The PERL toolkit: Using sand flies to identify leishmaniasis reservoirs

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.