomography angiogram with no beading of renal vasculature (arrows)</p></caption><graphic xlink:href="IJU-30-452-g003"/></fig></sec><sec sec-type="discussion" id="sec1-2"><title>DISCUSSION</title><p>Spontaneous perinephric hemorrhage (Wunderlich syndrome) is a rare condition with a wide-ranging etiology. Zhang <italic>et al</italic>. in their meta-analysis reported on 165 such cases.[<xref rid="ref1" ref-type="bibr">1</xref>] Of these, 101 (62.2%) were secondary to the rupture of renal tumors, with angiomyolipoma being the most common cause. Vasculitis accounted for 28 cases, and PAN was the cause in 20. They reported five cases of bilateral spontaneous hemorrhage and all of these were secondary to PAN.[<xref rid="ref1" ref-type="bibr">1</xref>]</p><p>PAN is a multi-system necrotizing vasculitis that involves small and medium sized vessels. The kidneys are affected in 80% cases with hypertension, proteinuria and ultimately renal functional deterioration being the most common manifestations.[<xref rid="ref2" ref-type="bibr">2</xref><xref rid="ref3" ref-type="bibr">3</xref><xref rid="ref4" ref-type="bibr">4</xref>] Spontaneous perinephric hemorrhage is a rare, but potentially devastating complication of PAN. About 60 cases have been reported in the literature.[<xref rid="ref2" ref-type="bibr">2</xref>] It is secondary to the rupture of arterial aneurysms that usually involve the renal artery, and its segmental and interlobar divisions.[<xref rid="ref3" ref-type="bibr">3</xref>] Nephrectomy is associated with a high mortality rate (50%) in the acute setting.[<xref rid="ref2" ref-type="bibr">2</xref>] Angiography with selective embolization allows preservation of renal parenchyma and is now the treatment of choice in cases of active bleeding.[<xref rid="ref2" ref-type="bibr">2</xref>] A reduction or resolution in aneurysms has been noted following immunosuppressive therapy and is probably due to the decline in inflammation of the vessel wall.[<xref rid="ref5" ref-type="bibr">5</xref>]</p><p>Per-rectal bleeding is an uncommon manifestation (<4%) and Pagnoux <italic>et al</italic>. identified gastrointestinal manifestations, and the need for a surgical consult as an independent predictor of mortality in PAN.[<xref rid="ref4" ref-type="bibr">4</xref>]</p></sec> |
“The creeping tumor:” An unusual presentation of upper urinary tract malignancy | <p>Upper urinary tract urothelial malignancy accounts for 5-10% of urothelial carcinomas. Synchronous bladder carcinoma occurs in 2-4% of patients with upper urinary tract tumors. Urothelial malignancy involving the entire upper urinary tract is an extremely rare entity. Most upper urinary tract malignancies are transitional cell carcinomas (TCC), of which the sarcomatoid variant is very rare. These tumors pose a challenge to the radiologist. We herein report a case of TCC involving the entire collecting system of the left kidney, extending down along the ureter and projecting as a mass in the bladder.</p> | <contrib contrib-type="author"><name><surname>Selvaraj</surname><given-names>Velaiyan</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Govindarajan</surname><given-names>Periasamy</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Deepak</surname><given-names>M.</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Sivaraj</surname><given-names>Manoharan</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib> | Indian Journal of Urology : IJU : Journal of the Urological Society of India | <sec sec-type="intro" id="sec1-1"><title>INTRODUCTION</title><p>Transitional cell carcinomas (TCC) spread along the urothelial cellular lining. However, presentation of TCC involving the entire upper urinary tract is extremely rare, and, to the best of our knowledge, this is the first case reported.[<xref rid="ref1" ref-type="bibr">1</xref>] Our patient had the sarcomatoid variant of TCC, a rare entity.[<xref rid="ref2" ref-type="bibr">2</xref>]</p></sec><sec id="sec1-2"><title>CASE REPORT</title><p>A 42-year-old male was admitted with history of painless hematuria and left loin pain for 4 months. Clinical examination revealed anill-defined hard mass palpable in the left lumbar region. Radiological studies revealed a heterogeneous mass lesion measuring 15 cm × 10 cm in the collecting system of the left kidney with hydronephrosis and perinephric urinoma. Infiltration into the left renal cortex with extension of the lesion into the left pararenal space and left latissimus dorsi muscles were seen. The mass extended into the left ureter up to the vesico-ureteric junction (VUJ) and into the bladder, presenting as an intravesical exophytic mass lesion. Extension of the lesion into the periureteric soft tissue was noted 8 cm above the VUJ [<xref ref-type="fig" rid="F1">Figure 1</xref>]. Cystoscopy and biopsy from the bladder growth was performed which suggested -high grade TCC.</p><fig id="F1" position="float"><label>Figure 1</label><caption><p>“The creeping tumour” – tumour extending from the left renal pelvis and whole of the left ureter and projecting into the bladder</p></caption><graphic xlink:href="IJU-30-454-g001"/></fig><p>Neoadjuvant chemotherapy consisting of three cycles of Cisplatin/Gemcitabine was administered.[<xref rid="ref3" ref-type="bibr">3</xref>] Clinically and radiologically the mass decreased in size [<xref ref-type="fig" rid="F2">Figure 2</xref>]. Open left nephroureterectomy with bladder cuff excision was carried out. Final histopathology revealed a sarcomatoid variant of TCC, pT4 N0 M0. The patient was administered adjuvant chemotherapy and is on follow up.</p><fig id="F2" position="float"><label>Figure 2</label><caption><p>Response to neoadjuvant chemotherapy – tumor has considerably reduced in size in the left kidney (right side image) following a course of neoadjuvant chemotherapy</p></caption><graphic xlink:href="IJU-30-454-g002"/></fig></sec><sec sec-type="discussion" id="sec1-3"><title>DISCUSSION</title><p>An upper urinary tract tumor can be defined as any neoplastic growth that affects the lining of the urinary tract from the calyces to the distal ureter. Unlike bladder urothelial tumors, they are relatively less common and may have a different prognosis. Unusual imaging features of urothelial carcinoma of the upper urinary tract can be challenging for any radiologist.[<xref rid="ref4" ref-type="bibr">4</xref>] For staging purposes, computed tomography or magnetic resonance imaging is most useful in determining the extent of invasion and metastasis. TCCs are invasive and epithelial spread may occur in both antegrade and retrograde manner. Involvement of the entire upper urinary tract is rare.</p></sec> |
Re: Parikh GP, Sonde SR, Kadam P. Venous air embolism: A complication during percutaneous nephrolithotomy. Indian J Urol 2014;30:348-9 | Could not extract abstract | <contrib contrib-type="author"><name><surname>Ozturk</surname><given-names>Hakan</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib> | Indian Journal of Urology : IJU : Journal of the Urological Society of India | <p>Dear Editor,</p><p>With reference to the recent article on venous air embolism during percutaneous nephrolithotomy published in your journal,[<xref rid="ref1" ref-type="bibr">1</xref>] we would like to reiterate that air embolism is a very rare and important complication that should always be kept in mind by endourologists performing percutaneous nephrolithotomy (PCNL). The position of the air embolism in the Clavien-Dindo classification system is not clear because it can presents a wide spectrum ranging from Grade I complication with spontaneously resolution to Grade V complication that may be fatal. It was first defined by Miller <italic>et al</italic>. in 1984[<xref rid="ref2" ref-type="bibr">2</xref>] While sudden death following air embolism during PCNL was first reported by Hobin <italic>et al</italic>. in 1985.[<xref rid="ref3" ref-type="bibr">3</xref>] Air embolism occurring in PCNL has potential consequences involving the respiratory, cardiovascular and central nervous systems such as hypoxia, ST-T depression on the ECG, hypotension and tachycardia. However, if the embolus passes into the arterial system, the complications make become worse and its diagnosis may be possible only with echocardiography. This situation, also called paradoxical air embolism and was first described during PCNL by Seung-Hun Song <italic>et al</italic>. in 2007.[<xref rid="ref4" ref-type="bibr">4</xref>] It developed in this case through a patent foramen ovalebut may also be possible through an undiagnosed atrial or ventricular septal defect. Air in the arterial system can cause neurological complications such as altered consciousness, loss of sensation and seizures. The most significant complication is “cryptogenic stroke.”[<xref rid="ref5" ref-type="bibr">5</xref>] Other possible complications include aphasia, coma, paraplegia, hemiplegia and quadriplegia. As discussed in this article, mortality and morbidity of the air embolism is determined by velocity, volume, pressure of the air, position of the patient during the procedure and situation of the heart. It should be known that other medical gases such as nitrogen, nitrogen dioxide, carbon dioxide and helium may also cause air embolism. The complication rate is relatively lower with soluble carbon dioxide. The volume of the collecting system is about 10-12 mL. Usually, 20-50 mL of air is used for pyelography. Most cases of air embolism during PCNL have been described for patients under general anesthesia, in the prone position and who have undergone pyelography with air. In the present case, 40 mL of air was used and the patient underwent the procedure in the prone position under general anesthesia. In 2013, Abbas Basiri <italic>et al</italic>. evaluated a total of 30,666 patients from 13 centers. In that series of patients, neurological complications were reported in 11 patients. In all cases, room air was used for pyelography with the patients being in the prone position under general anesthesia. Cases with complications were reported even though the amount of air used was close to the physiological volume (17 mL).[<xref rid="ref5" ref-type="bibr">5</xref>]</p><p>In conclusion, air embolism occurring during PCNL may result in serious complications such as death, coma, hypoxic encephalopathy, cryptogenic stroke, paraplegia, hemiplegia and quadriplegia, causing financial and emotional stress in the patients and their relatives. Urologists who prefer using air for pyelography during PCNL operation under general anesthesia should be aware of the potential risk of air embolism. They should especially know this rare complication and should be cautious on potential results in making diagnosis and planning the treatment.</p> |
Re: Sabharwal S, Macaden AR, Abrol N, Mukha RP, Kekre NS. A novel computer based stent registry to prevent retained stents: Will patient directed automated short message service and letter generator help? Indian J Urol 2014;30:150-2 | Could not extract abstract | <contrib contrib-type="author"><name><surname>Modi</surname><given-names>Jayesh</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1"/></contrib><contrib contrib-type="author"><name><surname>Nagarajan</surname><given-names>Ramya</given-names></name><xref ref-type="aff" rid="aff1"/></contrib><contrib contrib-type="author"><name><surname>Kumar</surname><given-names>Suresh</given-names></name><xref ref-type="aff" rid="aff1"/></contrib> | Indian Journal of Urology : IJU : Journal of the Urological Society of India | <p>Dear Editor,</p><p>We have read the original article titled “A novel computer based stent registry to prevent retained stents: Will patient directed automated short message service and letter generator help?” by Sabharwal <italic>et al</italic>., with great interest.[<xref rid="ref1" ref-type="bibr">1</xref>] Forgotten stents produce encrustations and stones, which may require either a retrograde or antegrade approach to remove them. Most importantly, these can be prevented by taking certain precautions. We would like to congratulate the authors for applying a novel computer-based stent registry to minimize this issue, especially in a big country like India.</p><p>However, certain problems may occur during this patient-directed automated information system. The targeted population for this novel registry mainly resides in villages. Frequent change of mobile numbers, network connectivity issues, or inadequate balance in the SIM-card can make this population difficult to reach. Additionally, short message service (SMS) generated in English language may not be that useful for people knowing only regional languages.</p><p>Lack of documentation and patient's unawareness about the presence of stent were the common reasons for retained stents as mentioned by authors in their 10-year retrospective study.[<xref rid="ref1" ref-type="bibr">1</xref>] We believe that one important cause of forgotten stents is inadequate explanation to the patient at the time of discharge by the house officers/junior resident doctors as they write up the discharge summary with explanations for further follow-up. We suggest certain simple but effective means to combat these pitfalls.</p><p>As visual impressions have a lasting impact on memory, it is the practice at our institute to get an X-ray KUB postoperatively and show the images of the stent to the patient (after removal of other tubes/catheters), stressing that he/she needs to get it removed. Second, as most patients requiring stent insertion usually have urolithiasis, we send a specimen of the calculus for structural and morphological stone analysis. The patient is told that he will have to pay us a second visit, at which time his stent would be removed, and strategies for preventing stone formation can be planned based on the results of the stone analysis. This review date is calculated in advance and written in the discharge summary. Third, involving the local doctor (or referring physician) into the whole process with a phone call/letter would help, as he or his family members would often visit the family physician for primary care, enabling us to trace the patient easily.</p><p>We like the authors’ idea of sending letters, and the advantage is that it can be written in the regional language, and it is unusual that people change their permanent address frequently. Postal mail is still frequently used in our country despite technological advances such as email and mobile messaging. However, technology is changing very fast, and we should certainly use it to our advantage in health care delivery, as proposed by the authors. We conclude that by adding certain simple measures mentioned above to the novel computer-based stent registry we can make it even more effective to decrease the incidence of retained “double J” stents.</p> |