A nanopore biosensor for leveling Mtb antigens in blood

结核病是一种全球性的健康威胁，但由于一线诊断分析的性能不佳和非定量性质，可能难以诊断和管理，这需要进行痰液或组织活检，并且在用于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、非基于痰的检测方法的需求尚未得到满足，这种方法可以在临床不同的人群中敏感地诊断活动性结核病并测量治疗反应。我们之前报道过，从结核分枝杆菌（Mtb）毒力因子CFP-10和ESAT-6中提取的两种肽的血清水平可以作为活动性结核病的特异性生物标志物，并使用质谱（MS）测定验证了它们在成人和儿童相关队列中的诊断性能。这包括HIV+/HIV-，肺/肺外，培养阳性/阴性结核病例，以及潜伏结核和非结核分枝杆菌感染的病例。该MS检测在成人和儿童中具有相似的诊断敏感性（88.6 vs 88.2%）和特异性（93.8 vs 97.2%），并且超过了在可比人群中报道的一线检测的性能。血清结核分枝杆菌抗原水平也是监测抗结核治疗反应的信息。然而，这种质谱分析不适合在资源有限的环境中使用。因此，拟议的研究将改进和评估基于蛋白质的纳米孔生物传感器测定的性能，以使用相同的血清生物标志物诊断活动性结核病病例。该系统操作简单，制造成本低，仪器成本低，可实现高通量和超灵敏的特定mtb衍生肽的测量。它的坚固性和可携带性也使其能够在结核病高流行的资源有限地区使用。我们的研究结果表明，纳米孔法可以准确地检测CFP-10和ESAT-6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质纳米孔生物传感器测定可以改善成人和儿童的结核病诊断，特别是在结核病高患病率的资源有限地区。我们将利用该系统测量结核分枝杆菌抗原衍生肽生物标志物的血清水平，以便：(1)开发一种敏感且强大的基于纳米孔的结核诊断检测；(2)在组织良好的队列中验证该检测方法，包括肺部和肺外结核患者；(3)评价抗结核治疗期间成人肺结核患者血清Mtb抗原水平的变化。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构建原型设备，并修改和扩展该系统以检测其他疾病生物标志物。该研究项目将加速将一个有前途的生物传感器平台转化为一个实用的检测方法，适合快速转化为疾病诊断的临床应用。

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.