A nanopore biosensor for leveling Mtb antigens in blood

用于平衡血液中 Mtb 抗原的纳米孔生物传感器

• 批准号: 10646134
• 负责人: Tony Y. Hu
• 金额: $ 75.26万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-15 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10646134
• 关键词: Accounting; Adult; Algorithms; Antibodies; Antigens; Antitubercular Agents; Bacteriology; Biological Assay; Biological Markers; Biopsy; Biosensor; Blood; Blood specimen; Cause of Death; Cessation of life; Child; Childhood; Clinical; Clinical Data; Communicable Diseases; Containment; Data; Detection; Development; Devices; Diagnosis; Diagnostic; Diagnostic Sensitivity; Diagnostic Specificity; Diagnostic tests; Disease; Early Diagnosis; Early treatment; Epidemic; Event; Exhibits; Generations; Goals; Guidelines; HIV; Immunoprecipitation; Individual; Infection; Longterm Follow-up; Lung; Mass Spectrum Analysis; Measurement; Measures; Monitor; Mycobacterium Infections; Mycobacterium tuberculosis; Mycobacterium tuberculosis antigens; Nature; Nucleic Acids; Organizational Objectives; Patient Monitoring; Patient-Focused Outcomes; Patients; Peptides; Performance; Persons; Population; Population Heterogeneity; Prevalence; Procedures; Proteins; Protocols documentation; Pulmonary Tuberculosis; Radiology Specialty; Recommendation; Reporting; Research; Resource-limited setting; Resources; Sampling; Sensitivity and Specificity; Serum; Specificity; Specimen; Sputum; System; Technology; Test Result; Testing; Tissues; Translations; Tuberculosis; Tuberculosis diagnosis; Validation; Virulence Factors; Work; World Health Organization; biosignature; clinical application; clinical research site; cohort; cost; diagnostic assay; diagnostic platform; disease diagnosis; disorder control; fabrication; global health; improved; instrument; nanopore; non-tuberculosis mycobacteria; patient population; patient response; peripheral blood; portability; predictive modeling; programs; prototype; rapid diagnosis; research clinical testing; response; specific biomarkers; success; treatment response; trend; tuberculosis diagnostics; tuberculosis treatment; validation studies

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.

结核病（TB）是全球健康威胁，但由于一线诊断测定的次优性能和非定量性质而可能难以诊断和管理，一线诊断测定需要痰或组织活检，并且当应用于诊断少杆菌或肺外TB病例时表现出降低的性能。对于可以灵敏地诊断活动性TB并测量临床上不同人群中的治疗反应的快速、非基于痰的测定存在未满足的需求。我们以前曾报道，血清水平的两种肽衍生自结核分枝杆菌（Mtb）的毒力因子CFP-10和ESAT-6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，验证其在成人和儿童的相关队列的诊断性能。这包括HIV+/HIV-、肺/肺外和培养阳性/阴性结核病例，以及潜伏性结核和非结核分枝杆菌感染病例。该MS检测在成人和儿童中具有相似的诊断灵敏度（88.6 vs 88.2%）和特异性（93.8 vs 97.2%），超过了可比人群中一线检测的报告性能。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，该MS测定不适用于资源有限的环境。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。该系统易于操作，具有低的制造和仪器成本，并且可以进行特定Mtb衍生肽的高通量和超灵敏测量。其坚固的性质和便携性也使其能够在结核病高发的资源有限地区使用。我们的研究结果表明，纳米孔检测可以准确地检测CFP-10和ESAT-6肽，并且该数据具有重要的诊断前景。基于这些发现，我们提出，将在这些研究中分析的便携式蛋白质纳米孔生物传感器检测可以改善成人和儿童的结核病诊断，特别是在结核病流行率高的资源有限地区。我们将利用该系统来测量Mtb抗原衍生的肽生物标志物的血清水平，以便：（1）开发灵敏且稳健的基于纳米孔的TB诊断测定;（2）在包含具有肺和肺外TB病例的患者的组织良好的群组中验证该测定;以及（3）评估在抗TB治疗期间成人PTB病例中Mtb抗原的血清水平如何变化。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。这项研究计划应该加速将一个有前途的生物传感器平台转化为一种实用的检测方法，适合快速转化为疾病诊断的临床应用。