Identification ofTrypanosoma brucei gambienseandT. b. rhodesiensein vectors using multiplexed high-resolution melt analysis

Abstract

AbstractBackgroundHuman African Trypanosomiasis (HAT) is a potentially fatal parasitic infection caused by the trypanosome sub-speciesTrypanosoma brucei gambienseandT. b. rhodesiensetransmitted by tsetse flies. Currently, global HAT case numbers are reaching less than 1 case per 10,000 people in many disease foci. As such, there is a need for simple screening tools and strategies to replace active screening of the human population which can be maintained post-elimination for Gambian HAT and long-term Rhodesian HAT. Here we describe the development of a novel high-resolution melt assay for the xenomonitoring ofTrypanosoma brucei gambienseandT. b. rhodesiensein tsetse.MethodsPrimers forT. b. rhodesienseandT. b. gambiensewere designed to target species-specific single copy genes. An additional primer set was included in the multiplex to determine if samples have sufficient genomic material for detecting low copy number targets. The assay was evaluated on 96 wild-caught tsetse previously identified to be positive forT. brucei s. l.of which two were infected withT. b. rhodesiense.ResultsThe assay was found to be highly specific with no cross-reactivity with non-target trypanosome species and the assay limit of detection was 104tryps/mL. HRM successfully identified threeT. b. rhodesiensepositive flies and was in agreement with the reference sub-species-specific PCRs. This assay provides an alternative to running multiple PCRs when screening for pathogenic sub-species ofT. bruceis. l and produces results in ~2 hours, avoiding gel electrophoresis.ConclusionsThis method could provide a component of a simple and efficient method of screening large numbers of tsetse flies in known HAT foci or in areas at risk of recrudescence or threatened by the changing distribution of both forms of HAT.