An integrated molecular approach for the detection and species-level identification of Leishmania spp. in clinical samples.

Leishmaniases are parasitic diseases transmitted by infected sand flies, present in over 98 countries. Accurate diagnostic tools are critical for prevention and control, especially in non-endemic areas. We present a standardized molecular workflow combining two PCR-based strategies: sensitive detection through amplification of conserved Leishmania kinetoplast DNA (kDNA) minicircles and species identification via sequencing of a 1,116-bp hsp70 gene fragment. The optimized kDNA-PCR achieved detection limits of 0.05 parasites/mL for Leishmania (Leishmania) infantum and 0.5 parasites/mL for Leishmania (Viannia) braziliensis, the most prevalent species in Argentina. The method also enabled differential diagnosis with Chagas disease and endemic mycoses. Evaluation with 150 clinical samples from 83 patients showed 69.7% sensitivity and 68% specificity, reflecting improved performance compared to microscopy. Eight kDNA-PCR "false positives" were confirmed as true infections by hsp70 sequencing. Four species were identified: Leishmania (Viannia) braziliensis, Leishmania (Viannia) guyanensis, Leishmania (Viannia) panamensis, and Leishmania (Leishmania) mexicana. This workflow provides a reliable diagnostic and species-level tool in resource-limited, endemic settings.IMPORTANCELeishmaniases are neglected tropical diseases that remain a major public health problem in many parts of the world. Accurate diagnosis is essential for guiding treatment and controlling transmission, yet current methods often fail to detect low parasite loads or identify the infecting species. This study introduces a standardized molecular workflow that combines two complementary PCR-based strategies for the detection and species identification of Leishmania directly from clinical samples. The approach increases sensitivity, allows discrimination from other endemic infections, and identifies the circulating species with high reliability. Its implementation revealed multiple Leishmania species in Argentina and neighboring countries, highlighting its value for epidemiological surveillance. The workflow is simple, adaptable, and cost-effective, making it suitable for laboratories in resource-limited settings. By improving diagnostic accuracy, this method supports better patient management and strengthens regional capacity to monitor and control leishmaniasis.