Ultrasensitive detection of lipoarabinomannan with plasmonic grating biosensors in clinical samples of HIV negative patients with tuberculosis
Ultrasensitive detection of lipoarabinomannan with plasmonic grating biosensors in clinical samples of HIV negative patients with tuberculosis
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DOI：
10.1371/journal.pone.0214161
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发表时间：
2019-03-26
期刊：
影响因子：
3.7
通讯作者：
Gangopadhyay, Shubhra
中科院分区：
文献类型：
--
作者：
Wood, Aaron;Barizuddin, Syed;Gangopadhyay, Shubhra
背景结核病的及时诊断对于患者的积极治疗效果至关重要，但每年可能有数百万人未得到诊断或未报告。痰液被广泛用作检测输入，但受到其复杂性、异质性和来源问题的限制。寻找非侵入性、非痰临床标本的方法对于改善结核病诊断和护理的可及性至关重要。在这项工作中，使用经济型等离子体光栅通过单分子荧光测定 (FLISA) 分析临床尿液样本中的结核病生物标志物阿拉伯脂甘露聚糖 (LAM)，并与金标准痰 GeneXpert MTB/RIF、培养物和参考 ELISA 检测结果进行比较。 方法和结果在这项研究中，从开始抗结核治疗前收集的 HIV 阴性患者样本库中回顾性选择了 20 组痰液和尿液样本治疗。 GeneXpert MTB/RIF 和患者痰液培养检测证实是否存在肺结核，而所有患者尿液的参考 ELISA LAM 均为阴性。通过低成本软光刻技术生产的等离子光栅与抗 LAM 捕获抗体结合，与患者尿液样本和生物素化检测抗体一起孵育。荧光标记的链霉亲和素通过落射荧光显微镜显示单分子发射。使用 1 fg/mL 基线作为检测限，单分子 FLISA 在 20 名患者中的 19 名中表现出与金标准测试良好的定性一致性，包括准确预测金标准阴性患者，而一名金标准阳性患者在尿液中没有产生可观察到的 LAM。 结论 通过等离子体光栅的单分子 FLISA 证明能够从高流行环境中的患者的复杂尿液样本中定量结核病 LAM，而复合物的干扰可以忽略不计媒体本身。 Moreover, agreement with patient diagnoses by gold standard testing suggests that single molecule FLISA could be used as a highly sensitive test to diagnose tuberculosis noninvasively.
BackgroundTimely diagnosis of tuberculosis disease is critical for positive patient outcomes, yet potentially millions go undiagnosed or unreported each year. Sputum is widely used as the testing input, but limited by its complexity, heterogeneity, and sourcing problems. Finding methods to interrogate noninvasive, non-sputum clinical specimens is indispensable to improving access to tuberculosis diagnosis and care. In this work, economical plasmonic gratings were used to analyze tuberculosis biomarker lipoarabinomannan (LAM) from clinical urine samples by single molecule fluorescence assay (FLISA) and compared with gold standard sputum GeneXpert MTB/ RIF, culture, and reference ELISA testing results.Methods and FindingsIn this study, twenty sputum and urine sample sets were selected retrospectively from a repository of HIV-negative patient samples collected before initiation of anti-tuberculosis therapy. GeneXpert MTB/RIF and culture testing of patient sputum confirmed the presence or absence of pulmonary tuberculosis while all patient urines were reference ELISA LAM-negative. Plasmonic gratings produced by low-cost soft lithography were bound with anti-LAM capture antibody, incubated with patient urine samples, and biotinylated detection antibody. Fluorescently labeled streptavidin revealed single molecule emission by epifluorescence microscope. Using a 1 fg/mL baseline for limit of detection, single molecule FLISA demonstrated good qualitative agreement with gold standard tests on 19 of 20 patients, including accurately predicting the gold-standard-negative patients, while one gold-standard-positive patient produced no observable LAM in urine.ConclusionsSingle molecule FLISA by plasmonic grating demonstrated the ability to quantify tuberculosis LAM from complex urine samples of patients from a high endemic setting with negligible interference from the complex media itself. Moreover, agreement with patient diagnoses by gold standard testing suggests that single molecule FLISA could be used as a highly sensitive test to diagnose tuberculosis noninvasively.