Abstract
<jats:p>Leishmaniasis continues to be a major neglected tropical disease, particularly in endemic areas where reliable diagnosis remains challenging. Many existing diagnostic methods are invasive, lack sufficient sensitivity, or require equipment and expertise that may not be available in resource-limited settings. Parasite-derived spliced leader (SL) RNA has recently gained attention as a promising molecular marker because it may enable the sensitive detection of viable Leishmania parasites and help track changes in infection over time. Extracellular vesicles (EVs) are also of growing interest because they protect and transport stable biomolecules that may provide valuable diagnostic information and influence interactions between the parasite and its host. In this study, we evaluated the feasibility of detecting Leishmania-derived SL-RNA within EV-associated samples. EVs were isolated from parasite-conditioned media (PCM) and infected THP-1 macrophage (I-MP) cell-conditioned medium (CCM), followed by nanoparticle tracking analysis for EV characterization. RNA was extracted from EV preparations, reverse transcribed into cDNA and analyzed for SL-RNA amplification using quantitative polymerase chain reaction (qPCR) and droplet digital PCR (ddPCR) approaches. Successful amplification of SL-RNA was detected in PCM and I-MP CCM EV samples, whereas uninfected controls showed no specific amplification. ddPCR analysis demonstrated sensitive detection across infected samples which supports the potential utility of this approach for low-abundance target detection. These findings support the feasibility of EV-associated SL-RNA as a molecular biomarker for Leishmania infection.</jats:p>