Abstract
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 for Rhodesian HAT. Here, we describe the proof of principle application of a novel high-resolution melt assay for the xenomonitoring of Trypanosoma brucei gambiense and T. b. rhodesiense in tsetse. Both novel and previously described primers which target species-specific single copy genes were used as part of a multiplex qPCR. An additional primer set was included in the multiplex to determine if samples had sufficient genomic material for detecting genes present in low copy number. The assay was evaluated 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 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 less than 2 hours, avoiding gel electrophoresis and subjective analysis. 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.
Highlights
Human African Trypanosomiasis (HAT) is a potentially fatal disease caused by subspecies of Trypanosoma brucei s. l. transmitted by the bite of an infected tsetse fly (Glossina spp)
Gambian HAT can remain asymptomatic for months to years with symptoms often presenting once the infection has significantly advanced
Due to the zoonotic nature of Rhodesian HAT (rHAT), this World Health Organisation (WHO) target is applicable to Gambian HAT (gHAT) only
Summary
Human African Trypanosomiasis (HAT) is a potentially fatal disease caused by subspecies of Trypanosoma brucei s. l. transmitted by the bite of an infected tsetse fly (Glossina spp). Gambian HAT (gHAT), caused by Trypanosoma brucei gambiense, is a largely anthroponotic disease found across central and west Africa and accounts for the large majority of HAT cases (>97%) [1]. Rhodesian HAT (rHAT), caused by Trypanosoma brucei rhodesiense, is a zoonosis with occasional human infection, and represents less than 3% of all HAT cases. Monitoring gHAT is largely reliant on the screening of the at-risk human population and treatment of cases [4]. In comparison to gHAT, there has been little progress or investment into the development of a screening tool for rHAT with current emphasis on passive case detection and control of the vector population. A reliance on passive detection results in a delay in the identification and treatment of infected individuals, both of which are crucial for the control of disease transmission
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