Lipoarabinomannan in sputum to detect bacterial load and treatment response in patients with pulmonary tuberculosis: Analytic validation and evaluation in two cohorts

BackgroundLipoarabinomannan (LAM) is a major antigen of Mycobacterium tuberculosis (MTB). In this report, we evaluated the ability of a novel immunoassay to measure concentrations of LAM in sputum as a biomarker of bacterial load prior to and during treatment in pulmonary tuberculosis (TB) patients.Methods and findingsPhage display technology was used to isolate monoclonal antibodies binding to epitopes unique in LAM from MTB and slow-growing nontuberculous mycobacteria (NTM). Using these antibodies, a sandwich enzyme-linked immunosorbent assay (LAM-ELISA) was developed to quantitate LAM concentration. The LAM-ELISA had a lower limit of quantification of 15 pg/mL LAM, corresponding to 121 colony-forming units (CFUs)/mL of MTB strain H37Rv. It detected slow-growing NTMs but without cross-reacting to common oral bacteria. Two clinical studies were performed between the years 2013 and 2016 in Manila, Philippines, in patients without known human immunodeficiency virus (HIV) coinfection. In a case-control cohort diagnostic study, sputum specimens were collected from 308 patients (aged 17-69 years; 62% male) diagnosed as having pulmonary TB diseases or non-TB diseases, but who could expectorate sputum, and were then evaluated by smear microscopy, BACTEC MGIT 960 Mycobacterial Detection System (MGIT) and Lowenstein-Jensen (LJ) culture, and LAM-ELISA. Some sputum specimens were also examined by Xpert MTB/RIF. The LAM-ELISA detected all smear- and MTB-culture–positive samples (n = 70) and 50% (n = 29) of smear-negative but culture-positive samples (n = 58) (versus 79.3%; 46 positive cases by the Xpert MTB/RIF), but none from non-TB patients (n = 56). Among both LAM and MGIT MTB-culture-positive samples, log10-transformed LAM concentration and MGIT time to detection (TTD) showed a good inverse relationship (r = −0.803, p 10 decrease of sputum LAM concentration, corresponding to an increase of 221 hours for MGIT TTD during the first 14 days of treatment, a treatment duration often used in early bactericidal activity (EBA) trials. Major limitations of this study include a relatively small number of patients, treatment duration up to only 56 days, lack of quantitative sputum culture CFU count data, and no examination of the correlation of sputum LAM to clinical cure.ConclusionsThese results indicate that the LAM-ELISA can determine LAM concentration in sputum, and sputum LAM measured by the assay may be used as a biomarker of bacterial load prior to and during TB treatment. Additional studies are needed to examine the predictive value of this novel biomarker on treatment outcomes.背景：脂阿拉伯甘露聚糖（Lipoarabinomannan, LAM）是结核分枝杆菌（Mycobacterium tuberculosis, MTB）的主要抗原。本研究评估了一种新型免疫检测方法，用于测定痰标本中LAM的浓度，以此作为肺结核（TB）患者治疗前及治疗期间的细菌负荷生物标志物。 方法与研究结果：本研究采用噬菌体展示技术，从MTB及缓慢生长型非结核分枝杆菌（nontuberculous mycobacteria, NTM）中分离出针对LAM独特表位的单克隆抗体。基于上述抗体，我们开发了夹心酶联免疫吸附试验（sandwich enzyme-linked immunosorbent assay, 简称LAM-ELISA）以定量检测LAM浓度。该LAM-ELISA的定量下限为15 pg/mL LAM，对应结核分枝杆菌标准株H37Rv的121个菌落形成单位（colony-forming units, CFU）/mL。该检测方法可识别缓慢生长型NTM，且不会与常见口腔菌群发生交叉反应。 2013年至2016年间，研究团队在菲律宾马尼拉开展了两项临床研究，纳入对象为未合并人类免疫缺陷病毒（human immunodeficiency virus, HIV）感染的患者。在一项病例对照队列诊断研究中，我们从308例患者（年龄17~69岁；男性占比62%）中收集痰标本，这些患者被确诊为肺结核或非肺结核疾病，且可自行咳出痰液；随后采用涂片显微镜检查、BACTEC MGIT 960分枝杆菌检测系统（MGIT）、洛温斯坦-詹森（Lowenstein-Jensen, LJ）培养法及LAM-ELISA对标本进行检测。部分痰标本还通过Xpert MTB/RIF进行了检测。 LAM-ELISA可检出所有涂片及MTB培养阳性的样本（n=70），以及50%（n=29）的涂片阴性但培养阳性的样本（n=58）；而Xpert MTB/RIF的检出率为79.3%，共检出46例阳性病例。此外，该检测未在非肺结核患者的标本中检出阳性结果（n=56）。 在LAM及MGIT MTB培养均为阳性的样本中，经log₁₀转换的LAM浓度与MGIT检测时间（time to detection, TTD）呈显著负相关（r=-0.803，P<0.001）。在治疗的前14天内，痰LAM浓度每降低1个对数级（对应浓度下降10倍），MGIT TTD则延长221小时——这一治疗周期常用于早期杀菌活性（early bactericidal activity, EBA）试验。 本研究的主要局限性包括：患者队列规模相对较小、治疗周期仅长达56天、缺乏定量痰培养CFU计数数据，且未考察痰LAM水平与临床治愈结局的相关性。 结论：上述研究结果表明，LAM-ELISA可准确定量检测痰标本中的LAM浓度，通过该方法测得的痰LAM水平可作为肺结核患者治疗前及治疗期间的细菌负荷生物标志物。未来需开展更多研究，以明确这一新型生物标志物对肺结核治疗结局的预测价值。