PT  - JOURNAL ARTICLE
AU  - Gala Garrod
AU  - Emily R. Adams
AU  - Jessica K. Lingley
AU  - Isabel Saldanha
AU  - Stephen J. Torr
AU  - Lucas J. Cunningham
TI  - Identification of <em>Trypanosoma brucei gambiense</em> and <em>T. b. rhodesiense</em> in vectors using multiplexed high-resolution melt analysis
AID  - 10.1101/2020.04.21.052928
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.04.21.052928
4099  - http://biorxiv.org/content/early/2020/04/21/2020.04.21.052928.short
4100  - http://biorxiv.org/content/early/2020/04/21/2020.04.21.052928.full
AB  - Background 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. 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 of Trypanosoma brucei gambiense and T. b. rhodesiense in tsetse.Methods Primers for T. b. rhodesiense and T. b. gambiense were 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 for T. brucei s. l. of which two were infected with T. b. rhodesiense.Results 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. HRM successfully identified three T. b. rhodesiense positive 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 of T. brucei s. l and produces results in ~2 hours, avoiding gel electrophoresis.Conclusions This 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.