Elucidating features of leishmaniasis transmission through analysis of single blood fed sand flies

Leishmaniasis is a vector-borne neglected parasitic disease transmitted by female phlebotomine sand fly bites. With many features of leishmaniasis transmission dynamics poorly understood, in-depth analysis of field-collected blood-fed sand flies can offer valuable insights. We hypothesize that linking the source of the sand fly bloodmeal to Leishmania infection status will shed light on the directionality of parasite transmission and point to potential animal reservoirs in a natural setting. We are developing a field-applicable toolbox based on analysis of single blood-fed sand flies preserved on FTA cards. Sand flies fed on Leishmania-infected animals were processed at discrete timepoints post-infection. We optimized DNA/RNA recovery per individual midgut, enabling us to run concurrent assays. Using a TaqMan probe-based qPCR targeting kinetoplast DNA, we detected and quantified the number of Leishmania in single infected midguts, with a clear distinction in parasite burden between immature (median 64 parasites) and mature (median 59,054 parasites) infections. Using RT-PCR, we analyzed the extracted RNA for expression of an infectious metacyclic stage-enriched gene, further distinguishing mature from immature infections. Using a multiplex PCR based on vertebrate-specific gene regions, we were able to establish host identity from sand flies experimentally fed on blood from humans and prevalent domestic animals. We are further optimizing the bloodmeal repertoire to include sylvatic animals relevant in leishmaniasis foci of East Africa to assess the performance of the toolbox in endemic areas. Once validated, this toolbox can be adapted to various localities, informing targeted control efforts in active or emerging foci of leishmaniasis.