Multilocus sequence typing of the clinical isolates from Indian Kala-azar reveals intraspecies genetic diversity

Visceral Leishmaniasis (VL) or Kala-azar (KA), a protozoan disease caused by Leishmania sp. complex exhibits significant genetic diversity that has impacts on the epidemiology of the disease. Multilocus Sequence Typing (MLST), a powerful molecular technique to characterize and differentiate Leishmania isolates has been employed in the present study. Multiple housekeeping genes sequences were analyzed at interspecies and intraspecies levels by assessing the genetic diversity among fifteen clinical isolates collected from confirmed Indian KA patients and para-Kala-Azar Dermal Leishmaniasis (para-KDL) respectively. In our previous study by using ITS1-RFLP and ITS1 sequencing, one clinical isolate was confirmed as L. tropica and fourteen (14) were L. donovani. MLST analyses were grouped in three schemes taking into account of studied housekeeping genes in our clinical isolates and their respective available reference isolates sequences retrieved from NCBI-BLAST. We observed distinct genetic parameters at interspecies and intraspecies levels indicating significant genetic diversity among the Leishmania population. Using six (6) concatenated sequence loci, phylogenetic analyses of thirty five (35) isolates clearly differentiated two species within the Leishmania species complex and L. (Viannia) subgenus level respectively. The present study revealed distinct monophyletic and paraphyletic phylogeny clusters in the Indian clinical isolates corroborating earlier study. Interestingly, the spermidine synthase (spdsyn) gene showed no intraspecies polymorphism and is conserved locus in the Indian L. donovani population. Several Sequence Types (STs) were identified and by unique sequence type, ST3 of Alat in L. tropica isolates, Indian KA isolates of L. donovani can be differentiated from Indian KA isolate of L. tropica.