<p>Primers and probes used in the study.</p>

Background
Tsetse 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 Findings
We 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/Significance
This 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.，Tsetse flies）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，该方法亦称分子宿主监测（molecular xenomonitoring）。尽管分子检测技术可提升病原体检出的效率与灵敏度，但该方法依赖受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提取与样本筛查工作会被外包至国内或海外大城市的中央实验室，而这些实验室往往远离舌蝇样本的原始采集点。此举会延长检测结果的周转时长，增加运输成本，还可能导致样本丢失或损坏。

方法/主要发现
本研究旨在开发、优化并验证适用于低资源现场需求场景的舌蝇分子宿主监测方法。研究团队构建了一套低成本实验方案，涵盖基于碱法的快速DNA提取技术，以及采用风干试剂混合物的锥虫qPCR检测体系。我们在乌干达阿鲁阿的一处野外工作站搭建了配置极简的实验室。完成一次培训工作坊后，3名昆虫学技术员对为期9周研究期间采集的286只舌蝇开展了样本筛查。技术员们稳定获得高质量DNA，提取成功率达98%，并成功在总样本中检出：布氏锥虫广义群（T. brucei sensu lato）占比4.3%（95%置信区间[CI]：2.23~7.37），刚果锥虫（T. congolense）占比3.6%（95%CI：1.73~6.47），vivax锥虫（T. vivax）占比3.9%（95%CI：1.98~6.92）；整体锥虫属检出率达10.7%（95%CI：9.6~11.8）。

结论/意义
本研究证实，无需冷链储存或高性能设备，即可开展针对HAT与AAT病原体的高灵敏度分子宿主监测。后续可通过以下方向进一步优化该系统：调整DNA提取方案以适配高通量检测或混合样本分析，提升冈比亚锥虫（T. b. gambiense）DNA检测的灵敏度，并探索更可持续的电力供应来源。