Extracellular Vesicle-Derived Spliced Leader RNA as a Minimally Invasive Molecular Biomarker for Leishmaniasis...

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 proof-of-concept study, we evaluated the feasibility of detecting Leishmania-derived SL-RNA in EV-enriched preparations from experimental leishmaniasis model systems. EV-enriched preparations were isolated from parasite-conditioned media (PCM), infected THP-1 macrophage (I-MP) cell-conditioned medium (CCM), and serum from experimentally infected mice using a precipitation method. As a complementary isolation approach, additional preparations were isolated from PCM by ultracentrifugation. An aliquot of each preparation was analyzed by nanoparticle tracking analysis to determine particle size and concentration, and the remaining portion was used for RNA extraction. RNA was reverse transcribed into cDNA and analyzed for SL-RNA amplification using quantitative polymerase chain reaction (qPCR). SL-RNA amplification was successfully detected in PCM, I-MP CCM, and serum from experimentally infected mice, while uninfected controls showed no specific amplification. These findings were independently confirmed via ddPCR analysis. Notably, EV-enriched preparations from infected mice yielded a stronger SL-RNA signal than serum alone, highlighting the diagnostic potential of targeting SL-RNA in EV-enriched human clinical samples, particularly in low-burden infections. Although these findings support the feasibility of EV-associated SL-RNA as a molecular biomarker for Leishmania infection, clinical validation with human samples is required.