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

尽管出现了多种广泛耐药的菌株，但结核病在很大程度上是可以治愈的 疾病，如果治疗得当的话。然而，在一些结核病流行地区，只有不到40%的结核病病例 由于缺乏准确和易于使用的诊断分析而被诊断，这些继续推动 流行病。目前，诊断依赖于临床上对结核分枝杆菌的证明。 标本经痰涂片镜检和重复培养。文化的局限性往往包括增长缓慢。 (长达8周)，由于所需的净化程序导致灵敏度低，难以获得连续的 儿童和非痰生产商的样本，以及化验的高昂成本。一种无痰、无生长 基于生物标记物的检测可以取代微生物中间终点，可以改变速度和 结核病药物开发和全球结核病控制的范围。它还可以用来达到一个 少菌病是儿童、肺外结核病和艾滋病毒/结核病的常见病。 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来应对 血清和尿液中细胞膜脂多糖阿拉伯甘露糖(LAM)定量检测的临床意义 诊断准确率。本项目的目标是：1)通过试验优化尿液和血清的前处理方案 蛋白水解酶和杂色剂，从而使最大数量的LAM可用于检测；2) 产生针对“体内”形式的LAM的高度特异和独特的单抗；以及3) 通过捕获ELISA推进基于非痰的LAM检测，并在特征良好的 结核病和艾滋病毒高负担国家的疑似结核病成人队列(南非、泰国、 艾滋病毒暴露低的国家(肯尼亚)和(秘鲁)。鉴于文化作为黄金标准的公认缺陷，在 单独的探索性分析我们将使用LAM气体检测来确定ELISA的敏感性和特异性 以气相色谱/质谱仪(GC/MS)为参比标准。我们的捕获酶联免疫吸附试验准确率目标 ≥的敏感性为85%，≥的特异性为95%。此外，我们还将测定尿液和血清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样本，旨在 确定尿液LAM是否可作为治疗反应的生物标志物。我们的最终目标是 开发基于捕获化学和定量工程的准确、简单的关注点测试 非痰标本LAM检测条件的探讨检测应覆盖所有结核病病例，无论他们是谁 艾滋病毒状况。选定的演示不是最终的设备，但将演示原则证明，即 与设计目标保持一致。

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.