Development and performance evaluation of an easy-to-use multiplex qPCR assay for the diagnosis of human visceral leishmaniasis

Laboratory diagnosis of human visceral leishmaniasis (VL) is essential for the timely initiation of appropriate treatment and the prevention of severe complications, including death. Molecular assays are recognized for their high diagnostic accuracy, particularly multiplex real-time quantitative PCR (qPCR) assay, which allow the simultaneous detection of multiple molecular targets and the amplification of endogenous controls. This study aimed to develop and evaluate the clinical performance of a multiplex qPCR assay for the diagnosis of VL by simultaneously detecting two Leishmania molecular targets: 18S rDNA for the detection of Leishmania spp. and kDNA for the specific detection of L. infantum , in addition to an endogenous control (GAPDH). This study was designed as a retrospective diagnostic accuracy study using stored DNA samples (peripheral blood), including 73 confirmed VL cases and 102 non-VL cases. Diagnostic accuracy was determined using a composite reference standard based on parasitological test, rk39 immunochromatographic test, and/or kDNA PCR assay. The multiplex qPCR assay showed limits of detection of 0.58 parasites/reaction for the 18S rDNA target and 0.16 parasites/reaction for the kDNA target. The kDNA primers and probe were specific for L. infantum , whereas the 18S rDNA primers and probe were specific for the genus Leishmania . During the clinical evaluation, both molecular targets yielded concordant results. The assay showed a sensitivity of 98.6%, specificity of 100%, and overall diagnostic accuracy of 99.4%. These findings support the use of this multiplex qPCR assay as a reliable molecular tool for the laboratory diagnosis of VL.