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 .
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 .
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 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 38e ; 13,162 km ) for evaluation of skin diseases .
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 ,
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  1c .
the isolated promastigotes were subcultured in rpmi-1640 medium supplemented with 15% fetal bovine serum ( fbs ) , 100 g / ml streptomycin , and 100 u / ml penicillin .
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  1c .
parasites from a 15 ml mid - logarithmic phase of the bulk culture were harvested by centrifugation ( 700  g for 20 min at 4c ) and washed three times in ice - cold sterile pbs ( ph 7.2 ) . 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 .
the target its1-dna was amplified in a reaction mixture that consisted of 1 l of litsr : 5-cttg gatcattttccgatg-3 and 1 l 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  l of total reaction , 15 l of deionized water , 5 l of target dna , and 25 l of master mix .
amplification products were separated in 1% agar gel and visualized under ultraviolet light after staining with ethidium bromide .
major ( mhom / ir/15/er ) and l. tropica ( mhm / su/79/k27 ) ( 13 ) were used as positive controls
the designed program for leishmania dna amplification was an initial denaturation cycle at 95c for 5 minutes ; 30 cycles with cycle 1 at 95c for 20 seconds ( denaturation ) , cycle 2 at 53c for 30 seconds ( annealing ) , cycle 3 at 72c for 1 minute ( extension ) , and a final extension at 72c for 7 minutes .
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 .
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 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 38e ; 13,162 km ) for evaluation of skin diseases .
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 ,
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  1c .
the isolated promastigotes were subcultured in rpmi-1640 medium supplemented with 15% fetal bovine serum ( fbs ) , 100 g / ml streptomycin , and 100 u / ml penicillin .
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  1c .
parasites from a 15 ml mid - logarithmic phase of the bulk culture were harvested by centrifugation ( 700  g for 20 min at 4c ) and washed three times in ice - cold sterile pbs ( ph 7.2 ) .
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 .
the target its1-dna was amplified in a reaction mixture that consisted of 1 l of litsr : 5-cttg gatcattttccgatg-3 and 1 l 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  l of total reaction , 15 l of deionized water , 5 l of target dna , and 25 l of master mix .
amplification products were separated in 1% agar gel and visualized under ultraviolet light after staining with ethidium bromide .
major ( mhom / ir/15/er ) and l. tropica ( mhm / su/79/k27 ) ( 13 ) were used as positive controls .
the designed program for leishmania dna amplification was an initial denaturation cycle at 95c for 5 minutes ; 30 cycles with cycle 1 at 95c for 20 seconds ( denaturation ) , cycle 2 at 53c for 30 seconds ( annealing ) , cycle 3 at 72c for 1 minute ( extension ) , and a final extension at 72c for 7 minutes .
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 .
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 )
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 .
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 .
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 ,
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 .
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 ) .
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 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 ,
( 20 ) , two profile bandings ( 220 bp and 140 bp ) in haeiii digestion were detected , similar to the standard .
taq1 digestion showed two patterns with 150 bp and 200 bp bands . in contrary , in a study in yazd ( 20 ) on molecular identification using taq1 restriction enzymes , four patterns were produced .
it should be emphasized that leishmaniasis in chabahar is new , and genetic variation is limited .
the results of the present study showed that cl infections were more common in males ( 48.4% ) than in females ( 24.2% ) ( table 2 ) , probably because males spend more time outdoors than females do in this region .
some studies have shown that pcr methods achieved positive results in more than 90% of cl cases ( 13 ) .
the crithidias are able to endosymbiotically live within some parasites related to the trypanosomatidae family and sub - families of leishmania , and move to different hosts along with them .
some researchers believe that the only way to isolate crithidia from leishmania is through kdna and/or ribosomal rna genes .
it has been suggested that by extracting kdna and/or rdna , then amplifying them with pcr using the pcr - rflp technique with the haeiii enzyme and isolation of bands at 350 bp for leishmania and at 450 bp for crithidia , these two parasites can be isolated from each other ( 21 ) .
however , its1-rflp pcr showed good sensitivity , and two its1 analyses showed the same results , as in a recent study by doudi et al .
thus , the results of the present study confirm that in the border area of sistan va baluchestan , on the peninsula of chabahar , l. major is the causative agent of cl , with genotypic variation in this area .
 backgroundzoonotic cutaneous leishmaniasis ( zcl ) is polymorphic disease that may show various clinical manifestations.objectivesthis 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 methodsa 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.resultsof 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.conclusionsthe 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
2. Objectives
3. Materials and Methods
3.1. Study Area and Population
3.2. Parasite Samples
3.3. DNA Preparation
3.4. Restriction Fragment Length Polymorphism
4. Results
5. Discussion