Development of a highly-sensitive urine test for tuberculosis (TB) that detects diverse forms of urinary TB lipoarabinomannan (uLAM)

项目摘要 结核病(TB)仍然是一个主要的世界性健康问题， 对活动性结核病感染的敏感护理点(POC)诊断是全球公共卫生的优先事项。 基于分枝杆菌脂阿拉伯甘露聚糖(LAM)检测的侧向流动(LF)分析 患者尿液中的抗原可能是结核病的POC检测方法，但目前的检测方法还不够理想。 敏感度高，并不适合所有人群。这种试验的早期形式是Alere 测定™TB LAM Ag试验，被世界卫生组织强烈推荐用于诊断活动性 HIV阳性患者中的结核病，其CD4细胞计数为200细胞/毫米，并患有晚期艾滋病毒疾病。 一种新的基于新型单抗的检测方法(Fujifilm SILVAMP TB LAM法) 在我的实验室分离或鉴定的(单抗)对这两种情况的敏感性都要高得多 HIV阳性和HIV阴性人群，但仍符合世卫组织的目标产品概况 以获得广泛的实用价值。FujiLAM检测使用一种独特的mAb组合(S4-20)，该组合 对结核分枝杆菌LAM和另一种广泛与LAM反应的单抗(A194-01)具有高度特异性 从许多分枝杆菌物种中分离出来。最近在我的实验室进行的研究，使用了25人的标准小组 从乌干达患者身上收集的大规模尿样，这些患者预先选择了一系列艾滋病毒 临床状态，CD4细胞计数和LAM尿液浓度已确定 ManLAM和ULAM免疫化学性质的差异，并显示出 ULAM在不同患者尿样中的免疫反应性存在显著差异。 这些研究包括一种新的单抗组合(FDX01/A194-01)，它具有 在我们的标准尿液分析仪中提高了ULAM的敏感度和检测广度，但可能有 结核分枝杆菌的特异性较低。上述所有单抗均用ManLAM抗原分离 从培养的结核分枝杆菌中提纯。基于这些结果，我们现在建议将抗原性 ULAM在包括肺结核病和肺外结核病在内的一系列结核病表型上的多样性， HIV+和HIV-TB，并确定这些不同形式的结核病相对于其他形式的特异性 呼吸道疾病、潜伏性结核病和未感染结核病的队列。同时，我们将分离出符合以下条件的单抗 通过B细胞分选和抗体克隆，对不同形式的ULAM具有高度的敏感性和特异性。我们 然后将选择并验证用于结核病检测的最高性能的mAb组合 通过一大组不同的结核病和非结核病临床样本，比较它们的敏感性和 特异性与新的FDX01/A194-01检测方法一致。这些研究将解决这些假设 在患者尿液中检测到的不同LAM免疫类型反映了重要的细菌学和 主机功能是可通用化诊断测试的组成部分，利用这些形式将导致 对ULAM的增强检测将提高尿液检测的敏感性和特异性 用于检测活动性结核病感染。

PROJECT ABSTRACT Tuberculosis (TB) remains a major world-wide health problem and the development of a sensitive point-of-care (POC) diagnostic for active TB infection is a global public health priority. Lateral flow (LF) assays based on the detection of mycobacterial lipoarabinomannan (LAM) antigen in patient urines are potential POC tests for TB, but current assays have suboptimal sensitivity, and are not suitable for all populations. An early form of this assay, the Alere Determine™ TB LAM Ag test, was strongly recommended by the WHO for diagnosis of active TB in HIV-positive patients with a CD4 cell count of <200 cells/mm and advanced HIV disease. A newer assay (the Fujifilm SILVAMP TB LAM assay) based on novel monoclonal antibodies (mAbs) isolated or characterized in my lab was found to be considerably more sensitive for both HIV-positive and HIV-negative populations, but still did meet the WHO’s Target Product Profile for wide-spread utility. The FujiLAM assay utilizes a unique combination of a mAb (S4-20) that is highly specific for M.tb LAM and a second mAb (A194-01) that is widely reactive with LAM from many mycobacterial species. More recent studies in my lab using a standard panel of 25 large-scale urine samples collected from Ugandan patients preselected for a range of HIV clinical status, CD4 cell count and LAM urinary concentrations have identified profound differences in the immunochemical properties between ManLAM and uLAM, and have shown significant diversity in the immunoreactivity of uLAM present in different patient urine samples. These studies have included a novel combination of mAbs (FDX01/A194-01) that possesses increased sensitivity and breadth of detection of uLAM in our standard urine panel, but may have lower specificity for M.tb. All of the mAbs described above were isolated using ManLAM antigen purified from cultured M.tb. Based on these results we now propose to characterize antigenic diversity of uLAM across a range of TB phenotypes, including pulmonary and extrapulmonary TB, HIV+ and HIV- TB, and determine the specificity of these different forms for TB against other respiratory diseases, latent TB, and TB-uninfected cohorts. In parallel we will isolate mAbs that are highly sensitive and specific for different forms of uLAM by B cell sorting and antibody cloning. We will then select and validate the highest performing mAb combinations for TB disease detection across a large panel of diverse TB and non-TB clinical samples, and compare their sensitivity and specificity with that ofthe novel FDX01/A194-01 assay. These studies will address the hypotheses that different LAM immunotypes detected in patient urines reflect important bacteriological and host features integral to a generalizable diagnostic test, and that exploiting these forms will lead to the enhanced detection of uLAM that will improve the sensitivity and specificity of urine assays for detection of active TB infection.