leishmaniasis is one of the most important tropical diseases worldwide , that appears in three main forms including cutaneous , visceral ( kalaazar ) and mucocutaneous .
this disease occurs in more than 98 countries across asia , europe , africa , america and australia ( 1 , 2 ) .
cutaneous leishmaniasis ( cl ) is considered as one of the major health problems in many countries around the world including iran .
approximately 11.5 million new cases of cl are reported worldwide annually , and about 90% of cases occur in eight countries : brazil , afghanistan , peru , iran , saudi arabia , syria , sudan and algeria ( 2 , 3 ) .
cl is one of the most important parasitic diseases in iran , and is the most important arthropod - borne disease .
the major causative agent of wet type is l. major , and rodents are major reservoir of its causative agent .
this disease is endemic in rural regions of 17 out of the 31 provinces of iran ( 4 ) . in iran golestan
province and lotf - abad in northeast , abar dezh , varamin , isfahan and yazd in the center , fars and sistan - o - baluchestan provinces in south and southeast , and ilam and khuzestan provinces in southwest of the country are reported as endemic foci of the disease ( 1 ) .
l. tropical is causative agent of dry type of cl and primarily human and in the next level dogs are its reservoir .
although , microscopic observation of parasites on direct stained direct smears from wounds is the simplest method to detect leishmania , but low sensitivity for the detection of parasites in low - grade parasitemia is the problem with this method ( 3 , 5 ) . to date , species identification of leishmania spp . is based on molecular approaches such as pcr methods .
pcr methods have advantages such as need to small amounts of dna , it is not affected by confounding conditions of environmental and host , examination of numerous samples in a short time and its high sensitivity ( 6 , 7 ) .
kalaleh county is located in the east and it is the fifth populous county of the province
. meanwhile , the county is located near endemic counties ( such as gonbad - e kavus , marave tappe , minoo dasht ) and other provinces ( such as north khorasan and semnan ) for cl , so increasing trend in incidence of cl , is probable .
a research carried out a study on vectors and reservoirs of leishmaniasis in the kalaleh county during 20067 , in this study phlebotomus papatasi was found predominant species of sandflies ( 8) . a study conducted on identification of leishmania spp . in border villages of gonbad - e - qabus county using pcr method on its1 gene in 2007 , in this study l. major was reported as the predominant species ( 9 ) .
moreover , ph . papatasi was reported as the predominant sandflies in the marave tappe , in 20112012 ( 10 ) . due to endemicity of the cl in neighboring regions of the kalaleh county ,
complex and various features of the cutaneous lesions and different treatment protocols for different species , exact identifications of the causative agent is essential , on the other hand there is no published study for this aim .
the objectives of this study were a better understanding of the demographic situation of the disease and molecular identification of the parasite species on stained slides of the referred patients to health center of the studied region , during 201314 .
in this descriptive cross - sectional study , samples were collected from 70-suspected individuals to cl that referred to laboratory of kalaleh health center , kalaleh county , golestan province , northen iran , from sep 2013 to nov 2014 .
demographic information of each patient including age , sex , locality , the onset of disease , number of lesions and location of lesions were recorded .
after disinfection of sampling sites , serous were collected from the margin of the lesions by sterile vaccine style .
thin smears were stained and slides were microscopically examined for the presence of leishman bodies ( inside or out of macrophages ) .
total genomic dna was extracted from positive samples by phenol - chloroform method ( 11 ) .
this fragment was amplified by using litsr ( 5 ctggatcattttccgatg 3 ) as forward and l5.8s ( 5tgataccacttatcgcactt3 ) as reverse primer .
study standard strains , l. tropica ( standard ) mhom / ir/02/mhom / r and l. major ( standard ) mhom / ir/75/er , obtained from school of public health , tehran university of medical sciences were used as positive controls .
pcr was prepared using amplicon ( taq dna polymerase master mix ) as ready - made mixture .
pcr was performed in a 15 l mixture containing 7.5 l taq master mix , 5 ng dna , 10 pmol of each primer and 5.5 l distilled water .
all of the pcrs were carried out under following conditions : an initial denaturation step at 95 c for 5 min , 30 repetitions at 94 c for 30 sec , an annealing step at 47 c for 30 sec , extension step at 72 c for 1 min , and final extension 72 c for 7 min .
pcr products were examined using 1.5% gel agarose , stained with ethidium bromide solution , and were seen under uv light .
rflp examination : rflp was carried out on positive pcr products to determine the parasite species .
the enzyme can cut pcr products to 200 and 140 bp fragments , and 200 , 60 and 40 bp fragments in l. major and l. tropica , respectively .
for this purpose 30 l reactions were prepared containing 10 l pcr product ( equivalent 1 g ) , 3 l enzyme - specific buffer , 17 l distilled water and 1 l fast - acting haeiii .
after a very short centrifugation , reactions were incubated at 37 c for 15 min to cut its-1 gene by the enzyme function . to confirm pcr - rflp results , beside 100 bp dna ladder ( product of sina gene company , iran , cat no :
finally , obtained data were analyzed using spss software ( version 11 ) and chi - squared test .
thirty - eight ( 54.3% ) specimens out of the 70 specimens were found positive for leishman body by microscopic examination of thin smears . among patients , 22 cases ( 57.9% )
frequency of cl cases based on sex , age group and location of lesion in kalaleh county , during 201314 there was no significant difference between the two sexes .
the youngest patient was 3 yr old and the oldest patient was 60-yr - old ( table 1 ) .
the most lesions were observed on hand , foot and face , respectively , but there was no statically significant difference in involvement of different organs ( table 1 ) .
the highest frequency was in the age group of 30-to 50-yr - old and the lowest frequency was in the age group of 19 yr old .
37% of patients were from urban regions ( kalaleh city ) and 67% of patients were from rural regions ( table 1 ) .
all of the 38 positive smears were found positive by pcr analysis , too , and an approximately 340 bp bands were detected under uv light after transferring pcr products of both of positive specimens and standard strains to 1.5% agarose gel ( fig .
agarose ( 1.5% ) gel showing the pcr products ( 340 bp ) of amplified from standard strain and samples .
lane m is dna size marker , lane 1 is standard strain of l. major , lanes 210 are positive samples and lane 11 is negative control agarose ( 2.5% ) gel showing the pcr - rflp pattern of its-1 gene cut with haeiii endonuclease ( approximately 200 bp and 140 bp bands ) .
lane m indicating dna size marker , lanes 14 indicating rflp pattern of l. major treatment protocol in the studied region was five times and weekly oblique injection of 5 ml glucantim ( lab osterop company , belgium ) under the edge of lesion and at four points , among the aforementioned patients , 10 were not admitted for treatment due to commuting costs , and presence of lesion in the unexposed parts of body .
because of increased awareness of endemic people about establishment of immunity against cl while lacking treatment , if lesions locate in the unexposed parts of the body , majority of patients are not eager to complete treatment .
the present study is the first study on frequency of cl in kalaleh county , northern iran . in this study , 70 specimens were examined during 15 months , by both microscopic and molecular methods 38 specimens ( 54.3% ) were found positive .
the most affected parts of body were hand , foot , and face , respectively . generally ,
the most affected parts of body are hands and feet in rural cl and face in urban cl ( 1 ) . in this study , most patients ( 47.4% ) had only one lesion .
about 2 , 3 and 7 lesions were seen in 16 ( 42.1% ) , 3 and 1 cases , respectively . these findings
( number of lesions ) are in agreement with results of l. major lesions ( 1214 ) .
the highest prevalence was seen in the age group of 3050 yr old , as 50% of the patients were in this category ; this can be caused by more contact of this age group with vector of the disease , because they spend more time outdoors .
these findings are in consistent with results of studies in gonbad - e - qabus , in kermanshah , and kashan ( 9 , 15 , 16 ) .
one study determined l. major as major causative agent of cl by pcr method in gonbad - e - qavus ( 9 ) .
papatasi is predominant species of sandflies in marave tappe county , golestan province ( 10 ) .
the large scale entrance of non - immune individuals ( such as emigrants , tourists and soldiers ) to epidemic regions result in significant increase in the disease incidence and epidemic leishmaniasis ( 17 , 18 ) .
interestingly , an epidemic event occurs in 2011 , 85% of patients were emigrants ( labors of border villages and soldiers ) . on the other hand
the main reason for decrease of the cases in 2012 , was preventive measures of county and province health centers , including awareness of the people about transmission of the disease and health education , vector control ( spraying of insecticide particularly in husbandries ) and rodent control using zinc phosphide grain baits ( 8) . in this study ,
dna extraction for all smears was done successfully , in accordance with another study ( 19 ) .
thus , molecular identification could be done on stained smears . however , the limitation of our study was lack of dna extraction on negative smears , which should be done in future .
however , smears without parasites should also submit to molecular identification , as they may be positive .
after adding haeiii endonuclease ( carrying out rflp ) to pcr products of litsr and l5.8s primers , and running electrophoresis , 200 and 140 bp bands , and 200 and 60 bp bands are detectable under uv light for l. major and l. tropica , respectively ( 19 ) .
researchers used its - pcr - rflp and introduced as an appropriate method to distinguish species of leishmania ( 20 , 21 ) .
l. major is found causative agent of cl in this case , however , more studies on investigation of reservoir of the parasite and natural infection in vectors is highly recommended .
ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc . ) have been completely observed by the authors .
 background : leishmaniasis is a zoonotic disease caused by species of protozoa of the genus leishmania . in recent years , incidence of cutaneous leishmaniasis has increasing trend in golestan province , north of iran . 
 the aim of the present study was to identify the frequency of cutaneous leishmaniasis using pcr - rflp in patients referred to kalaleh health center , during 201314.methods:this descriptive cross - sectional study was conducted on 70 individuals with suspected cutaneous leishmaniasis that referred to health center of kalaleh county , golestan province , northern iran , from sep 2013 to nov 2014 . 
 samples of cutaneous lesions were examined microscopically . 
 dna was extracted from all of the positive smears and pcr was done on its-1 gene . 
 rflp was performed using haeiii enzyme for species identification.results:totally , 38 out of the 70 ( 54.3% ) suspected individuals including 22 males ( 57.9% ) were found positive by microscopic examination . 
 all of microscopically positive samples were confirmed to be positive for leishmania dna ( approximately 340 bp bands were detected ) . 
 rflp revealed 140 bp and 200 bp bands ( approximate size ) , indicative of l. major.conclusion:the detected species of studied region was l. major . 
 cutaneous leishmaniasis has high prevalence in kalaleh county , thus more studies on leishmaniasis in the animal reservoirs , comparison of homology of animal and human isolates and a survey regarding natural infection of vectors in this region is highly recommended . 
Introduction
Materials and Methods
Results
Discussion
Conclusion
Ethical considerations