Development and evaluation of different PCR-based typing methods for discrimination of Leishmania donovani isolates from Nepal

Introduction. Leishmania donovani, the causative agent of visceral leishmaniasis in the Indian subcontinent, has been reported to be genetically homogeneous. In order to support ongoing initiatives to eliminate the disease, highly discriminative tools are required for documenting the parasite population and dynamics. Methods. Thirty-four clinical isolates of L. donovani from Nepal were analysed on the basis of size and restriction endonuclease polymorphisms of PCR amplicons from kinetoplast minicircle DNA, 5 nuclear microsatellites, and nuclear loci encoding glycoprotein 63, cysteine proteinase B, and hydrophilic acylated surface protein B. We present and validate a procedure allowing standardized analysis of kDNA fingerprint patterns. Results. Our results show that parasites are best discriminated on the basis of kinetoplast minicircle DNA (14 genotypes) and 1 microsatellite defining 7 genotypes, while the remaining markers discriminated 2 groups or were monomorphic. Combination of all nuclear markers revealed 8 genotypes, while extension with kDNA data yielded 18 genotypes. Conclusion. We present tools that allow discrimination of closely related L. donovani strains circulating in the Terai region of Nepal. These can be used to study the micro-epidemiology of parasite populations, determine the geographical origin of infections, distinguish relapses from re-infection, and monitor the spread of particular variants