Sequencing of hsp70 for discernment of species from the Leishmania (Viannia) guyanensis complex from endemic areas in Colombia.

BACKGROUND: Colombia is ranked very high among countries with the highest numbers of endemic Leishmania species (n = 9) causing human disease. Although much effort has been devoted to generating simple and specific tools for Leishmania species identification, challenges remain in the discrimination of species belonging to the Leishmania (Viannia) guyanensis complex: L. (V.) guyanensis and L. (V.) panamensis. METHODS: A set of seven reference strains of species belonging to the L. (Leishmania) and L. (Viannia) subgenera, clinical strains from human cases of cutaneous leishmaniasis (CL; n = 26) and samples collected from sylvatic mammals and sand flies (n = 7) from endemic areas in Colombia were analyzed in this study. The heat-shock protein 70 gene (hsp70) was amplified by PCR from DNA extracted from logarithmic-phase promastigotes or tissue samples, and the PCR products were sequenced. Sequence alignment was performed against a set of previously published and curated sequences, and phylogenetic analysis based on the maximum-likelihood and Bayesian inference approaches was conducted. Haplotype diversity among strains and species of the L. (V.) guyanensis complex was explored using a median-joining network. RESULTS: Sequencing of the hsp70 gene for L. (Viannia) spp. typing was comparable to species identification using isoenzyme electrophoresis or monoclonal antibodies. Complete species matching was found, except for one sylvatic sample with an identity yet unsolved. Among the L. (V.) panamensis clinical strains, two distinctive phylogenetic clusters were found to correlate with two different zymodemes: L. (V.) panamensis Z2.2 and Z2.3. Analysis of samples from sylvatic environments identified novel records of naturally infected wild mammal and sand fly species. CONCLUSIONS: Our results support the adequacy of hsp70 gene sequencing as a single-locus approach for discrimination of L. (Viannia) spp., as well as for exploring the genetic diversity within the L. (V.) guyanensis complex.