Multiplexed detection of cell-free M. Tuberculosis DNA and its drug-resistant variants in blood

血液中无细胞结核分枝杆菌 DNA 及其耐药变异体的多重检测

• 批准号: 10639855
• 负责人: Tony Y. Hu
• 金额: $ 75.34万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-11 至 2028-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10639855
• 关键词: 2019-nCoV; Address; Affect; Aftercare; Antitubercular Agents; Archives; Area; Bacillus; Biological Assay; Biopsy; Blood; Blood specimen; Body Fluids; COVID-19 detection; COVID-19 diagnosis; COVID-19 pandemic; Cause of Death; Cells; Child; Circulation; Clinical; Clinical Data; Clustered Regularly Interspaced Short Palindromic Repeats; Communicable Diseases; DNA; DNA amplification; DNA analysis; Data; Detection; Diagnosis; Diagnostic; Diagnostic tests; Disease; Drug Evaluation; Drug resistance; Drug resistance in tuberculosis; Early Diagnosis; Equipment; Evaluation; Exhibits; Extreme drug resistant tuberculosis; Face; Genetic Recombination; Goals; HIV; Half-Life; Health Resources; Healthcare; Immune response; Immune system; India; Individual; Infection; Interferon Type II; Isoniazid resistance; Laboratories; Longitudinal cohort study; Lung; Measures; Mediating; Methods; Modeling; Monitor; Multidrug-Resistant Tuberculosis; Mycobacterium tuberculosis; Mycobacterium tuberculosis antigens; Paper; Pathologic; Patients; Performance; Persons; Pharmaceutical Preparations; Physiological; Plasma; Polymerase; Population; Prediction of Response to Therapy; Predisposition; Prevalence; Prognosis; Prospective cohort; Public Health; RNA; Reaction; Reader; Relapse; Resistance; Resolution; Resource-limited setting; Resources; Respiratory Disease; Retrospective cohort; Rifampicin resistance; Sampling; Sensitivity and Specificity; Serum; Severities; Severity of illness; South Africa; Special Equipment; Specificity; Sputum; Testing; Time; Tissues; Treatment Efficacy; Treatment Failure; Tuberculosis; Tuberculosis diagnosis; United States National Institutes of Health; Urine; Validation; Variant; X Inactivation; accurate diagnosis; biobank; biomedical referral center; cell free DNA; clinical research site; cohort; detection assay; detection limit; detection platform; diagnostic assay; disorder control; drug-sensitive; falls; follow-up; immune function; improved; lipoarabinomannan; mortality; multiplex detection; novel; patient population; point of care; point of care testing; point-of-care diagnosis; prognostic performance; quinolone resistance; rapid diagnosis; rapid testing; reproductive; response; sample collection; treatment response; tuberculosis diagnostics; tuberculosis treatment; viral RNA

ABSTRACT Every year 10 million people fall ill with tuberculosis (TB) and 1.5 million people die from TB – making it the leading cause of death from infectious disease – but TB can be difficult to diagnose. Extended M. tuberculosis (Mtb) cultures are still often used for diagnosis. PCR-based assays (e.g. Xpert MTB/RIF) that detect TB DNA can provide more rapid results, but require special equipment and, like Mtb culture, exhibit reduced sensitivity when employed to analyze sputum from individuals with extrapulmonary TB or compromised immune systems. Blood-based TB assays should detect all forms of TB, but current tests analyze the immune response to Mtb antigens and cannot distinguish active and latent TB. Sensitive detection of Mtb-derived cell-free DNA (cfDNA) in the circulation, however, represents a potential new means for enhanced TB diagnosis. Circulating cfDNA is rapidly degraded after its release and disease-associated cfDNA levels in blood can rapidly change in response to pathologic changes and physiologic responses. This short half-life can enable “real-time” cfDNA analyses required for accurate evaluation of the current status of active Mtb infections and rapid granular evaluation of Mtb treatment responses. However, the limit of detection (LoD) of current PCR-based assays are not sufficient to reliably detect Mtb cfDNA in blood. We have established a CRISPR-Cas12a-based detection system that can ultra-sensitively detect trace amount of SARS-CoV-2 RNA in blood to diagnose COVID-19, predict disease severity, and evaluate infection resolution. We have adapted this approach to develop a blood-based multiplexed CRISPR-mediated TB diagnosis (CRISPR-TBD) assay that can detect circulating Mtb cfDNA, including SNPs responsible for drug-resistant TB. Our preliminary data from longitudinal serum samples of patients undergoing TB treatment provide strong proof-of-principle evidence for the clinical utility of this platform. We have adapted this approach to a paper strip-based point of care (POC) CRISPR-TBD detection platform suitable for use in resource-limited regions with high TB burden, without decreasing assay sensitivity. We now propose to: 1) systematically optimize all CRISPR-TBD paper strip assay steps to improve quantitative detection of Mtb-cfDNA in POC settings; 2) evaluate the performance of this POC assay to diagnose pulmonary and extrapulmonary TB and to identify drug-sensitive and -resistant TB cases; 3) quantify Mtb-cfDNA changes in serum during TB treatment as a measure of treatment efficacy or failure and for early detection of nascent drug resistance; and 4) in-field validate the diagnostic performance of this POC assay in an independent TB patient cohort when performed in a clinical laboratory in a high endemic TB region and evaluate in parallel the performance our predictive Mtb-cfDNA model for TB treatment.

抽象的 每年有 1000 万人患上结核病 (TB)，150 万人死于结核病——这使其成为全球最严重的结核病。 传染病导致死亡的主要原因——但结核病很难诊断。 (Mtb) 培养物仍常用于检测 TB DNA 的 PCR 检测（例如 Xpert MTB/RIF）。 可以提供更快速的结果，但需要特殊设备，并且与 Mtb 培养一样，灵敏度较低 当用于分析患有肺外结核或免疫系统受损的个体的痰液时。 基于血液的结核病检测应检测所有形式的结核病，但目前的检测分析对结核分枝杆菌的免疫反应 抗原，无法区分活动性和潜伏性结核病，对 Mtb 衍生的游离 DNA (cfDNA) 进行灵敏检测。 然而，循环中的 cfDNA 代表了增强结核病诊断的潜在新方法。 释放后迅速降解，血液中与疾病相关的 cfDNA 水平会迅速发生变化 这种短半衰期可以实现“实时”cfDNA 分析。 需要准确评估活动性结核分枝杆菌感染的当前状态并快速进行细粒度评估 然而，目前基于 PCR 的检测方法的检测限 (LoD) 还不够。 为了可靠地检测血液中的 Mtb cfDNA，我们建立了基于 CRISPR-Cas12a 的检测系统。 超灵敏检测血液中的微量 SARS-CoV-2 RNA 以诊断 COVID-19、预测疾病 我们已经采用这种方法来开发基于血液的多重检测。 CRISPR 介导的结核病诊断 (CRISPR-TBD) 检测可检测循环 Mtb cfDNA，包括 SNP 我们的初步数据来自接受治疗的患者的纵向血清样本。 结核病治疗为该平台的临床效用提供了强有力的原理证明证据。 这种基于纸条的护理点 (POC) CRISPR-TBD 检测平台的方法适用于 资源有限、结核病负担高的地区，在不降低检测灵敏度的情况下，我们现在建议：1) 系统地优化所有 CRISPR-TBD 试纸条检测步骤，以改进 Mtb-cfDNA 的定量检测 在 POC 设置中；2) 评估该 POC 诊断肺部和肺外结核病的性能 并识别药物敏感和耐药结核病病例；3) 量化结核病期间血清中的 Mtb-cfDNA 变化； 治疗作为治疗效果或失败的衡量标准以及早期检测新生耐药性；和 4) 在独立的结核病患者队列中现场验证该 POC 检测的诊断性能 在结核病高发地区的临床实验室进行，并并行评估我们的表现 结核病治疗的预测 Mtb-cfDNA 模型。