Validation of urine/serum LAM in HIV/nonHIV TB suspects and POC Test Development

HIV/非 HIV 结核病疑似者的尿液/血清 LAM 验证和 POC 测试开发

• 批准号: 9925722
• 负责人: DELPHI CHATTERJEE
• 金额: $ 59.14万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-06-08 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9925722
• 关键词: Acid Fast Bacillae Staining Method; Address; Adult; Affinity; Antibodies; Area; Bacillus; Bacteria; Bacterial DNA; Bedside Testings; Binding; Biological Assay; Biological Markers; Blood; Cell Wall; Chemistry; Child; Clinical; Collaborations; Complex; Country; DNA; Decontamination; Detection; Detergents; Development; Devices; Diagnosis; Diagnostic; Diagnostic tests; Disease; Drug resistance in tuberculosis; Engineering; Enzyme-Linked Immunosorbent Assay; Epidemic; Epitopes; Evaluation; Goals; Gold; Growth; HIV; HIV Seronegativity; HIV Seropositivity; HIV/TB; Immune response; Kenya; Knowledge; Lateral; Mass Fragmentography; Memory B-Lymphocyte; Methodology; Methods; Microbiology; Microscopy; Molecular Cloning; Monoclonal Antibodies; Mycobacterium tuberculosis; Nature; Patients; Peptide Hydrolases; Peru; Population; Procedures; Production; Property; Protein Denaturation; Proteins; Protocols documentation; Reagent; Reference Standards; Reporting; Research; Sampling; Sensitivity and Specificity; Serum; South Africa; Specificity; Specimen; Sputum; Testing; Thailand; Tuberculosis; Urea; Urine; Validation; Work; base; cell envelope; clinical Diagnosis; clinical diagnostics; cohort; cost; design; diagnostic accuracy; diagnostic assay; drug development; extensive drug resistance; human subject; immunosuppressed; improved; in vivo; interest; lipoarabinomannan; pandemic disease; point of care; prevent; protein complex; resistant strain; treatment response; tuberculosis diagnostics; tuberculosis drugs; tuberculosis treatment; urinary

Despite the emergence of multiple and extensively drug resistant strains, tuberculosis (TB) is largely a curable disease if treated appropriately. However, in some tuberculosis-endemic areas, fewer than 40% of TB cases are diagnosed due to the lack of accurate and easy-to-use diagnostic assays, and these continue to drive the epidemic. Currently, diagnosis relies on demonstration of the bacteria, Mycobacterium tuberculosis, in clinical specimens by sputum smear microscopy and repeat culture. Limitations of culture often include slow growth (up to 8 weeks), low sensitivity as a result of required decontamination procedures, difficulty in obtaining serial samples in children and non-sputum producers, and the high cost of the assay. A non-sputum, non-growth based biomarker assay could replace microbiologic intermediate endpoints, could transform the pace and scope of TB drug development and of global TB control. It may additionally have utility for reaching a population of paucibacillary disease as is often seen in children, extrapulmonary TB, and HIV/TB. This proposed research will establish our goal towards developing a highly sensitive approach to the quantitative detection of lipoarabinomannan (LAM), a cell envelope lipoglycan in serum and urine with clinical diagnostic accuracy. The project aims are 1) to optimize the urine and serum pretreatment protocol by testing proteases and chaotropic agents, thus making maximal amount of the LAM available for detection; 2) to generate highly specific and unique monoclonal antibodies directed against “in vivo” forms of LAM; and 3) to take forward non-sputum based LAM detection via a capture ELISA with further validation in well-characterized adult cohorts with suspected TB in countries which have high burdens of TB and HIV (South Africa, Thailand, Kenya) and (Peru) with low HIV exposure. Given the recognized imperfection of culture as a gold standard, in a separate exploratory analysis we will determine ELISA sensitivity and specificity using LAM detection by Gas Chromatography/Mass Spectrometry (GC/MS) as the reference standard. Our capture ELISA accuracy targets are ≥85% sensitivity and ≥95% specificity. In addition, we will determine, the changes in urine and serum LAM concentrations during TB treatment; this will use a separate set of existing specimens and is intended to establish whether LAM in urine might be useful as a biomarker of treatment response. Our ultimate goal is to develop an accurate, simple Point-of-Care test based on quantitative engineering of capture chemistry and conditions for LAM detection in non-sputum specimens. The test should reach all TB cases irrespective of their HIV status. The chosen demonstration will not be a final device but will demonstrate a proof-of-principle that is aligned with the design goals.

尽管出现了多种和广泛耐药菌株，但结核病（TB）在很大程度上是可治愈的。 疾病如果治疗得当。然而，在一些结核病流行地区， 由于缺乏准确和易于使用的诊断测定，这些继续推动 疫情目前，诊断依赖于在临床上展示细菌结核分枝杆菌， 痰涂片显微镜检查标本并重复培养。文化的局限性通常包括生长缓慢 (up至8周），由于所需的去污程序而导致的低灵敏度，难以获得连续的 儿童和非痰液生产者的样本，以及高成本的分析。一种不生痰、不生长 基于生物标志物的测定可以取代微生物中间终点，可以改变步伐， 结核病药物开发和全球结核病控制的范围。它还可以具有用于达到 在儿童中常见的少杆菌病人群、肺外结核和艾滋病毒/结核。 这项拟议的研究将确立我们的目标，即开发一种高度敏感的方法， 定量检测血清和尿液中的脂阿拉伯甘露聚糖（LAM），一种细胞包膜脂聚糖， 诊断准确性。本课题的主要目的是：1）通过试验优化尿液和血清的预处理方案 蛋白酶和离液剂，从而使最大量的LAM可用于检测; 2） 产生针对LAM的“体内”形式的高度特异性和独特的单克隆抗体;和3） 通过捕获ELISA进行基于非痰的LAM检测，并在充分表征的 结核病和艾滋病毒负担重的国家（南非、泰国， 肯尼亚）和（秘鲁），艾滋病毒感染率较低。鉴于文化作为金本位的公认缺陷， 在单独的探索性分析中，我们将使用Gas检测LAM来确定ELISA的灵敏度和特异性。 色谱/质谱法（GC/MS）作为参比标准品。我们的捕获ELISA准确度目标 敏感性≥85%，特异性≥95%。此外，我们还将测定尿和血清LAM的变化 结核病治疗期间的浓度;这将使用一组单独的现有标本， 确定尿中LAM是否可用作治疗反应的生物标志物。我们的最终目标是 基于捕获化学的定量工程开发一种准确、简单的床旁检测， 非痰标本中LAM检测的条件。该检测应覆盖所有结核病例，无论其 艾滋病毒感染状况。所选演示将不是最终器械，但将演示原理证明， 符合设计目标。