Multi-site DNA polymorphism analyses of Leishmania isolates define their genotypes predicting clinical epidemiology of leishmaniasis in a specific region

Leishmania isolates from 57 cases of human cutaneous (CL), human visceral (VL), and canine visceral (CVL) leishmaniasis in Turkey were grouped by multi-site DNA polymorphism analyses into five genotypes. The initial grouping was based on DNA heterogeneity of the faster-evolving mitochondrion (kinetoplast) minicircles and the intergenic regions of two nuclear repetitive genes. Taxonomic affiliation and phylogenetic relationships of the five genotypes were inferred by comparing them with reference species for sequence heterogeneity in a approximately 1.4 kb conserved single-copy gene, encoding N-acetylglucosamine-1-phosphate transferase (NAGT). Alignment of the available sequences revealed no gap, but up to 7% scattered base substitutions, suggesting that this functionally important gene is a suitable marker. Three genotypes are completely identical to the NAGTs of the reference species, identifying them as L. infantum, L. tropica. and L. major, respectively. The remaining two are recognized as L. major NAGT variants with one and four base substitutions, respectively. As expected, Maximum Likelihood analysis of the NAGT sequences separates them into three clades, corresponding to the three species. The majority of the isolates obtained are L. infantum and L. tropica, which have been known to cause infantile VL and anthroponotic CL in western and southeastern Turkey, respectively. Unexpected is the finding of Leishmania major variants and their dispersal, possibly as previously unrecognized clinico-epidemiologic entities of CL and VL.