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background zoonotic cutaneous leishmaniasis zcl is polymorphic disease that may show various clinical manifestations

objectives this study investigates the determination of genetic variation within the species of leishmania major isolates from new cases in chabahar a port city in southeast iran situated at the iran pakistan border

migration in this region indicates that leishmaniasis is spreading gradually and a new micro habitat focus appears each year

materials and methods a variety of nucleic acid detection methods that target both dna and rna have been developed

the restriction fragment length polymorphism analysis of amplified internal transcribed spacer 1 with polymerase chain reaction its1 rflp pcr assay is a multipurpose tool for the diagnosis of leishmania from clinical samples and for enabling the determination of the infecting leishmania species

the goal of this study was the identification of species based on its1 rflp in the ribosomal operon of l major from clinically different forms of zcl amplified by pcr followed by the digestion of the pcr product with restriction enzymes

the profiles were observed and visualized in agarose gel under uv light

we used direct smears to identify the parasites

while taking the smear samples were collected for culture or direct pcr

we used the pcr rflp assay of the its1 genes for direct identification of leishmania species in 24 out of 33 suspected patients

pcr its1 amplification was done on the 24 samples confirmed by culture via growth and parasitological methods

results of the 24 isolates 21 had 350 bp bands 87 5 and three had 450 bp bands 12 5

after using the restriction enzyme banding patterns including fragments of 210 and 140 bp for l major were detected in 19 cases

conclusions the l major species causing zcl in chabahar have limited genetic variation

there seems to be little manifestation of diversity between these lesions as a new focus of disease and new micro habitats for the disease are appearing in parts of this region

1

background zoonotic cutaneous leishmaniasis zcl is polymorphic disease with various clinical manifestations 1 2

leishmaniasis a group of parasitic infections has a worldwide distribution 3 the world health organization estimates a prevalence of approximately 12 million cases with an annual mortality rate of 60 000 and an at risk population of approximately 350 million 4

approximately 90 of cl cases occur in just seven countries including iran 5

several molecular targets for diagnostic pcr testing have been evaluated in leishmania including minicircle kinetoplast dna kdna 5 6 the mini exon spliced leader rna gene 7 gp63 polymerase chain reaction analysis of restriction fragment length polymorphism pcr rflp 8 and the internal transcribed spacer its 1 3 9 10

pcr is a new alternative to existing diagnostic procedures such as direct smear of parasites from clinical specimens or by cultivation with microscopic examination

2

objectives in the present study as described previously 1 11 12 we used the rflp analysis of amplified its1 in ribosomal operons to investigate the parasite that causes zcl and the genetic variations among l major isolates from chabahar a port city in southeast iran situated at the iran pakistan border

in this region the manifestation of zcl as a new focus of disease was in doubt but our findings showed that the species is l major with variable genes

the disease was correlated with the clinical manifestations of zcl in chabahar so this is a new major public health problem

3 1

study area and population a variety of nucleic acid detection methods that target both dna and rna have been developed 1

the its1 rflp pcr assay is a multipurpose tool for the diagnosis of leishmania from clinical samples and it enables determination of the infecting species 3

the procedure was performed as described by schonian et al

the study was approved by the institutional ethics committee and all patients signed informed consent forms

we evaluated the pcr rflp assay of the its1 genes for direct identification of leishmania species in 24 out of 33 suspected patients

these patients were referred for diagnosis to the central laboratory of chabahar 25 17 n 70 38 e 13 162 km2 for evaluation of skin diseases

the average annual temperature and humidity in chabahar are 36 4 c and 75 9 respectively

the population migration into and emigration out of this port have important medical implications

the clinical samples were taken from patients suspected to have cl in different endemic areas of chabahar including negor and other districts and villages

each diagnosis was confirmed by the demonstration of amastigotes on the slit smear and or the presence of flagellated promastigotes in novy macneal nicolle nnn medium from skin samples of 24 patients with typical and atypical lesions

for the parasitological investigation three skin samples were taken from the edges of lesions using sterile surgical blades

two were used for direct smears and were stained with giemsa and the third was inoculated into sterile screw tap tubes that contained blood agar slanted in nnn medium then incubated at 25 1 c

the isolated promastigotes were subcultured in rpmi 1640 medium supplemented with 15 fetal bovine serum fbs 100 μg ml streptomycin and 100 u ml penicillin

finally 24 positive culture isolates were selected for molecular examination

3 2

parasite samples the diagnosis of leishmaniasis was confirmed by the presence of amastigotes in slit smears and or the presence of flagellated promastigotes in nnn medium from typical and atypical lesions

for parasitological investigations three skin samples were collected from the edges of the lesions using sterile surgical blades

two of the samples were used for direct smears and were stained with giemsa and the third was inoculated into sterile screw top tubes containing blood agar slanted in nnn medium then incubated at 25 1 c

the isolated promastigotes were subcultured in rpmi 1640 medium supplemented with 15 fbs

they were washed twice by resuspension in buffered saline then centrifuged

the pellets were stored at 70 c until further processing

parasites from a 15 ml mid logarithmic phase of the bulk culture were harvested by centrifugation 700 g for 20 min at 4 c and washed three times in ice cold sterile pbs ph 7 2

3 3

dna preparation for species identification the dna isolated from amastigotes in ulcers and promastigotes in cultures was extracted using a commercial extraction kit high pure template dna preparation kit roche germany according to the manufacturer s instructions

leishmania reference strains were obtained from isfahan and tehran universities

the target its1 dna was amplified in a reaction mixture that consisted of 1 μl of litsr 5 cttg gatcattttccgatg 3 and 1 μl of l5 8s 5 tga tac cac tta tcg cat t 3 12

the reaction was carried out with the pcr ready supreme mix in 47 μl of total reaction 15 μl of deionized water 5 μl of target dna and 25 μl of master mix

amplification products were separated in 1 agar gel and visualized under ultraviolet light after staining with ethidium bromide

leishmania

major mhom ir 15 er and l tropica mhm su 79 k27 13 were used as positive controls

distilled water instead of the dna template was used as the negative control

the designed program for leishmania dna amplification was an initial denaturation cycle at 95 c for 5 minutes 30 cycles with cycle 1 at 95 c for 20 seconds denaturation cycle 2 at 53 c for 30 seconds annealing cycle 3 at 72 c for 1 minute extension and a final extension at 72 c for 7 minutes

3 4

restriction fragment length polymorphism the pcr products from its1 dna pcr were digested using the haeiii restriction endonuclease enzyme and the taq1 restriction enzyme as recommended by the manufacture new england biolabs inc

the its1 sequence was amplified from all isolates and reference stains were digested separately with restriction enzymes according to the manufacturer s instructions

4

results we used the pcr rflp assay of its1 genes for the direct identification of leishmania species in 24 out of 33 patients suspected to have cl

these 24 patients who were positive on smears and cultures for l donovani bodies demonstrated microscopically were selected for the study

the patient characteristics are shown in table 1 including gender age number of lesions ulcer duration and distribution of ulcers on the body

with the use of litsr l5 8s primer for pcr amplification single products of the expected size of 350 bp of l major were detected in 21 patients figure 1

in three isolates a nonspecific 450 bp band was seen

rflp was performed on the its1 region in the ribosomal operons of 24 isolates of l major

after using the restriction enzyme haeiii banding patterns including fragments of 140 bp and 210 bp bands were observed in 19 cases lma

in three cases with this enzyme 300 bp and 150 bp bands figure 2 were observed probably flagellates

no digestion was observed in two cases

electrophoresis results of its1 pcr from smears m molecular size marker 50 bp lane 1 standard l major mrho ir 75 er lane 2 standard l tropica mhom ir 99 yaz1 lane 3 pcr products 350 bp as a standard lane 4 pcr products 450 bp different from standard lanes 5 10 pcr products lane 11 negative control

restriction enzyme digestion profile of amplified its1 region with the haeiii restriction enzyme m molecular marker 50 bp lane 1 standard l major lane 2 standard l tropica lanes 3 9 pcr products lane 10 the only sample different from the normal samples and standard patterns probably flagellates lane 11 negative control

4

results digestion of its1 from ribosomal dna with taq1 enzymes enabled us to identify two different patterns

using the taq1 restriction enzyme banding patterns including 150 bp and 200 bp were observed in 19 cases same as the standard of l major genotype pattern lma

in three isolates digestion with this enzyme showed 300 bp and 150 bp banding patterns dissimilar to the l major reference strain

in two cases no digestion was observed lmb figures 3 and 4

restriction enzyme digestion profile of amplified its1 region from l major with the taqi enzyme m molecular marker 50 bp lane 1 standard l major lane 2 l tropica lanes 3 9 pcr products lane 10 the only sample different from the normal samples and standard patterns probably flagellates lane 11 negative control

m molecular marker 50 bp lane 1 standard l major lane 2 l tropica lane 3 pcr products lane 4 pcr products lane 5 pcr products lane 6 the only sample different from the normal samples and standard patterns probably flagellates lane 7 pcr products lanes 8 10 pcr products lane 11 negative control

5

discussion leishmaniasis is a major societal health problem that mostly affects populations where essential health services are not easily accessible and the detection of cl is based on clinical characteristics 14

the present study shows that cl is caused by l major in chabahar iran

parasitological methods are the gold standard for the diagnosis of cl with a high dependence on the number of parasites in the samples and a requirement of technical skill for sampling

the sensitivity of these methods varies from 27 to 85 for the diagnosis of leishmaniasis which is their main disadvantage 15

in addition contamination of the cultured medium is an occasional problem such material must be discarded

molecular techniques have proved to be sensitive and powerful tools for detecting leishmania directly in clinical samples as well as for parasite characterization using pcr 1

when pcr is appropriately applied genetic information can be obtained 16

some studies on viannia isolates from different hosts and geographic areas have found high levels of intra and interspecific variation

schonian et al

established a diagnostic its1 pcr rflp method using the restriction enzyme haeiii for leishmaniasis combining high sensitivity for detecting leishmania directly in clinical materials and the ability to identify all medically relevant species groups 12

in our study using the haeiii restriction enzyme the restriction analysis of the amplified its1 ribosomal dna revealed two different schizodeme patterns lma and lmb in l major

on the other hand some researchers have found that haeiii was not sufficient to distinguish all species of viannia 16 17

we applied more restriction enzymes in the diagnosis of leishmania species using its1 rflp pcr

we found that haeiii rsa and hinf1 can produce two profile bandings alu1 produces three dde1 and scrf1 produce four and taq1 produces five 1

in this study similar to the studies of de almeida et al

18 doudi et al

19 and mirzaie et al

20 two profile bandings 220 bp and 140 bp in haeiii digestion were detected similar to the standard

the most prevalent genotypes related to isolates in chabahar were lma