Ahmed Madeeha, Pollak Nina M, Hugo Leon E, van den Hurk Andrew F, Hobson-Peters Jody, Macdonald Joanne
Centre for Bioinnovation, University of the Sunshine Coast, Sippy Downs, QLD, 4556, Australia.
School of Science, Technology and Engineering, University of the Sunshine Coast, Sippy Downs, QLD, 4556, Australia.
Gates Open Res. 2022 Dec 22;6:81. doi: 10.12688/gatesopenres.13534.2. eCollection 2022.
The pantropic emergence of severe dengue disease can partly be attributed to the co-circulation of different dengue viruses (DENVs) in the same geographical location. Effective monitoring for circulation of each of the four DENVs is critical to inform disease mitigation strategies. In low resource settings, this can be effectively achieved by utilizing inexpensive, rapid, sensitive and specific assays to detect viruses in mosquito populations. In this study, we developed four rapid DENV tests with direct applicability for low-resource virus surveillance in mosquitoes. The test protocols utilize a novel sample preparation step, a single-temperature isothermal amplification, and a simple lateral flow detection. Analytical sensitivity testing demonstrated tests could detect down to 1,000 copies/µL of virus-specific DENV RNA, and analytical specificity testing indicated tests were highly specific for their respective virus, and did not detect closely related flaviviruses. All four DENV tests showed excellent diagnostic specificity and sensitivity when used for detection of both individually infected mosquitoes and infected mosquitoes in pools of uninfected mosquitoes. With individually infected mosquitoes, the rapid DENV-1, -2 and -3 tests showed 100% diagnostic sensitivity (95% CI = 69% to 100%, n=8 for DENV-1; n=10 for DENV 2,3) and the DENV-4 test showed 92% diagnostic sensitivity (CI: 62% to 100%, n=12) along with 100% diagnostic specificity (CI: 48-100%) for all four tests. Testing infected mosquito pools, the rapid DENV-2, -3 and -4 tests showed 100% diagnostic sensitivity (95% CI = 69% to 100%, n=10) and the DENV-1 test showed 90% diagnostic sensitivity (55.50% to 99.75%, n=10) together with 100% diagnostic specificity (CI: 48-100%). Our tests reduce the operational time required to perform mosquito infection status surveillance testing from > two hours to only 35 minutes, and have potential to improve accessibility of mosquito screening, improving monitoring and control strategies in low-income countries most affected by dengue outbreaks.
严重登革热疾病在全球范围的出现，部分原因可归结于不同登革病毒（DENV）在同一地理位置的共同传播。对四种DENV各自的传播情况进行有效监测，对于制定疾病缓解策略至关重要。在资源匮乏地区，通过使用廉价、快速、灵敏且特异的检测方法来检测蚊虫群体中的病毒，可有效实现这一目标。在本研究中，我们开发了四种快速DENV检测方法，可直接应用于资源匮乏地区的蚊虫病毒监测。检测方案采用了新颖的样本制备步骤、单温度等温扩增以及简单的侧向流动检测。分析灵敏度测试表明，这些检测方法能够检测低至1000拷贝/微升的病毒特异性DENV RNA，分析特异性测试表明，这些检测方法对各自的病毒具有高度特异性，且未检测到密切相关的黄病毒。当用于检测单独感染的蚊虫以及未感染蚊虫群体中的感染蚊虫时，所有四种DENV检测方法均显示出出色的诊断特异性和灵敏度。对于单独感染的蚊虫，快速DENV - 1、- 2和- 3检测方法显示出100%的诊断灵敏度（95%置信区间 = 69%至100%，DENV - 1为n = 8；DENV 2、3为n = 10），DENV - 4检测方法显示出92%的诊断灵敏度（置信区间：62%至100%，n = 12），并且所有四种检测方法的诊断特异性均为100%（置信区间：48 - 100%）。在检测感染蚊虫群体时，快速DENV - 2、- 3和- 4检测方法显示出100%的诊断灵敏度（95%置信区间 = 69%至100%，n = 10），DENV - 1检测方法显示出90%的诊断灵敏度（55.50%至99.75%，n = 10），同时诊断特异性为100%（置信区间：48 - 100%）。我们的检测方法将进行蚊虫感染状态监测检测所需的操作时间从超过两小时缩短至仅35分钟，并且有潜力提高蚊虫筛查的可及性，改善在受登革热疫情影响最严重的低收入国家的监测和控制策略。
