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

摘要 每年有1000万人感染结核病，150万人死于结核病--使其成为世界上最大的结核病 传染病导致死亡的主要原因--但结核病可能很难诊断。扩大的结核分枝杆菌 (结核分枝杆菌)培养仍经常用于诊断。基于PCR的检测结核DNA的方法(例如Xpert MTB/RIF) 可以提供更快的结果，但需要特殊的设备，并且像结核分枝杆菌培养一样，表现出较低的敏感性 当被用于分析患有肺外结核病或免疫系统受损的人的痰时。 基于血液的结核病检测应该能检测出所有形式的结核病，但目前的检测分析的是对结核杆菌的免疫反应。 抗原，不能区分活动性和潜伏性结核病。结核分枝杆菌来源的游离DNA(CfDNA)的灵敏检测 然而，在循环中，它代表了一种潜在的新手段，用于加强结核病诊断。循环中的cfDNA是 释放后迅速降解，血液中与疾病相关的cfDNA水平会迅速变化 对病理变化和生理反应的反应。这种短暂的半衰期可以实现对cfDNA的实时分析 准确评估活动性结核分枝杆菌感染的当前状态和快速细粒度评估 结核分枝杆菌治疗反应。然而，目前基于聚合酶链式反应的检测方法的检测限(LOD)是不够的 目的：可靠地检测血液中结核分枝杆菌cfDNA。我们已经建立了一个基于CRISPR-CAS12a的检测系统，可以 超敏检测血中微量SARS-CoV-2RNA诊断新冠肺炎、预测疾病 严重程度，并评估感染解决方案。我们已经采用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)方法可以检测包括SNPs在内的循环Mtb cfDNA 对耐药结核病负责。我们从患者纵向血清样本中获得的初步数据 结核病治疗为该平台的临床应用提供了强有力的原则性证据。我们已经适应了 该方法是一种基于纸条的护理点(POC)CRISPR-TBD检测平台，适用于 资源有限、结核病负担高的地区，而不降低化验灵敏度。我们现在建议：1) 系统优化CRISPR-TBD纸条分析步骤以提高Mtb-cfDNA的定量检测 在POC环境中；2)评估该POC检测诊断肺和肺外结核的性能 3)定量检测结核病患者血清中mtb-cfDNA的变化。 将治疗作为治疗效果或失败的衡量标准，并及早发现新生的耐药性；以及 4)现场验证此POC检测在独立的结核病患者队列中的诊断性能 在结核病高发地区的临床实验室进行，并同时评估我们的 结核分枝杆菌-cfDNA结核分枝杆菌治疗预测模型。

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.