Multiplexed point-of-care molecular detection for multiple infections in co-endemic settings

项目摘要：热带贫困地区不成比例的传染病 对人类健康和经济发展造成毁灭性影响，数十亿人面临风险。这些 疾病包括艾滋病毒/艾滋病、疟疾和结核病，但也包括被忽视的热带疾病（例如， 血吸虫病、丝虫病和其它蠕虫、微生物和原生动物感染）。那些导致 这些疾病往往是共同流行的。伴随感染可改变宿主反应，疾病预后， 传播动力学和治疗结果，以及对准确解释 数据集中于单一感染。一个单一的，简单的诊断测试， 以高灵敏度检测存在于每个个体中的那些感染将是成本方面的突破， 时间和准确的诊断。不幸的是，目前对疾病传播的诊断和监测， 治疗效果很大程度上取决于通常不准确、劳动密集或不可靠方法。这些 在大规模药物管理计划中，限制获得了额外的意义，其中有效性的措施 需要准确监测感染（和合并感染）状态、治疗成功率、疾病传播 率和耐药性的出现。宿主体液中病原体核酸的分子检测或 组织提供了可靠性、灵敏度和特异性，但目前的方法需要基础设施的支持，高度- 训练有素的员工和昂贵精密的设备在这个项目中，我们将建立在我们的实质性进展， 开发微流体、护理点/接触（POC）装置，其能够实现现场、廉价的分子生物学， 由受过最低限度培训的人员进行感染诊断和疾病传播监测。我们特别 将：目标1）开发、优化和验证新的2阶段扩增病原体检测方案 目的2）转移和调整开发的台式RAMP测定法 在目标1中，设计了一种新的微流体多路复用芯片，并验证该芯片允许同时进行POC 多种病原体核酸的检测。这些目标的实现将作为概念的证明 并为未来使用感染动物模型进行更广泛的改进和测试奠定了基础， 共感染、临床样本和现场研究。这种方法将很容易转化为突破性的新的 热带疾病和合并感染的现场可用POC分子诊断和监测技术。

Project summary: Infectious diseases that disproportionately impact impoverished regions in the tropics have devastating impacts on human health and economic development, with billions of people at risk. These diseases include HIV/AIDS, malaria, and tuberculosis, but also the neglected tropical diseases (eg, schistosomiasis, filariasis, and other helminth, microbial, and protozoan infections). The agents that cause these diseases are often co-endemic. Concomitant infections can alter host responses, disease prognosis, transmission dynamics, and treatment outcomes, as well as posing difficulties for accurate interpretation of data focused on single infections. A single, simple diagnostic test that could specifically and concurrently detect those infections present in each individual with high sensitivity would be a breakthrough in terms of cost, time, and accurate diagnosis. Unfortunately, current diagnosis and monitoring of disease transmission and efficacy of treatment depend largely on methods that are often inaccurate, labor-intensive, or unreliable. These limitations acquire added significance in mass drug administration programs, where measures of effectiveness require accurate monitoring of infection (and coinfection) status, treatment success, disease transmission rates, and emergence of drug resistance. Molecular detection of pathogen nucleic acids in host fluids or tissues offers reliability, sensitivity, and specificity, but current methods require infrastructural support, highly- trained staff, and expensive, delicate equipment. In this project, we will build upon our substantial advances in development of microfluidic, point-of-care/contact (POC) devices that enable on-site, inexpensive molecular diagnosis of infection and monitoring of disease transmission by minimally-trained personnel. Specifically, we will: Aim 1) develop, optimize, and validate a new 2-stage amplification pathogen detection protocol (RAMP) using benchtop assays; and Aim 2) transfer and adapt the benchtop RAMP assays developed in Aim 1 to a new microfluidic, multiplexing chip, and verify that the chip allows for simultaneous POC detection of multiple pathogen nucleic acids. Accomplishment of these aims will serve as proof of concept and set the stage for more extensive refinement and testing in future work using animal models of infection and coinfection, clinical samples, and field studies. The approach will readily translate into groundbreaking new technology for field-ready, POC molecular diagnosis and monitoring of tropical diseases and coinfections.