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1000万人因结核病（TB）而生病，有150万人死于结核病 - 使其成为 传染病的主要死亡原因 - 但结核病可能很难诊断。结核分枝杆菌 （MTB）文化仍然经常用于诊断。基于PCR的测定（例如XPERT MTB/RIF）检测TB DNA 可以提供更快速的结果，但需要特殊设备，例如MTB培养，暴露了降低的灵敏度 当被用来分析肺内结核病或免疫系统受损的人的痰液时。 基于血液的结核病测定应检测所有形式的结核 抗原，无法区分活性和潜在结核。 MTB衍生的无细胞DNA（CFDNA）的敏感检测 但是，在循环中，代表了增强结核病诊断的潜在新手段。循环cfDNA是 释放后迅速退化，血液中与疾病相关的CFDNA水平可能会迅速改变 病理变化和生理反应。这个短的半衰期可以实现“实时” CFDNA分析 准确评估活动MTB感染的当前状态和快速颗粒状评估所必需的 MTB治疗反应。但是，当前基于PCR的测定的检测极限（LOD）不够 可靠地检测到血液中的mtb cfDNA。我们已经建立了一个基于CRISPR-CAS12A的检测系统 超敏性检测血液中的痕量SARS-COV-2 RNA诊断Covid-19，预测疾病 严重程度并评估感染分辨率。我们已经改编了这种方法来开发基于血液的多路复用 CRISPR介导的结核病诊断（CRISPR-TBD）测定法，可以检测到循环的MTB CFDNA，包括SNP 负责耐药结核病。我们来自接受的患者纵向血清样本的初步数据 结核病治疗为该平台的临床实用性提供了有力的原理证据。我们已经适应了 这种方法用于基于纸条的护理点（POC）CRISPR-TBD检测平台适合用于 资源有限的区域具有较高的结核病负担，而不会降低评估敏感性。我们现在建议：1） 系统地优化所有CRISPR-TBD纸条测定步骤，以改善MTB-CFDNA的定量检测 在POC设置中； 2）评估该POC分析的性能以诊断肺和肺外结核 并确定对药物敏感和耐药性结核病病例； 3）量化TB期间血清中的MTB-CFDNA变化 治疗作为治疗效率或失败的量度，以及早期检测新生耐药性；和 4）现场验证该POC分析在独立结核病患者队列中的诊断性能 在高流行结核病区域的临床实验室中进行，并同时评估我们 用于结核病处理的预测MTB-CFDNA模型。