PDF(Pubmed)
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.
摘要：
人类非洲锥虫病（HAT）是由锥虫亚种布氏锥虫和T.b引起的潜在致命的寄生虫感染。目前,在许多疾病病灶中，全球HAT病例数达到每10,000人中不到1例。因此，需要简单的筛选工具和策略来代替对GambianHAT和RhodesianHAT的长期消除后可以维持的人群的主动筛选。这里,我们描述了一种新颖的高分辨率熔解测定法在采采录中的异种监测中的原理应用证明。将靶向物种特异性单拷贝基因的新的和先前描述的引物用作多重qPCR的一部分。在多重中包括另外的引物组以确定样品是否具有足够的基因组材料用于检测以低拷贝数存在的基因。对先前鉴定为T.bruceis.l阳性的96个野生捕获的采采采进行评估。已知其中两个对T.b.Rhodesiense呈阳性。发现该测定是高度特异性的,没有与非靶锥虫物种的交叉反应性,并且测定检测极限为104胰蛋白酶/mL。qPCR成功鉴定出三只罗得西亚氏杆菌阳性果蝇，与参考物种特异性PCR一致。该测定提供了在筛选T.bruceis.l的致病性亚种时运行多个PCR的替代方案，并在不到2小时内产生结果，避免凝胶电泳和主观分析。该方法可以提供一种简单有效的方法的组成部分，该方法可以在已知的HAT病灶中或在有复发风险或受到两种形式的HAT分布变化威胁的区域中筛选大量的采采蝇。
