Amin Anita G, De Prithwiraj, Spencer John S, Brennan Patrick J, Daum Joshua, Andre Barbara G, Joe Maju, Bai Yu, Laurentius Lars, Porter Marc D, Honnen William J, Choudhary Alok, Lowary Todd L, Pinter Abraham, Chatterjee Delphi
Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, 1682 Campus Delivery, Fort Collins, CO, 80523, USA.
Alberta Glycomics Centre and Department of Chemistry, University of Alberta, Edmonton, AB, T6G 2G2, Canada.
Tuberculosis (Edinb). 2018 Jul;111:178-187. doi: 10.1016/j.tube.2018.06.004. Epub 2018 Jun 6.
TB diagnosis and treatment monitoring in resource limited regions rely heavily on serial sputum smear microscopy and bacterial culture. These microbiological methods are time-consuming, expensive and lack adequate sensitivity. The WHO states that improved TB diagnosis and treatment is imperative to achieve an end to the TB epidemic by 2030. Commercially available lipoarabinomannan (LAM) detection tools perform at low sensitivity that are highly dependent on the underlying immunological status of the patient; those with advanced HIV infection perform well. In this study, we have applied two novel strategies towards the sensitive diagnosis of TB infection based on LAM: Capture ELISA to detect LAM in paired urine and serum samples using murine and human monoclonal antibodies, essentially relying on LAM as an 'immuno-marker'; and, secondly, detection of α-d-arabinofuranose and tuberculostearic acid (TBSA)- 'chemical-markers' unique to mycobacterial cell wall polysaccharides/lipoglycans by our recently developed gas chromatography/mass spectrometry (GC/MS) method. Blinded urine specimens, with microbiologically confirmed active pulmonary TB or non TB (HIV+/HIV-) were tested by the aforementioned assays. LAM in patient urine was detected in a concentration range of 3-28 ng/mL based on GC/MS detection of the two LAM-surrogates, d-arabinose and tuberculostearic acid (TBSA) correctly classifying TB status with sensitivity > 99% and specificity = 84%. The ELISA assay had high sensitivity (98%) and specificity (92%) and the results were in agreement with GC/MS analysis. Both tests performed well in their present form particularly for HIV-negative/TB-positive urine samples. Among the HIV+/TB+ samples, 52% were found to have >10 ng/mL urinary LAM. The detected amounts of LAM present in the urine samples also appears to be associated with the gradation of the sputum smear, linking elevated LAM levels with higher mycobacterial burden (odds ratio = 1.08-1.43; p = 0.002). In this small set, ELISA was also applied to parallel serum samples confirming that serum could be an additional reservoir for developing a LAM-based immunoassay for diagnosis of TB.
资源有限地区的结核病诊断和治疗监测严重依赖于系列痰涂片显微镜检查和细菌培养。这些微生物学方法耗时、昂贵且灵敏度不足。世界卫生组织指出，改善结核病诊断和治疗对于在2030年前终结结核病流行至关重要。市售的脂阿拉伯甘露聚糖（LAM）检测工具灵敏度较低，且高度依赖患者的基础免疫状态；晚期HIV感染者检测效果较好。在本研究中，我们基于LAM应用了两种新型策略用于结核病感染的灵敏诊断：捕获ELISA法，使用鼠源和人源单克隆抗体检测配对尿液和血清样本中的LAM，本质上是将LAM作为一种“免疫标志物”；其次，通过我们最近开发的气相色谱/质谱（GC/MS）方法检测α - D - 阿拉伯呋喃糖和结核硬脂酸（TBSA）——分枝杆菌细胞壁多糖/脂多糖特有的“化学标志物”。对经微生物学确诊为活动性肺结核或非肺结核（HIV阳性/HIV阴性）的盲法尿液标本进行上述检测。基于对两种LAM替代物——D - 阿拉伯糖和结核硬脂酸（TBSA）的GC/MS检测，患者尿液中LAM的检测浓度范围为3 - 28 ng/mL，正确分类结核病状态的灵敏度>99%，特异性 = 84%。ELISA检测具有高灵敏度（98%）和特异性（92%），结果与GC/MS分析一致。两种检测方法在当前形式下表现良好，尤其对于HIV阴性/结核阳性尿液样本。在HIV阳性/结核阳性样本中，发现52%的样本尿LAM>10 ng/mL。尿液样本中检测到的LAM量似乎也与痰涂片分级相关，LAM水平升高与更高的分枝杆菌负荷相关（优势比 = 1.08 - 1.43；p = 0.002）。在这个小样本中，ELISA也应用于平行血清样本，证实血清可能是开发基于LAM的结核病诊断免疫测定的另一个样本来源。
