Feasibility of feeding Aedes aegypti mosquitoes on dengue virus-infected human volunteers for vector competence studies in Iquitos, Peru

BackgroundTransmission of dengue virus (DENV) from humans to mosquitoes represents a critical component of dengue epidemiology. Examinations of this process have generally been hampered by a lack of methods that adequately represent natural acquisition of DENV by mosquitoes from humans. In this study, we assessed artificial and natural blood feeding methods based on rates of DENV infection and dissemination within mosquitoes for use in a field-based epidemiological cohort study in Iquitos, Peru.Methodology/Principal findingsOur study was implemented, stepwise, between 2011 and 2015. Participants who were 5 years and older with 5 or fewer days of fever were enrolled from ongoing clinic- and neighborhood-based studies on dengue in Iquitos. Wild type, laboratory-reared Aedes aegypti were fed directly on febrile individuals or on blood collected from participants that was either untreated or treated with EDTA. Mosquitoes were tested after approximately 14 days of extrinsic incubation for DENV infection and dissemination. A total of 58 participants, with viremias ranging from 1.3 × 102 to 2.9 × 106 focus-forming units per mL of serum, participated in one or more feeding methods. DENV infection and dissemination rates were not significantly different following direct and indirect-EDTA feeding; however, they were significantly lower for mosquitoes that fed indirectly on blood with no additive. Relative to direct feeding, infection rates showed greater variation following indirect-EDTA than indirect-no additive feeding. Dissemination rates were similar across all feeding methods. No differences were detected in DENV infection or dissemination rates in mosquitoes fed directly on participants with different dengue illness severity.Conclusions/SignificanceOur study demonstrates the feasibility of using direct and indirect feeding methods for field-based studies on vector competence. Direct mosquito feeding is preferable in terms of logistical ease, biosecurity, and reliability.背景 登革病毒（DENV）由人向蚊的传播，是登革热流行病学研究的核心环节。长期以来，由于缺乏能够精准模拟蚊虫从人体自然获取登革病毒的实验方法，该过程的相关研究始终难以深入推进。本研究以秘鲁伊基托斯地区的现场流行病学队列研究为应用场景，基于蚊虫体内登革病毒感染率与播散率，评估了人工吸血与自然吸血两种实验方法的适用性。 研究方法与主要结果 本研究于2011年至2015年间分阶段实施。研究对象招募自伊基托斯地区正在进行的登革热临床与社区监测队列，纳入标准为年龄≥5岁、发热时长≤5天的参与者。实验所用的野生型实验室饲养埃及伊蚊（Aedes aegypti），分别通过两种方式吸血：直接叮咬发热参与者，或叮咬采集自参与者的未处理血液或经乙二胺四乙酸（EDTA）处理的血液。在体外潜伏期（extrinsic incubation）孵育约14天后，检测蚊虫体内的登革病毒感染与播散情况。 本研究共纳入58名参与者，其血清病毒血症水平介于1.3×10²至2.9×10⁶焦点形成单位（focus-forming units）/毫升之间，所有参与者均接受了一种或多种吸血方式处理。结果显示，直接吸血与经EDTA处理的间接吸血方式下，蚊虫的登革病毒感染率与播散率无显著差异；而经未添加任何试剂的间接吸血的蚊虫，其感染率与播散率均显著更低。与直接吸血相比，经EDTA处理的间接吸血后的感染率变异程度，高于未添加试剂的间接吸血组。不同吸血方式间的病毒播散率无显著差异。直接叮咬不同病情严重程度的登革热患者后，蚊虫的感染率与播散率均未出现显著差异。 研究结论与意义 本研究证实，在登革热媒介传播能力（vector competence）的现场研究中，直接与间接吸血实验方法均具备可行性。从操作便利性、生物安全性与结果可靠性角度而言，直接蚊虫叮咬法更为优选。