Primers and probes used in the study.

BackgroundTsetse flies (Glossina sp.) are the primary vectors of trypanosomes causing human African trypanosomiasis (HAT) and animal African trypanosomiasis (AAT). Disease surveillance can be carried out by detecting Trypanosoma DNA in tsetse, also known as molecular xenomonitoring. Whilst molecular methods can increase the efficiency and sensitivity of pathogen detection, trained staff and a well-equipped laboratory are required. In many cases, DNA extraction and screening is outsourced to a central laboratory in a major city either in-country or abroad, far removed from original tsetse collection sites. This increases results turnaround time, incurs transportation costs, and can lead to sample loss or damage.Methodology/Principle FindingsWe set out to develop, optimise and trial methods for tsetse xenomonitoring in a low-resource point-of-need setting. A low-cost protocol was developed consisting of rapid alkali-based DNA extraction and Trypanosoma detection qPCR assays using air-dryable reagent mixes. A minimally-equipped laboratory was established in a field station in Arua, Uganda. Following a training workshop, three entomology technicians carried out screening on 286 tsetse collected over a nine-week study period. The technicians consistently extracted high quality DNA (98% success rate) and were able to successfully detect T. brucei sensu lato in 4.3% (95% confidence interval (CI) [2.23 - 7.37]), T. congolense in 3.6% (95% CI [1.73 – 6.47]) and T. vivax in 3.9% (95% CI [1.98 – 6.92]) of total tsetse, representing a total Trypanosoma sp detection prevalence of 10.7% (95% CI [9.6 – 11.8]).Conclusions/SignificanceThis study demonstrated that sensitive molecular xenomonitoring of HAT and AAT pathogens can be carried out without the need for cold-chain storage or high-powered equipment. Further improvements to the system might be achieved by modifying the DNA extraction protocol to enable high-throughput or pooled samples, increasing the sensitivity of the T. b. gambiense DNA detection assay and exploring more sustainable power sources.背景 舌蝇（Glossina sp.）是引发人类非洲锥虫病（HAT）与动物非洲锥虫病（AAT）的锥虫的主要传播媒介。可通过检测舌蝇体内的锥虫DNA开展疾病监测，该方法亦称分子媒介监测（molecular xenomonitoring）。尽管分子检测方法可提升病原体检测的效率与灵敏度，但该技术依赖经过专业培训的操作人员与配置完备的实验室。在多数场景中，DNA提取与筛选工作会被外包至国内或海外大城市的中心实验室，而这些实验室往往远离舌蝇样本采集点。此举不仅延长了检测结果的周转时长、增加了运输成本，还可能导致样本丢失或损坏。 研究方法与主要发现 本研究旨在开发、优化并测试适用于低资源现场即时检测场景的舌蝇分子媒介监测方法。研究团队开发了一套低成本实验方案，该方案基于快速碱裂解法提取DNA，并使用可风干试剂组合开展锥虫检测的实时荧光定量PCR（qPCR）实验。研究团队在乌干达阿鲁阿的一处野外工作站搭建了一套简易配置实验室。经过专题培训后，三名昆虫学技术人员对为期九周的研究期间采集的286只舌蝇开展了检测工作。该团队技术人员始终能提取到高质量DNA（成功率达98%），并成功在总样本中检测出布氏锥虫复合体（T. brucei sensu lato）阳性率为4.3%（95%置信区间[CI]：2.23~7.37）、刚果锥虫（T. congolense）阳性率为3.6%（95% CI：1.73~6.47）、vivax锥虫（T. vivax）阳性率为3.9%（95% CI：1.98~6.92），整体锥虫属病原体检测阳性率达10.7%（95% CI：9.6~11.8）。 结论与意义 本研究证实，无需冷链储存或高精度设备，即可开展针对HAT与AAT病原体的高灵敏度分子媒介监测。后续可通过以下方式进一步优化该系统：调整DNA提取方案以适配高通量检测或混合样本检测、提升冈比亚锥虫（T. b. gambiense）DNA检测的灵敏度，以及探索更可持续的电力供应方案。