The PEARL toolkit: Using sand flies to identify leishmaniasis animal reservoirs

Iniguez, E.; Huffcutt, P.; Serafim, T.; Cecilio, P.; Doh, S.; Pugh, A.; Doehl, J.; Meneses, C.; Lambert, B.; Valenzuela, J.; Kamhawi, S.

doi:10.64898/2026.06.27.734966

bioRxiv · 10.64898/2026.06.27.734966

The PEARL toolkit: Using sand flies to identify leishmaniasis animal reservoirs

Iniguez, E.·Huffcutt, P.·Serafim, T.·Cecilio, P.·Doh, S.·Pugh, A.·Doehl, J.·Meneses, C.·Lambert, B.·Valenzuela, J.·Kamhawi, S.

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Abstract

Animal reservoirs remain unknown in emerging and most endemic leishmaniasis foci hindering control efforts. Here, we developed a field-applicable PERL (Phlebotomines Establish Reservoirs of Leishmania) toolkit using individual blood fed sand flies (IBF) to identify leishmaniasis reservoirs. Using IBF, we optimized DNA and RNA co-extraction and parasite detection of [≥]1 parasite/s by kDNA qPCR and ssu rRNA RT-qPCR. We then screened IBF for expression of two parasite genes, sherp and HPB, identified by RNAseq as having low-to-absent expression in IBF given a first Leishmania donovani-infected blood meal (IBF-iBM1) and high expression in specimens provided subsequent uninfected blood meals (IBF-BMS+). Linear discriminant analysis of target gene expression classified iBM1 parasites with a predictive accuracy of ~87% and ~82% in membrane- or naturally-fed on hamsters sand flies, respectively. By determining the blood source in specimens determined as IBF-iBM1, the PERL toolkit provides an innovative and practical approach to identification of leishmaniasis reservoirs.

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Iniguez, E., Huffcutt, P., Serafim, T., Cecilio, P., Doh, S., Pugh, A., Doehl, J., Meneses, C., Lambert, B., Valenzuela, J., Kamhawi, S.. 2026-06-29. The PEARL toolkit: Using sand flies to identify leishmaniasis animal reservoirs. https://doi.org/10.64898/2026.06.27.734966

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