Study of Genetic Variation of Leishmania major Based on Internal Transcribed Spacer 1 (ITS1) in Chabahar, Iran

Abstract

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.

Molecular Identification of Agents of Human Cutaneous Leishmaniasis and Canine Visceral Leishmaniasis in Different Areas of Iran Using Internal Transcribed Spacer 1 PCR-RFLP

“…These findings are similar to studies reported that L. tropica isolates included the highest divergence in ITS1 genes ( 26 ). Similar to a study on L. major with 98.2% similarity, results of the current study showed a limited genetic variation with seven haplotypes seen in L. major (H1–H7) ( 25 ). Four haplotypes of L. major were found in a distinct clade in nine rodent isolates in Central Iran ( 23 ) and six haplotypes of L. major were identified through PCR-RFLP of rodent samples using ITS1 markers ( 8 ).…”

“…Several studies have amplified ITS-1 region of the ribosomal DNA repeat unit (rDNA-ITS1) for the recognition of Iranian Leishmania spp. ( 8 , 23 – 25 ). In the present study, a number of unknown Leishmania spp.…”

The Geographical Distribution of Human Cutaneous and Visceral Leishmania Species Identified by Molecular Methods in Iran: A Systematic Review With Meta-Analysis

“…This region is highly conserved among species (178). The ITS region as a target for differentiation of Leishmania at species and strain level has been used in different studies (102,105,132). As a whole, it should be noted that apply of two genetic markers simultaneously could provide more data regarding genetic map of the Leishmania parasite particularly in an endemic focus.…”

A rare encounter in nonsuspecting circumstances: First congenital visceral leishmaniasis (kala-azar) in Libya

“…The PCR products were visualized using ultraviolet transillumination with GelRed TM dye (GIBCO BRL Life Technologies, Inc., Gaithersburg, MD, USA) on 1.55 agarose gel (SIGMA, St. Louis) in 1X TAE buffer for 45 min at 100 V after electrophoresis; the size of the amplified product was determined by comparison with a 100 bp ladder (Sigma-Aldrich, USA). [2] The results were positive for Leishmania infantum [Figure 2].…”