Genetic polymorphism analysis of Leishmania tropica isolated from three endemic regions (Bam, Kermanshah and Mashhad) in Iran by PCR-RFLP technique and based on ITS1 sequences

Abstract

Anthroponotic Cutaneous Leishmaniasis (ACL) is a parasitic disease caused by a single-celled parasitic protozoan Leishmania tropica with an overall prevalence of approximately 12 million cases worldwide. In Iran it is an increasing public health problem with an endemic focus in three regions of Bam, Kermanshah and Mashhad. This study was designed to characterize Leishmania spp. by polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP) technique, regarding to both high incidence rates of the disease in Iran and the variety of different species of Leishmaniawhich might affect the prevention strategies. A total of 330 samples were taken directly from skin scars of confirmed cases of leishmaniasis derived from three endemic region of Iran. The samples were used for smear-slide preparations and microbial culture to confirm the infection and then they were subjected to PCR-RFLP detection. DNA from each slide was extracted separately and was examined by means of PCR-RFLP. The information revealed in DNA sequencing was achieved from the amplification of ribosomal internal transcribed spacer 1 (ITS1) which was done using LITSr and L5.8s primers. Six different genotype groups of L. tropica were obtained in these three studied regions. In total six genotypic groups LtA, LtB, LtC, LtD, LtE, LtF were identified among L. tropicaisolates. The most frequent genotype, LtA, belonged to the isolates collected from all three endemic regions of ACL in Iran. Genotypes LtF and LtE were found in the isolate that came from Bam and LtD, LtC and LtB were identified exclusively among isolates of Mashhad. L. tropica is the pathogenic agents responsible for ACL in endemic region of Iran. This infectious agent is genetically polymorphism and it was hypothesized that there could be a probable correlation between the prevalence rate of the infection and genotypic variations of the studied stains and also their geographic origin. Key words: Polymorphism, Leishmania tropica, polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP), internal transcribed spacer 1 (ITS1).