Molecular Diagnosis of Leishmaniasis: Development of a qPCR Assay for Genus Detection and Differentiation of Leishmania (L.) amazonensis and Leishmania (V.) braziliensis.

Background/

Objective

Leishmaniasis is a neglected tropical disease caused by species of the genus Leishmania, with a broad clinical spectrum that can overlap with other infectious and non-infectious conditions. Accurate species identification is critical for appropriate treatment and prognosis; however, parasitological methods are limited by suboptimal sensitivity, specificity, and inability to reliably differentiate species. This study aimed to develop and validate a real-time PCR assay based on melting-curve analysis (Leish-qPCR) for the detection of Leishmania spp. and the differentiation of Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis. Methods and

Results

Genus-specific primers were designed based on the kDNA (kinetoplast DNA) minicircle consensus sequences of Leishmania species, while species-specific primers targeted the internal transcribed spacer 2 (ITS2) consensus regions of the ribosomal RNA locus of L. (L.) amazonensis and L. (V.) braziliensis. Analytical performance was evaluated in silico and in vitro using a panel of protozoa, fungi, and bacteria, exhibiting 100% specificity with no cross-amplification. The limit of detection was one copy per reaction for all targets using positive controls. Clinical validation was performed using skin biopsy specimens from patients with granulomatous lesions. The optimized Leish-qPCR assay, performed in separate reaction tubes within the same run, demonstrated reliable analytical specificity and sensitivity, with distinct and reproducible melting temperature (Tm) peaks across plasmid controls, parasite DNA, and clinical samples. Comparative analysis with histopathological examination demonstrated moderate agreement between the methods, supporting the applicability of the assay for sensitive detection and species-level discrimination of Leishmania spp. in clinical samples.

Conclusions

The Leish-qPCR assay presented high sensitivity, specificity, and diagnostic accuracy, representing a promising tool for routine diagnosis of leishmaniasis and for the differentiation of L. (L.) amazonensis and L. (V.) braziliensis in clinical samples.