利用低资源快速检测受感染按蚊体内的恶性疟原虫

Leon E. Hugo, Karla van Huyssteen, Olamide K. Oloniniyi, Laura Donnelly, Anna Conn, Katharine A. Collins, H. Mitchell, James S. McCarthy, Joanne Macdonald
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引用次数: 0

摘要

恶性疟原虫病媒监测对于监测和减少最严重的疟疾形式之一至关重要,这种疟疾会导致五岁以下儿童和孕妇的高发病率和高死亡率。在此,我们开发了一种用于检测恶性疟原虫(Pf)感染蚊子的快速、高灵敏度检测方法(快速 Pf 检测方法),非常适合在低资源条件下实施病媒监测。快速 Pf 检测法的分析灵敏度与实验室检测法相似,可检测到低至 4 个拷贝/μL 的 18S rRNA DNA 标准。此外,快速 Pf 检测可在 30 分钟内完成,操作时只需要液体样本制备试剂、研杵、试管和 39°C 的加热块,这表明该检测方法适合在低资源条件下使用。诊断检测使用未感染或喂过恶性疟原虫配子细胞培养物的按蚊进行。根据对 19 只蚊子子集进行的平行显微镜检查和 qPCR 检测,确定这些喂养过恶性疟原虫的蚊子的感染率为 79%。然而,我们的快速 Pf 试验在检测单个感染蚊子(n=30)时确定了 90% 的阳性检测率,并且未检测出 40 只未感染蚊子(n=40),无论其是否吸血,这表明蚊子的真实感染率可能高于 qPCR 和显微镜的计算值。实验证明,快速 Pf 试验可检测单只蚊子(新鲜蚊子和冷冻/解冻蚊子)、1 只感染蚊子与 19 只已知未感染蚊子混合的蚊子池以及在诱捕器中放置长达 8 天的单只蚊子的感染情况。在对感染和未感染蚊子(n=148)进行检测后,与显微镜和 qPCR 的综合结果估计流行率相比,保守估计快速 Pf 检测的诊断灵敏度为 100%(置信区间为 95%,CI:91%-100%),诊断特异性为 97%(CI:92%-99%)。这些结果表明,恶性疟原虫快速检测可为每周监测受感染蚊子提供一种高效工具,以协助恶性疟原虫监测和干预研究。
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Rapid low-resource detection of Plasmodium falciparum in infected Anopheles mosquitoes
Vector surveillance of Plasmodium falciparum is critical for monitoring and reducing one of the most severe forms of malaria, which causes high morbidity and mortality in children under five and pregnant women. Here we developed a rapid and highly sensitive test for the detection of P. falciparum (Pf)-infected mosquitoes (Rapid Pf test), with high suitability for low-resource vector surveillance implementation. The Rapid Pf test had similar analytical sensitivity to laboratory-based tests, detecting down to 4 copies/μL of a 18S rRNA DNA standard. In addition, the Rapid Pf test could be completed in less than 30 minutes, and only required a liquid sample preparation reagent, pestle, tube, and 39°C heating block for operation, indicating amenability for low-resource implementation. Diagnostic testing was performed using Anopheles stephensi mosquitoes, either uninfected, or fed with P. falciparum gametocyte cultures. These P. falciparum fed mosquitoes were determined to have 79% infection prevalence based on parallel microscopy and qPCR testing on a subset of 19 mosquitoes. However, our Rapid Pf test determined a 90% positive test rate when testing individual infected mosquitoes (n=30), and did not detect 40 uninfected mosquitoes regardless of blood-fed status (n=40), suggesting the true prevalence of infection in the mosquitoes may have been higher than calculated by qPCR and microscopy. The Rapid Pf test was demonstrated to detect infection in individual mosquitoes (both fresh and frozen/thawed), as well as pools of 1 infected mosquito mixed with 19 known uninfected mosquitoes, and individual mosquitoes left in traps for up to 8 days. After testing on infected and uninfected mosquitoes (n=148) the Rapid Pf test was conservatively estimated to achieve 100% diagnostic sensitivity (95% confidence interval, CI: 91%-100%) and 97% diagnostic specificity (CI: 92%-99%) compared to the estimated prevalence from combined microscopy and qPCR results. These results indicate the Rapid Pf test could provide a highly effective tool for weekly surveillance of infected mosquitoes, to assist with P. falciparum monitoring and intervention studies.
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