A 100-fold more sensitive TB diagnostic based on magnetic concentration and "coffee ring" formation

全球结核病(TB)的负担每年约为900万例；南非是最严重的 全球结核病负担沉重的国家。结核病也是艾滋病毒感染者最常见的机会性感染 它造成的死亡人数占南非死亡人数的21%。结核病活动性病例发现和药物治疗计划 为了阻断结核病的传播，需要在城市和农村社区进行耐药性分型，但我们目前 缺乏可获得的护理点诊断来实现这一目标。 特别是在南非农村，那里的结核病感染很普遍，对痰进行显微镜检查 涂片是最容易检测到结核病的方法。两大挑战是1)更容易接近 镜检法目前的检测极限是每毫升痰中检测10,000个细菌，这可以防止早期 检测并允许感染的持续传播，以及2)没有使用明场显微镜的方法 用于检测耐药结核分枝杆菌的基因型别。 在这项提案中，范德比尔特大学和开普敦大学建立了合作伙伴关系，以开发 并测试从痰中诊断结核病的明场显微镜方法。我们建议开发简单的方法 为了提高现有的和更容易获得的痰样本明视场显微镜的检测极限， 100杆菌/毫升。我们的方法是基于磁浓缩策略和高密度沉积到 使用咖啡环现象的标准显微镜载玻片(目标1)。第二，我们建议发展一套 树枝状大分子基因分型探针和硅胶缩合反应，使药物的鉴定成为可能 耐药表型(目标2)。这些实验室设计随后被打包成简单的试剂盒格式，用于初始 在实验室和南非农村对回溯性痰样本进行临床测试(目标3)。 完成后，这些对目前广泛使用的显微镜方法的简单修改将 识别痰中结核杆菌数量较少的个体，并更早地启用 治疗。此外，通过明场显微镜鉴定耐药杆菌将提供 更早的手段是开始更积极的治疗，进一步防止耐药结核病的传播。

The global burden of tuberculosis (TB) stands at ~9 million cases per year; and South Africa is the most heavily TB-burdened country globally. TB is also the most common opportunistic infection in HIV-infected persons and it is responsible for 21% of deaths in South Africa. A program of active TB case finding and drug resistance typing in both urban and rural communities is needed to disrupt TB transmission, yet we currently lack accessible point of care diagnostics to achieve this goal. Particularly in rural South Africa, where TB infection is widespread, microscopic examination of a sputum smear is the most accessible method for detection of TB. Two major challenges are 1) the more accessible microscopy methods have a current limit of detection of 10,000 bacilli per ml of sputum which prevents early detection and allows continued spread of infection, and 2) no methods using bright field microscopy are available for detecting drug resistant Mycobacterium tuberculosis genotypes. In this proposal Vanderbilt University and The University of Cape Town form a partnership to develop and test bright field microscopy methods for diagnosing TB from sputum. We propose to develop simple methods to improve the limit of detection of existing and more accessible bright field microscopy of sputum samples to 100 bacilli/ml. Our approach is based on a magnetic concentration strategy and high density deposition onto a standard microscope slide using the coffee ring phenomenon (Aim 1). Secondly, we propose to develop a dendrimer-genotyping probe followed by a silica condensation reaction to enable bright field identification of drug resistant phenotypes (Aim 2). These laboratory designs are then packaged into a simple kit format for initial clinical testing of retrospective sputum samples in a laboratory as well as in rural South Africa (Aim 3). When completed, these simple modifications to the current widely accessible microscopy methods will identify individuals with low numbers of M. tuberculosis bacilli present in their sputum and enable much earlier treatment. In addition, enabling the identification of drug resistant bacilli by bright field microscopy will provide an earlier means to begin more aggressive therapies and further prevent the spread of drug-resistant TB.