Evaluation of 4 polymerase chain reaction protocols for cultured Leishmania spp. typing

Leishmaniasis is a disease caused by the protozoan Leishmania resulting in a variety of clinical manifestations, from self-healing skin lesions to fatal visceral disease. The development of polymerase chain reaction (PCR)-based techniques has made species identification easier, faster, and less labor intensive. The main targets for PCR amplification include kinetoplastid DNA (kDNA), miniexon, and conserved regions such as the internal transcribed spacer. The objective of this work was to evaluate 4 different PCR techniques designed to type Leishmania using laboratory strains. Parasites were subjected to 4 PCR procedures using specific Leishmania primers for miniexon (designated A1 and A2) and kDNA (designated B1 and B2, C1 and C2, and D1, D2 and D3). Discrimination between some species and the 2 main subgenera Leishmania and Viannia was achieved. Unweighted pair group method analysis resulted in the expected clustering of the 2 species from the subgenus Leishmania. However, some species in the subgenus Viannia could not be distinguished, representing a continued challenge for PCR-based protocols. Results are discussed in terms of advantages, limitations, and reproducibility of these 4 PCR-based techniques in the taxonomy of Leishmania.