Real-time PCR to differentiate among Leishmania (Viannia) subgenus, Leishmania (Leishmania) infantum and Leishmania (Leishmania) amazonensis: Application on Brazilian clinical samples.

Leishmaniasis is a complex disease caused by Leishmania species belonging to subgenera Leishmania and Viannia. In South America, L. (L.) infantum is considered the most important causative agent of visceral leishmaniasis, while L. (L.) amazonensis and Viannia subgenus species are responsible for the different cutaneous or mucocutaneous forms. In our previous work, we developed a diagnostic approach for Leishmania species discrimination based on two qPCRs (qPCR-ML and qPCR-ama) targeting the minicircle kDNA followed by melting analysis. This approach allowed to (i) differentiate the subgenera Leishmania and Viannia, and (ii) distinguish between L. (L.) infantum and L. (L.) amazonensis. The aim of this work was to demonstrate the applicability of the approach previously described, using human and canine clinical samples and strains from a Brazilian region, where L. (L.) infantum, L. (L.) amazonensis and Viannia subgenus species coexist. After validation on New World strains, the diagnostic approach was applied blindly to 36 canine clinical samples (peripheral blood and bone marrow) and 11 human clinical samples (peripheral blood and bone marrow). The sensitivity was 95.6% (95% confidence interval 77.3-100%) and 100% (95% confidence interval 76.9-100%) in the canine bone marrow samples and human (peripheral blood and bone marrow) samples, respectively, compared to conventional PCR assays. Concerning the Leishmania species identification, the conventional and qPCR-based methods showed kappa value of 0.876 (95% confidence interval 0.638-1.000), indicating good agreement. Therefore, this approach proved to be useful in both veterinary and human clinical context in regions co-endemic for L. (L.) infantum, L. (L.) amazonensis, and Viannia subgenus, helping to provide rapid diagnosis and to allow studies of species distribution.