Assessment of High-Resolution Melting Curve Analysis for Leishmania spp. Detection in Different Clinical Manifestations of Leishmaniasis in India

The accurate diagnosis and identification of species are crucial for the therapeutic selection and effective treatment of leishmaniasis. This study aims to develop and evaluate the use of high-resolution melting curve analysis (HRM)-PCR for species identification causing visceral leishmaniasis (VL), post-kala-azar dermal leishmaniasis (PKDL) and cutaneous leishmaniasis (CL) in the Indian subcontinent. Two multi-copy targets (ITS-1 and 7SL-RNA genes) were selected, and an HRM-PCR assay was established using , , and standard strain DNA. The assay was applied on 93 clinical samples with confirmed infection, including VL ( = 30), PKDL ( = 50), and CL ( = 13) cases. The ITS-1 HRM-PCR assay detected as little as 0.01 pg of template DNA for and up to 0.1 pg for and . The detection limit for the 7SL-RNA HRM-PCR was 1 pg for and 10 pg for and . The ITS-1 HRM-PCR identified 68 out of 93 (73.11%) leishmaniasis cases, whereas 7SL-RNA HRM-PCR could only detect 18 out of 93 (19.35%) cases. A significant correlation was observed between the kDNA-based low Ct values and ITS-1 HRM-PCR positivity in the VL ( = 0.007), PKDL ( = 0.0002), and CL ( = 0.03) samples. The ITS-1 HRM-PCR assay could identify spp. causing different clinical forms of leishmaniasis in the Indian subcontinent, providing rapid and accurate results that can guide clinical management and treatment decisions.