Molecular characterization of Leishmania spp. causing cutaneous Leishmaniasis in Nepal

Abstract

Leishmaniasis is one of the leading vector borne disease to cause death world-wide. It is caused by more than 20 Leishmania species in 98 countries in five continents. The disease is categorized as one of the most “neglected tropical diseases'' and has a strong and complex association with poverty. Nepal, was formerly endemic for the visceral type where fewer cases of cutaneous leishmaniasis has been seen. Due to lack of facilities at all medical centers, diagnosing the disease is challenging. The main concern right now to determine the types of Leishmania spp. that are currently prevalent in Nepal and identifying whether or not the agent that causes visceral form is also responsible for cutaneous form. Patients with cutaneous lesions were sampled for parasitological diagnosis using direct examination (DE), kinetoplast DNA (kDNA) nested PCR (CSB1X/CSB2X and 13Z/LiR primers). Further, the kDNA positive samples were amplified for the ITS-1 region. The amplified ITS-1 region were subjected to Restriction Fragment Length Polymorphism (RFLP) using enzyme HaeIII. For the validation of the RFLP result Sequencing was performed. The data were statistically analyzed using graph pad prism. Only 22 (55%) were found to be positive for kDNA Nested PCR observed bands were 720bp, 600bp for Leishmania donovani complex and Leishmania major respectively and 12 (30%) on ITS-1 PCR. Following, ITS1 PCR-RFLP genotyping of ITS-1 with restriction enzyme HaeIII, results in two distinct patterns that clearly distinguished L. donovani (50,75,180 bp) from L. major (140, 210bp). The RFLP finding was then validated by sequencing the amplified ITS1 PCR products. This study finds that the parasite L. donovani which causes visceral form of the disease is also the causative agent for cutaneous form. Two species L. donovani and L. major are circulating species causing Cutaneous Leishmaniasis. As the CL is in increasing trend in Nepal. PCR based diagnostic facilities might help to prevent misdiagnose the disease. Molecular screening can be done by ITS1 PCR-RFLP followed by sequencing. Keywords: Cutaneous leishmaniasis, HaeIII, kinetoplast DNA, nested PCR, RFLP, Sequencing.