Differentiation of Leishmania(L.)infantum Leishmania(L.)amazonensis and Leishmania(L.)mexicana Using Sequential qPCR Assays and High-Resolution Melt Analysis

摘要

protozoa are the etiological agents of visceral, cutaneous and mucocutaneous leishmaniasis. In specific geographical regions, such as Latin America, several species are endemic and simultaneously present; therefore, a diagnostic method for species discrimination is warranted. In this attempt, many qPCR-based assays have been developed. Recently, we have shown that and can be distinguished through the comparison of the Cq values from two qPCR assays (qPCR-ML and qPCR-ama), designed to amplify kDNA minicircle subclasses more represented in and , respectively. This paper describes the application of this approach to and introduces a new qPCR-ITS1 assay followed by high-resolution melt (HRM) analysis to differentiate this species from . We show that can be distinguished from using the same approach we had previously validated . Moreover, it was also possible to reliably discriminate from by using qPCR-ITS1 followed by an HRM analysis. Therefore, a diagnostic algorithm based on sequential qPCR assays coupled with HRM analysis was established to identify/differentiate , , and subgenus. These findings update and extend previous data published by our research group, providing an additional diagnostic tool in endemic areas with co-existing species.