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

使用外泌体从血液中诊断结核病的新型护理点检测

• 批准号: 10596302
• 负责人: Richard S Garfein
• 金额: $ 75.42万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10596302
• 关键词: Address; Affect; Agreement; Antibodies; Antigens; Archives; Bacteriology; Biological; Biological Assay; Biological Markers; Blood; Blood Tests; Blood specimen; California; Cancer Detection; Cellular Phone; Cessation of life; Child; Clinical Sensitivity; Communicable Diseases; Complex; Cross Reactions; Detection; Diagnosis; Diagnostic; Diagnostic tests; Disease; Electrophoresis; Elements; Engineering; Evaluation; Fluorescent Antibody Technique; Fluorescent Probes; Foundations; Goals; HIV Infections; Image; Lipopolysaccharides; Lung; Lung diseases; Measures; Methods; Mexico; Microchip Electrophoresis; Microscopy; Monitor; Mycobacterium Infections; Mycobacterium tuberculosis; Mycobacterium tuberculosis antigens; National Institute of Allergy and Infectious Disease; Organizational Objectives; Outcome; Pakistan; Patient Triage; Patients; Performance; Process; Proteins; Reader; Reproducibility; Research; Research Personnel; Risk; Sampling; Sensitivity and Specificity; Series; Serum; Specificity; Sputum; Technology; Testing; Triage; Tuberculosis; Tuberculosis diagnosis; United States National Institutes of Health; Universities; Whole Blood; Work; World Health Organization; antigen binding; antigen test; base; biobank; cancer diagnosis; cost; detection limit; diagnostic assay; exosome; experimental study; field study; in-vitro diagnostics; innovation; meetings; microchip; non-tuberculosis mycobacteria; novel; novel marker; point of care; point-of-care diagnosis; point-of-care diagnostics; prognostic; prospective; prospective test; rapid diagnosis; rapid test; research clinical testing; screening; success; transmission process; tuberculosis diagnostics; tuberculosis treatment; uptake

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.

项目概要 结核病 (TB) 是全球头号传染病杀手，每年导致 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较低的痰检测，因此结核病 严重未被充分诊断，导致世界范围内的结果不佳。检测分枝杆菌的测定 血液中的结核病 (Mtb) 抗原可能会彻底改变结核病诊断方法，因为血液易于收集和检测 可以检测不限于肺部的结核病；然而，由于抗原超低，基于血液的检测仍然难以实现 血液中存在的浓度和交叉反应元素。该提案的目标是开发和 评估高度创新的基于血液的即时护理 (POC) 检测，以快速诊断活动性结核病 基于检测血液中外泌体结合的 Mtb 抗原来诊断疾病 (ATB)。我们将利用交替 POC 平台上的电流电泳 (ACE) 用于分离和浓缩外泌体结合的 Mtb 抗原 未经处理的患者血液样本可诊断活动性结核病，其准确度达到或超过世界卫生组织的要求 组织的结核病分类分析的目标产品概况 (TPP) 性能标准（灵敏度 >95%， 特异性 >80%）。这种突破性的检测最初是为癌症诊断而开发的，使用 ACE 从血液中捕获外泌体等小生物分子（~0.1 至 1 微米）以浓缩 Mtb 的技术 Ag，同时排除潜在的抑制剂，允许使用荧光抗体探针对它们进行成像。这 研究旨在：1) 评估 ExoVerita Flex 平台上 Mtb Ag 微芯片检测的概念验证 200 份存档的血清样本，来自经培养确认的 ATB、LTBI 和培养阴性对照患者 患者量化检测性能驱动因素并确定优化检测性能的机会， 可能带有额外的 Mtb 抗原； 2) 从 ExoVerita 转移优化的 Mtb Ag 微芯片检测 Flex 平台连接到 OmniVerita-M POC 阅读器，使用来自 UCSD 的相同 200 个存档血清样本 生物库并使用前瞻性收集的全血 (n=100) 表征测定的 POC 性能； 3) 在现场确定 POC 阅读器上优化测定的临床敏感性和特异性 基于前瞻性的研究，从结核病高危人群中采集了结核病风险患者的血液样本 （巴基斯坦）、中度（墨西哥）和低度（美国）结核病负担（n=1,500）。我们将评估和优化 Mtb Ag 使用来自充分表征的生物库的血清样本和疑似患者的全血进行检测 加州大学圣地亚哥分校的 ATB (n=100)，然后与标准相比评估其表现 在高感染率的现场环境中对疑似 ATB 患者进行 Mtb 细菌学确证方法 （巴基斯坦）、中度（墨西哥）和低度（美国）结核病负担（n=1,500）。拟议的工作在以下方面具有高度创新性 在全血中分离外泌体结合的 Mtb 抗原是结核病诊断的首创方法， 它还可以量化血液中的结核分枝杆菌抗原，以用于结核病治疗监测。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，这将有 通过广泛采用早期结核病诊断，对结核病治疗产生持续的积极影响。