Preliminary evaluation of a qPCR HRM assay for differentiation of Leishmania major and Leishmania tropica

Rapid and accurate identification of Leishmania species is important for the diagnosis and epidemiological surveillance of cutaneous leishmaniasis (CL). High-resolution melting (HRM) analysis has emerged as a rapid molecular approach for species discrimination. This study aimed to preliminarily evaluate a quantitative real-time PCR coupled with HRM (qPCR-HRM) assay targeting kinetoplast minicircle DNA (kDNA) for differentiation of Leishmania major and Leishmania tropica in northeastern Iran. A qPCR-HRM assay targeting the kDNA region was developed using species-specific primers. Clinical samples from 60 patients with suspected CL in North Khorasan and Razavi Khorasan provinces were examined microscopically and cultured. DNA extracted from cultured promastigotes was analyzed using qPCR-HRM, and the results were compared with conventional PCR and ITS1-rDNA sequencing. In addition, ITS1-rDNA sequence analysis was performed to evaluate the genetic diversity of L. major isolates. Among the 60 suspected CL samples, 13 were positive by molecular analysis. qPCR-HRM successfully differentiated reference strains of L. major and L. tropica based on distinct melting temperature profiles, with a mean ΔTm of approximately 0.86 °C ( L. major : 84.13 °C; L. tropica : 83.27 °C). All clinical isolates were identified as L. major by qPCR-HRM and confirmed by ITS1 sequencing, showing complete agreement between the two methods. ITS1-rDNA analysis of the 13 L. major isolates identified nine sequence types and seven haplotypes. The qPCR-HRM assay demonstrated promising performance for rapid differentiation of L. major and L. tropica under the conditions evaluated in this study. However, further validation using larger numbers of direct clinical specimens representing both species is required before routine diagnostic application.