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

基于磁集中和“咖啡环”形成的结核病诊断灵敏度提高 100 倍

• 批准号: 9754769
• 负责人: Jonathan Michael Blackburn
• 金额: $ 32.67万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-25 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9754769
• 关键词: Area; Bacillus (bacterium); Binding; Cessation of life; Coffee; Collaborations; Country; DNA Binding; Dendrimers; Deposition; Detection; Diagnosis; Diagnostic; Drops; Drug resistance; Drug resistance in tuberculosis; Early Diagnosis; Early treatment; Evaluation; Genotype; Goals; HIV; Individual; Infection; Laboratories; Liquid substance; Magnetism; Malaria; Mannose; Methodology; Methods; Microfluidics; Microscope; Microscopic; Microscopy; Modification; Mutation; Mycobacterium tuberculosis; Opportunistic Infections; Persons; Pharmaceutical Preparations; Pharmacotherapy; Phase; Phenotype; Physical condensation; Procedures; Reaction; Reagent; Resistance; Resources; Rural Community; Sampling; Sensitivity and Specificity; Silicon Dioxide; Site; Slide; South Africa; Sputum; Stains; Steel; Structure; Surface; Testing; Tuberculosis; Universities; Variant; Wool; aggressive therapy; base; case finding; density; design; global health; improved; magnetic beads; nanoparticle; nanoscale; point-of-care diagnostics; prevent; programs; research clinical testing; resistant strain; rural South Africa; tool; transmission process; tuberculosis diagnostics

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.

全球结核病负担每年约为900万例;南非是最多的国家。 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。 在南非，它造成了21%的死亡。活动性肺结核病人发现和药物治疗方案 需要在城市和农村社区进行耐药分型，以阻断结核病传播，但我们目前 缺乏实现这一目标所需的护理点诊断。 特别是在南非农村，结核病感染很普遍， 涂片检查是检测结核病最方便的方法。两个主要挑战是1）更容易获得 显微镜方法目前的检测极限为每毫升痰液中10，000个杆菌， 检测并允许感染继续传播，以及2）没有使用亮视野显微镜的方法可用 用于检测耐药结核分枝杆菌基因型。 在这项提案中，范德比尔特大学和开普敦大学结成伙伴关系， 并测试从痰液中诊断结核病的明视野显微镜方法。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品的明视野显微镜的检测极限， 100个杆菌/ml。我们的方法是基于磁浓度的战略和高密度沉积到一个 使用咖啡环现象的标准显微镜载玻片（目标1）。第二，我们建议发展一个 树枝状聚合物基因分型探针，然后进行二氧化硅缩合反应，以实现药物的明场鉴定 耐药表型（Aim 2）。这些实验室设计然后被包装成一个简单的套件格式， 在实验室和南非农村对回顾性痰液样本进行临床检测（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 识别具有低数量M的个体。结核杆菌存在于他们的痰中， 治疗此外，通过明场显微镜鉴定耐药杆菌将提供 尽早开始更积极的治疗并进一步预防耐药结核病的传播。