A Novel Point-of-Care Assay for Diagnosing TB from Blood using Exosomes

项目摘要 结核病（TB）是全球主要的传染病杀手，每年造成150万人死亡， 由于目前大多数结核病诊断依赖于复杂或敏感性低的结核病检测， 在世界范围内导致了很差的结果。检测分枝杆菌的试验 血液中的结核病（Mtb）抗原可能会彻底改变结核病诊断，因为血液很容易收集， 可以检测出不仅限于肺部的结核病;但由于抗原超低，血液检测仍然难以捉摸 血液中存在的浓度和交叉反应元素。本提案的目的是发展和 评估用于快速诊断活动性结核病的高度创新的基于血液的床旁（POC）检测方法 基于检测血液中的外泌体结合的Mtb抗原来检测结核病（ATB）。我们将利用交替 在POC平台上进行电流电泳（ACE）以分离和浓缩Mtb中的外泌体结合的Mtb抗原。 未经处理的患者血液样本诊断活动性结核病的准确性达到或超过世界卫生组织 结核病分类检测的组织目标产品概况（TPP）性能标准（灵敏度> 95%， 特异性>80%）。这种开创性的检测方法最初是为癌症诊断而开发的， 从血液中捕获小（~0.1至1微米）生物分子（如外来体）以浓缩结核分枝杆菌的技术 Ag，同时排除潜在的抑制剂，使他们能够使用荧光抗体探针成像。这 研究旨在：1）评价ExoVerita Flex平台上的Mtb Ag微芯片检测的概念验证， 200份存档血清样本，来自经培养确认的ATB、LTBI患者和培养阴性对照 患者量化分析性能驱动因素并确定优化分析性能的机会， 可能与其他Mtb抗原一起使用; 2）从ExoVerita转移优化的Mtb Ag微芯片检测试剂盒 使用来自UCSD的相同200份存档血清样本，将Flex平台连接到OmniVerita-M POC读取器 生物样本库，并使用前瞻性采集的全血（n=100）表征检测试剂盒的POC性能; 和3）在现场确定POC读取器上优化的测定的临床灵敏度和特异性， 基于前瞻性收集的高风险结核病患者血液样本的研究 （巴基斯坦）、中等（墨西哥）和低（美国）结核病负担（n= 1，500）。我们将评估和优化结核分枝杆菌抗原 使用来自良好表征的生物样本库的血清样品和来自疑似 ATB（n=100）在加州大学圣地亚哥分校，然后评估其性能相比，标准 在现场环境中疑似ATB患者中的Mtb细菌学确证方法， （巴基斯坦）、中等（墨西哥）和低（美国）结核病负担（n= 1，500）。这项工作具有很强的创新性， 在全血中分离外泌体结合的Mtb抗原是结核病诊断的第一种方法， 其还能够定量血液中的Mtb抗原以用于TB治疗监测。这个项目意义重大 因为它将为快速、低成本的血液结核病POC诊断奠定基础， 通过广泛采用结核病早期诊断，对结核病治疗产生持续的积极影响。

Project Summary Tuberculosis (TB) is the leading infectious disease killer globally, causing 1.5 million deaths annually, and because most TB diagnoses currently rely on sputum-based tests that are complex or have low sensitivity, TB is massively underdiagnosed leading to poor outcomes worldwide. Assays that detect Mycobacterium tuberculosis (Mtb) antigens in blood could revolutionize TB diagnostics, because blood is easy to collect and can detect TB not limited to the lungs; yet blood-base assays remain elusive due to ultra-low antigen concentrations and cross-reacting elements present in blood. The objective of this proposal is to develop and evaluate a highly innovative, blood-based point-of-care (POC) assay for rapid diagnosis of active TB disease (ATB) based on detection of exosome-bound Mtb antigens in blood. We will exploit Alternating Current Electrophoresis (ACE) on a POC platform to isolate and concentrate exosome-bound Mtb antigens in unprocessed patient blood samples to diagnose active TB with accuracy that meets or exceeds World Health Organization’s Target Product Profile (TPP) performance criteria for a TB triage assay (sensitivity >95%, specificity >80%). This groundbreaking assay, originally developed for cancer diagnoses, uses ACE technology to capture small (~0.1 to 1 micron) biomolecules such as exosomes from blood to concentrate Mtb Ag while excluding potential inhibitors allowing them to be imaged using fluorescent antibody probes. This study aims to: 1) evaluate the proof-of-concept Mtb Ag microchip assay on the ExoVerita Flex platform against 200 archived serum samples from patients with culture-confirmed ATB, LTBI, and culture-negative control patients to quantify assay performance drivers and identify opportunities for optimizing assay performance, potentially with additional Mtb antigens; 2) transfer the optimized Mtb Ag microchip assay from the ExoVerita Flex platform to the OmniVerita-M POC reader using the same 200 archived serum samples from UCSD’s biobank and characterizing the assay’s POC performance using prospectively collected whole blood (n=100); and 3) determine the clinical sensitivity and specificity of the optimized assay on the POC reader in a field- based study with prospectively collected blood samples from patients at risk for TB in settings with high (Pakistan), medium (Mexico) and low (U.S.) TB burden (n=1,500). We will evaluate and optimize the Mtb Ag assay using serum samples from a well-characterized Biobank and whole blood from patients with suspected ATB (n=100) at the University of California, San Diego, and then assess its performance compared to standard Mtb bacteriological confirmatory methods among patients with suspected ATB in field settings with a high (Pakistan), medium (Mexico) and low (U.S.) TB burden (n=1,500). The proposed work is highly innovative in that isolation of exosome-bound Mtb antigens in whole blood is a first-of-its-kind approach for TB diagnosis, which may also be able to quantify Mtb antigens in blood for TB treatment monitoring. This project is significant because it will lay the foundation for rapid, low-cost POC diagnosis of TB from blood, which will have a sustained positive impact on TB treatment by enabling broad uptake of early TB diagnosis.