A pilot study demonstrating the identification of Trypanosoma brucei gambiense and T. b. rhodesiense in vectors using a multiplexed high-resolution me

Human African Trypanosomiasis (HAT) is a potentially fatal parasitic infection caused by the trypanosome sub-species Trypanosoma brucei gambiense and T. b. rhodesiense transmitted by tsetse flies. HAT surveillance is reliant on active and passive case detection and with case numbers nearing elimination in many foci there is a need for simple and sustainable screening methods. Here, we describe a proof of principle application of a novel qPCR assay designed to detect both human pathogenic trypanosome species in tsetse. Assay evaluation was carried out on 96 wild-caught tsetse previously identified to be positive for T. brucei s. l. of which two were known to be positive for T. b. rhodesiense. The assay was found to be highly specific with no cross-reactivity with non-target trypanosome species and the assay limit of detection was 104 tryps/mL. The qPCR successfully identified three T. b. rhodesiense positive flies, in agreement with the reference PCRs. This assay provides an alternative to running multiple PCRs when screening for pathogenic sub-species of T. brucei s. l. and produces quantitative results in less than 2 hours. This method could provide a component of a simple and efficient method of screening large numbers of tsetse flies in known HAT foci, in areas at risk of recrudescence or threatened by the changing distribution of HAT.