부산대학교 도서관

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. Author summary: With global cases of Human African Trypanosomiasis (HAT) reaching pre-elimination numbers, identifying areas of trypanosome transmission becomes increasingly important. With decreasing numbers of cases, the cost of identifying each positive case increases. Screening for trypanosomes in tsetse flies instead of in the human host allows for a faster, more cost-effective and efficient method of detecting areas in which parasite transmission is on-going. Molecular tools such as PCR play a critical role in molecular identification of parasites in vectors. However, at present, multiple PCRs are required to confirm the presence or absence of human infective trypanosomes. Here we present a novel assay which allows for the screening of both species of trypanosomes responsible for HAT in one assay. This method eliminates the need for multiple assays per sample, produces results which are easier to interpret, and reduces the risk of contamination of samples. We found the assay to be as sensitive as the current gold-standard PCR when evaluated on a subset of wild tsetse flies. With further field evaluation, this method could provide an alternative monitoring method for HAT, which can be used leading up to elimination and maintained once elimination has been achieved. [ABSTRACT FROM AUTHOR]