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的抗药性菌株，但结核病（TB）在很大程度上是可以治愈的 疾病如果适当治疗。但是，在某些结核病 - 末端区域，不到40％的结核病病例 由于缺乏准确且易于使用的诊断测定法而被诊断出来，这些测定法继续推动 流行性。目前，诊断依赖于临床中细菌，结核分枝杆菌的证明 痰涂片显微镜和重复培养的标本。文化的局限性通常包括缓慢的增长 （长达8周），由于所需的净化程序而导致低灵敏度，难以获得串行 儿童和非投放生产者的样品以及测定的高成本。非增长，非增长 基于的生物标志物测定可以取代微生物中间端点，可以改变空间和 结核病药物开发和全球结核病控制的范围。它可能还具有实用性 儿童，肺外结核病和艾滋病毒/结核病经常看到的paucibaCillarry疾病的种群。 这项拟议的研究将确定我们的目标，以开发高度敏感的方法 脂肪牵联的定量检测（LAM），一种细胞包膜，血清中的细胞包膜和尿液中 诊断准确性。该项目的目的是1）通过测试优化尿液和血清预处理方案 蛋白酶和混合物剂，因此可用于检测的最​​大含量； 2）到 产生针对LAM“体内”形式的高度特异性和独特的单克隆抗体；和3）到 通过捕获ELISA进行非供应的LAM检测，并在良好的特征中进行进一步验证 成人队列在TB和HIV高燃烧的国家（南非，泰国，泰国， 肯尼亚）和（秘鲁）艾滋病毒暴露率低。鉴于将文化视为黄金标准的公认的不完美 单独的探索性分析我们将使用气体检测LAM检测来确定ELISA敏感性和特异性 色谱/质谱法（GC/MS）作为参考标准。我们捕获ELISA的精度目标 ≥85％的灵敏度和≥95％的特异性。此外，我们将确定尿液和血清的变化 结核病治疗期间的浓度；这将使用一组单独的现有标本，旨在 确定尿液中的lam是否可以作为治疗反应的生物标志物有用。我们的最终目标是 基于捕获化学的定量工程和 在非估计标本中检测的LAM检测条件。该测试应达到所有结核病案例 艾滋病毒状况。所选的演示将不是最终设备，但会证明原理证明是 与设计目标保持一致。