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

开发一种高灵敏度的结核病尿液检测方法，可检测多种形式的尿液结核菌脂阿拉伯甘露聚糖 (uLAM)

• 批准号: 10667871
• 负责人: ABRAHAM PINTER
• 金额: $ 78.32万
• 依托单位: RUTGERS BIOMEDICAL AND HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-23 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10667871
• 关键词: Address; Affinity; Antibodies; Antigenic Diversity; Antigens; B-Lymphocytes; Bacteriology; Biological; Biological Assay; Biological Markers; CD4 Lymphocyte Count; Cell Separation; Cell Wall; Cells; Characteristics; Clinical; Cloning; Data; Detection; Development; Diagnosis; Diagnostic; Diagnostic tests; Disease; Early Diagnosis; Enrollment; Epitopes; Genes; Glycolipids; Grant; HIV; HIV Seronegativity; HIV Seropositivity; HIV-1; HIV/TB; Health; Immunoassay; Immunoglobulin G; Immunologics; Individual; Label; Lung; Microbiology; Monoclonal Antibodies; Mycobacterium tuberculosis; Nature; New Jersey; Patients; Pattern; Performance; Phase; Phenotype; Polysaccharides; Population; Property; Reagent; Respiratory Disease; Sampling; Sensitivity and Specificity; Serum; Specificity; Sputum; Testing; Triage; Tuberculosis; Tuberculosis diagnosis; Uganda; Urine; Validation; Work; antibody detection; base; co-infection; cohort; comorbidity; detection assay; immunoreactivity; improved; insight; lateral flow assay; lipoarabinomannan; mortality; mycobacterial; non-tuberculosis mycobacteria; novel; patient screening; point of care; point of care testing; point-of-care diagnostics; prospective; public health priorities; rapid testing; reagent standard; tuberculosis diagnostics; urinary; vector

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.

项目摘要 结核病（TB）仍然是一个主要的世界性健康问题， 对活动性结核感染进行敏感的床旁（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（LF）测定 患者尿液中的抗原是潜在的结核病POC检测，但目前的检测方法并不理想。 敏感性，并不适合所有人群。该检测试剂盒的早期形式是Alere Determine™ TB LAM Ag检测是WHO强烈推荐用于诊断活动性 CD 4细胞计数<200个细胞/mm 2的HIV阳性患者和晚期HIV疾病中的结核病。 一种基于新型单克隆抗体的新检测方法（Fujifilm SILVAMP TB LAM检测方法） 在我的实验室中分离或鉴定的单克隆抗体被发现对这两种抗体都更敏感。 艾滋病毒阳性和阴性人群，但仍符合世卫组织的目标产品简介 用于广泛的用途。FujiLAM检测试剂盒采用了mAb（S4-20）的独特组合， 对结核分枝杆菌LAM具有高度特异性，第二种mAb（A194-01）与LAM具有广泛反应性 来自许多分枝杆菌物种。我实验室最近的研究使用了25个标准样本 从乌干达预选的一系列艾滋病毒患者中收集的大规模尿液样本 临床状态，CD 4细胞计数和LAM尿浓度已确定深刻的 ManLAM和uLAM之间的免疫化学性质的差异，并已显示 不同患者尿样中存在uLAM免疫反应性的显著差异。 这些研究包括一种新的mAb组合（FDX 01/A194-01）， 在我们的标准尿液面板中，uLAM的检测灵敏度和广度增加，但可能具有 结核分枝杆菌特异性较低。使用ManLAM抗原分离上述所有mAb 从培养的结核分枝杆菌中纯化。基于这些结果，我们现在提出表征抗原 uLAM在一系列TB表型中的多样性，包括肺和肺外TB， HIV+和HIV-结核病，并确定这些不同形式的结核病对其他结核病的特异性。 呼吸道疾病、潜伏性结核病和未感染结核病的人群。同时，我们将分离出 通过B细胞分选和抗体克隆对不同形式的uLAM高度敏感和特异。我们 然后将选择和验证用于结核病检测的最高性能mAb组合 在大量不同的结核病和非结核病临床样本中， 特异性与新FDX 01/A194-01测定法一致。这些研究将解决的假设 在患者尿液中检测到的不同LAM免疫型反映了重要的细菌学和 主机功能集成到一个通用的诊断测试，并利用这些形式将导致 uLAM的增强检测将提高尿液测定的灵敏度和特异性 用于检测活动性结核病感染。