A nanopore biosensor for leveling Mtb antigens in blood

结核病是一种全球健康威胁，但由于一线诊断分析的性能不佳和非定量性质，可能很难诊断和管理，这些分析需要痰或组织活检，在诊断少杆菌或肺外结核病例时表现出较低的性能。对一种快速的、非痰为基础的检测方法的需求尚未得到满足，这种检测方法可以灵敏地诊断活动性结核病并测量临床不同人群的治疗反应。我们此前曾报道，来自结核分枝杆菌(Mtb)毒力因子CFP-10和ESAT-6的两种多肽的血清水平可以作为活动性结核病的特定生物标志物，并使用质谱仪(MS)分析，在成人和儿童的相关队列中验证了它们的诊断性能。这包括HIV+/HIV-、肺/肺外、培养阳性/阴性结核病病例，以及潜伏性结核病和非结核分枝杆菌感染病例。这种MS检测在成人和儿童中具有相似的诊断敏感性(88.6vs88.2%)和特异性(93.8vs97.2%)，并超过了在可比人群中报道的一线检测的表现。血清结核分枝杆菌抗原水平也可作为监测抗结核治疗反应的参考指标。然而，这种MS分析不适合在资源有限的环境中使用。因此，拟议的研究将完善和评估基于蛋白质的纳米孔生物传感器试验的性能，以使用相同的血清生物标记物诊断活动性结核病病例。该系统操作简单，制造和仪器成本低，可以对特定的结核分枝杆菌衍生多肽进行高通量和超灵敏的测量。它的强健性质和便携性也使其能够在结核病高流行的资源有限的地区使用。我们的结果表明，纳米孔法可以准确地检测CFP-10和ESAT-6多肽，这一数据具有重要的诊断前景。基于这些发现，我们建议将在这些研究中分析的便携式蛋白纳米孔生物传感器试验可以改善成人和儿童的结核病诊断，特别是在结核病发病率高的资源有限的地区。我们将利用这一系统来测量血清结核分枝杆菌抗原衍生多肽生物标志物的水平，以便：(1)开发一种灵敏和可靠的基于纳米孔的结核病诊断方法；(2)在包含肺和肺外结核病例的组织良好的队列中验证该方法；以及(3)评估在抗结核治疗期间成年肺结核患者的血清结核杆菌抗原水平是如何变化的。鉴于这些概念验证研究的成功，拟议研究计划的长期目标是为结核病高负担地区的大规模现场临床验证研究建立原型设备，并修改和扩展该系统以检测其他疾病生物标志物。这一研究计划应该会加速将一个有前途的生物传感器平台转化为适合快速转化为疾病诊断临床应用的实用检测方法。

Tuberculosis (TB) is a global health threat but can be difficult to diagnose and manage due to the sub-optimal performance and non-quantitative nature of frontline diagnostic assays, which require sputum or tissue biopsies and exhibit reduced performance when applied to diagnose paucibacillary or extrapulmonary TB cases. There is an unmet need for a rapid, non-sputum-based assay that can sensitively diagnose active TB and measure treatment responses in clinically diverse populations. We have previously reported that serum levels of two peptides derived from the Mycobacterium tuberculosis (Mtb) virulence factors CFP-10 and ESAT-6 can act as specific biomarkers of active TB and, using a mass spectrometry (MS) assay, validated their diagnostic performance in relevant cohorts of adults and children. This included HIV+/HIV-, pulmonary / extrapulmonary, and culture-positive / negative TB cases, as well as those with latent TB and nontuberculous mycobacteria infections. This MS assay had similar diagnostic sensitivity (88.6 vs 88.2%) and specificity (93.8 vs 97.2%) in adults and children and exceeded the reported performance of frontline tests in comparable populations. Serum Mtb antigen levels were also informative in monitoring anti-TB treatment responses. However, this MS assay is not suitable for use in resource limited settings. The proposed studies will therefore refine and evaluate the performance of a protein-based nanopore biosensor assay to diagnose active TB cases using the same serum biomarkers. This system is easy to operate, has low fabrication and instrument costs, and can perform high- throughput and ultra-sensitive measurements of specific Mtb-derived peptides. Its robust nature and portability also allow its use in resource-limited areas subject to high TB prevalence. Our results show that a nanopore assay can accurately detect CFP-10 and ESAT-6 peptides, and this data has significant diagnostic promise. Based on these findings, we propose that the portable protein nanopore biosensor assay that will be analyzed in these studies can improve TB diagnosis in adults and children, particularly in resource-limited areas with high TB prevalence. We will utilize this system to measure serum levels of Mtb antigen-derived peptide biomarkers in order to: (1) develop a sensitive and robust nanopore-based TB diagnostic assay; (2) validate this assay in well-organized cohorts, containing patients with pulmonary and extrapulmonary TB cases; and (3) evaluate how serum levels of Mtb antigens change in adult PTB cases in during anti-TB therapy. Given the success of these proof-of-concept studies, the long-term goal of the proposed research program is to build prototype devices for large-scale on-site clinical validation studies in high TB burden regions, and to modify and extend this system to detect other disease biomarkers. This research program should hasten the translation of a promising biosensor platform into a practical assay suitable for rapid translation to clinical applications for disease diagnosis.