Physical Chemistry and Applied Spectroscopy, Chemistry Division, Los Alamos National Laboratory, Los Alamos, New Mexico, United States of America.
Actinide Analytical chemistry, Chemistry Division, Los Alamos National Laboratory, Los Alamos, New Mexico, United States of America.
PLoS One. 2021 Apr 7;16(4):e0243337. doi: 10.1371/journal.pone.0243337. eCollection 2021.
Lipoarabinomannan (LAM), an amphiphilic lipoglycan of the Mycobacterium tuberculosis cell wall, is a diagnostic target for tuberculosis. Previous work from our laboratory and others suggests that LAM is associated with host serum lipoproteins, which may in turn have implications for diagnostic assays. Our team has developed two serum assays for amphiphile detection: lipoprotein capture and membrane insertion. The lipoprotein capture assay relies on capture of the host lipoproteins, exploiting the biological association of host lipoprotein with microbial amphiphilic biomarkers to "concentrate" LAM. In contrast, the membrane insertion assay is independent of the association between pathogen amphiphiles and host lipoprotein association, and directly captures LAM based on its thermodynamic propensity for association with a supported lipid membrane, which forms the functional surface of an optical biosensor. In this manuscript, we explored the use of these assays for the detection of LAM in sera from adults whose tuberculosis status had been well-characterized using conventional microbiological tests, and endemic controls. Using the lipoprotein capture assay, LAM signal/noise ratios were >1.0 in 29/35 (83%) individuals with culture-confirmed active tuberculosis, 8/13 (62%) individuals with tuberculosis symptoms, but no positive culture for M. tuberculosis, and 0/6 (0%) symptom-free endemic controls. To evaluate serum LAM levels without bias associated with potential differences in circulating host lipoprotein concentrations between individuals, we subsequently processed available samples to liberate LAM from associated host lipoprotein assemblies followed by direct detection of the pathogen biomarker using the membrane insertion approach. Using the membrane insertion assay, signal/noise for detection of serum LAM was greater than that observed using the lipoprotein capture method for culture-confirmed TB patients (6/6), yet remained negative for controls (2/2). Taken together, these results suggest that detection of serum LAM is a promising TB diagnostic approach, but that further work is required to optimize assay performance and to decipher the implications of LAM/host lipoprotein associations for diagnostic assay performance and TB pathogenesis.
脂阿拉伯甘露聚糖（LAM）是分枝杆菌细胞壁的一种两亲性糖脂，是结核病的诊断靶标。我们实验室和其他实验室的先前工作表明，LAM 与宿主血清脂蛋白有关，这可能反过来对诊断检测有影响。我们的团队开发了两种用于检测两亲性物质的血清检测方法：脂蛋白捕获和膜插入。脂蛋白捕获检测法依赖于宿主脂蛋白的捕获，利用宿主脂蛋白与微生物两亲性生物标志物的生物学关联来“浓缩”LAM。相比之下，膜插入检测法不依赖于病原体两亲性物质与宿主脂蛋白的关联，而是直接基于 LAM 与支撑脂质膜的热力学亲和力来捕获 LAM，该脂质膜形成了光学生物传感器的功能表面。在本手稿中，我们探索了这些检测方法在使用传统微生物学检测方法充分描述结核病状况的成人血清中检测 LAM 的用途，以及在地方病对照中检测 LAM 的用途。使用脂蛋白捕获检测法，在 35 例经培养证实患有活动性结核病的个体中，有 29 例（83%）的 LAM 信号/噪声比>1.0，在 13 例（62%）有结核病症状但无结核分枝杆菌阳性培养的个体中，有 8 例（62%）的 LAM 信号/噪声比>1.0，在 6 例无症状地方病对照中，有 0 例（0%）的 LAM 信号/噪声比>1.0。为了评估血清 LAM 水平，而不受个体间循环宿主脂蛋白浓度差异的潜在偏差影响，我们随后处理了可用的样本，从相关的宿主脂蛋白组装体中释放 LAM，然后使用膜插入方法直接检测病原体生物标志物。使用膜插入检测法，对经培养证实的结核病患者进行 LAM 检测的信号/噪声比大于使用脂蛋白捕获法检测的信号/噪声比（6/6），而对对照（2/2）仍为阴性。总之，这些结果表明，检测血清 LAM 是一种很有前途的结核病诊断方法，但需要进一步工作来优化检测性能，并阐明 LAM/宿主脂蛋白的关联对诊断检测性能和结核病发病机制的影响。
