Department of Vector Biology, Liverpool School of Tropical Medicine, Liverpool, United Kingdom.
Vector and Vector-borne Diseases Research Institute, Tanga, Tanzania.
PLoS Negl Trop Dis. 2024 Aug 12;18(8):e0012095. doi: 10.1371/journal.pntd.0012095. eCollection 2024 Aug.
Tsetse flies (Glossina sp.) are vectors of Trypanosoma brucei subspecies that cause human African trypanosomiasis (HAT). Capturing and screening tsetse is critical for HAT surveillance. Classically, tsetse have been microscopically analysed to identify trypanosomes, but this is increasingly replaced with molecular xenomonitoring. Nonetheless, sensitive T. brucei-detection assays, such as TBR-PCR, are vulnerable to DNA cross-contamination. This may occur at capture, when often multiple live tsetse are retained temporarily in the cage of a trap. This study set out to determine whether infected tsetse can contaminate naïve tsetse with T. brucei DNA via faeces when co-housed.
METHODOLOGY/PRINCIPLE FINDINGS: Insectary-reared teneral G. morsitans morsitans were fed an infectious T. b. brucei-spiked bloodmeal. At 19 days post-infection, infected and naïve tsetse were caged together in the following ratios: (T1) 9:3, (T2) 6:6 (T3) 1:11 and a control (C0) 0:12 in triplicate. Following 24-hour incubation, DNA was extracted from each fly and screened for parasite DNA presence using PCR and qPCR. All insectary-reared infected flies were positive for T. brucei DNA using TBR-qPCR. However, naïve tsetse also tested positive. Even at a ratio of 1 infected to 11 naïve flies, 91% of naïve tsetse gave positive TBR-qPCR results. Furthermore, the quantity of T. brucei DNA detected in naïve tsetse was significantly correlated with cage infection ratio. With evidence of cross-contamination, field-caught tsetse from Tanzania were then assessed using the same screening protocol. End-point TBR-PCR predicted a sample population prevalence of 24.8%. Using qPCR and Cq cut-offs optimised on insectary-reared flies, we estimated that prevalence was 0.5% (95% confidence interval [0.36, 0.73]).
CONCLUSIONS/SIGNIFICANCE: Our results show that infected tsetse can contaminate naïve flies with T. brucei DNA when co-caged, and that the level of contamination can be extensive. Whilst simple PCR may overestimate infection prevalence, quantitative PCR offers a means of eliminating false positives.
采采蝇（ Glossina sp. ）是引起人类非洲锥虫病（ HAT ）的布氏锥虫亚种的媒介。捕获和筛选采采蝇对 HAT 监测至关重要。经典上，通过显微镜分析采采蝇来鉴定锥虫，但这正逐渐被分子 xenomonitoring 所取代。尽管如此，诸如 TBR-PCR 等敏感的布鲁斯锥虫检测检测方法容易受到 DNA 交叉污染。这种污染可能发生在捕获时，此时通常会有多个活的采采蝇被暂时保留在陷阱的笼子中。本研究旨在确定当共笼时，感染的采采蝇是否可以通过粪便将布鲁斯锥虫 DNA 污染给未感染的采采蝇。
方法/原理发现：在昆虫饲养室中饲养的幼龄 G. morsitans morsitans 被喂食含有传染性 T. b. brucei spikes 的血餐。在感染后 19 天，感染和未感染的采采蝇以以下比例（ T1 ）9:3、（ T2 ）6:6 （ T3 ）1:11 和对照（ C0 ）0:12 进行共笼，每种比例重复三次。孵育 24 小时后，从每只苍蝇中提取 DNA ，并使用 PCR 和 qPCR 筛查寄生虫 DNA 的存在。所有在昆虫饲养室中饲养的感染苍蝇均使用 TBR-qPCR 对布鲁斯锥虫 DNA 呈阳性。然而，未感染的采采蝇也呈阳性。即使感染与未感染的采采蝇比例为 1:11 ，仍有 91%的未感染采采蝇的 TBR-qPCR 结果呈阳性。此外，未感染采采蝇中检测到的布鲁斯锥虫 DNA 数量与笼内感染比例显著相关。由于存在交叉污染的证据，因此随后使用相同的筛选方案评估了来自坦桑尼亚的野外捕获的采采蝇。终点 TBR-PCR 预测样本人群的患病率为 24.8%。使用 qPCR 和在昆虫饲养室中饲养的苍蝇上优化的 Cq 截止值，我们估计患病率为 0.5%（95%置信区间[0.36,0.73]）。
结论/意义：我们的研究结果表明，当共笼时，感染的采采蝇可以将布鲁斯锥虫 DNA 污染给未感染的采采蝇，并且污染程度可能很严重。虽然简单的 PCR 可能会高估感染的流行率，但定量 PCR 提供了消除假阳性的方法。
