Rapid Detection of TB from Blood using Cell-Free DNA and CRISPR

使用无细胞 DNA 和 CRISPR 快速检测血液中的结核病

• 批准号: 10620065
• 负责人: Richard S Garfein
• 金额: $ 79.52万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-17 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10620065
• 关键词: Address; Adult; Affinity; Aftercare; Arkansas; Binding; Biological Assay; Biological Markers; Blood; Blood Tests; Blood specimen; COVID-19; California; Cessation of life; Child; Childhood; Clinical; Clinical Research; Clinical Sensitivity; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Communicable Diseases; Cryopreservation; DNA; DNA Binding; Detection; Diagnosis; Diagnostic; Diagnostic Sensitivity; Diagnostic Specificity; Disease; Drug resistance in tuberculosis; Extinction; Fluorescence; Goals; Growth; HIV; Immunocompromised Host; Laboratories; Lateral; Lung diseases; Mexico; Microscopy; Molecular; Monitor; Mycobacterium tuberculosis; Nonlinear Dynamics; Pakistan; Paper; Patient-Focused Outcomes; Patients; Pediatrics; Performance; Persons; Pharmaceutical Preparations; Pilot Projects; Predisposition; Process; Proteins; Reader; Reference Standards; Reporter; Reporting; Research; Resource-limited setting; Risk; Sampling; Sensitivity and Specificity; Signal Transduction; South Africa; Specificity; Sputum; Testing; Triage; Tuberculosis; Tuberculosis diagnosis; United States National Institutes of Health; Universities; accurate diagnosis; biobank; cell free DNA; co-infection; cohort; cost; cross reactivity; detection assay; detection limit; diagnostic criteria; innovation; isothermal amplification; lateral flow assay; molecular diagnostics; mortality; non-tuberculosis mycobacteria; novel; nuclease; peripheral blood; point-of-care diagnostics; prospective; rapid detection; rapid diagnosis; rapid test; technology platform; transmission process; tuberculosis diagnostics; tuberculosis treatment

Project Summary While >90% of TB cases are curable, treatment is dependent on diagnostics based exclusively on the detection of Mycobacterium tuberculosis (Mtb) in patient sputum, despite the limitations of sputum as a diagnostic sample. Sputum is difficult to process, almost impossible to obtain from patients with paucibacillary TB, such as people living with HIV (PLWH) and children and is not diagnostic for patients with extrapulmonary TB. A low- cost, rapid test using blood instead of sputum could transform TB diagnosis for ALL patients and reduce the current diagnostic gap between patients with and without HIV co-infection. Most active TB disease (ATB) is still diagnosed with sputum smear microscopy, despite its low sensitivity and specificity. This lack of diagnostic rigor contributes to the ongoing underdiagnosis of ~3 million TB cases/year, resulting in continued transmission and poor patient outcomes, especially among PLWH in whom mortality is highest. Our long-term goal is to transform TB diagnosis with a blood test that accurately distinguishes patients with ATB from latent TB infection (LTBI), and other pulmonary disorders regardless of co-infection with HIV. The objective of this study is to demonstrate the analytical and clinical performance of a novel assay for rapid diagnosis and treatment monitoring of ATB that exploits the precise molecular affinity of CRISPR and promiscuous nuclease activity of Cas12a to detect ultra-low concentrations of Mtb cell free DNA (cfDNA) circulating in blood. Preliminary analytical studies indicate assay detection limits of <1fg/µL of Mtb cfDNA in blood and no cross-reactivity with non-tuberculosis mycobacteria (NTM) DNA. Preliminary clinical studies including adults and children with presumptive TB, as well as samples from a diagnostically challenging cohort of symptomatic immunocompromised children living with HIV, demonstrated high diagnostic sensitivity and specificity in a pooled adult and pediatric group (>90%), and high diagnostic sensitivity (85%) in children. We will achieve our proposed objective through the following specific aims: AIM 1: Validate a lateral flow version of the CRISPR-TB assay for low resource settings. Our current working assay requires a PCR step and fluorescence reader. Preliminary studies indicate we can significantly simplify this workflow using isothermal amplification and a novel lateral flow (LF) strip for detection of Mtb cfDNA. We will expand on our pilot studies to validate the existing LF assay using contrived and clinical samples. AIM 2: Define the dynamics of the Mtb cfDNA signal for treatment monitoring. Preliminary studies indicate the Mtb cfDNA signal decreases to extinction after treatment initiation but can have non-linear dynamics. We will define the variability and trajectory of the Mtb cfDNA signal in biobanked serial samples from 34 patients treated for drug resistant TB over a year, and 50 prospectively collected patients with drug susceptible TB treated over six months. AIM 3: Determine the clinical sensitivity and specificity of the CRISPR-TB assay in field settings. We will prospectively collect and test blood samples from 450 patients at risk for TB in TB-endemic field settings with high (South Africa), medium (Pakistan) and low (Mexico) HIV burden.

项目概要 虽然 >90% 的结核病病例是可以治愈的，但治疗取决于完全基于检测的诊断 尽管痰作为诊断手段存在局限性，但患者痰中结核分枝杆菌 (Mtb) 的检测 样本。痰液很难处理，几乎不可能从少杆菌性结核病患者身上获得，例如 艾滋病毒感染者 (PLWH) 和儿童，不能诊断肺外结核患者。一个低 成本，使用血液代替痰进行快速检测可以改变所有患者的结核病诊断， 缩小目前感染艾滋病毒和未感染艾滋病毒的患者之间的诊断差距。最活跃的结核病 尽管其敏感性和特异性较低，但仍通过痰涂片镜检来诊断结核病（ATB）。这 缺乏严格的诊断导致每年约 300 万结核病病例的漏诊，导致 持续传播和患者预后不佳，尤其是死亡率最高的感染者和艾滋病病毒感染者。我们的 长期目标是通过准确区分 ATB 患者的血液检测来改变结核病诊断 潜伏性结核感染 (LTBI) 和其他肺部疾病，无论是否与 HIV 合并感染。这 本研究的目的是证明一种新型快速检测方法的分析和临床性能 利用 CRISPR 的精确分子亲和力对 ATB 进行诊断和治疗监测 Cas12a 的混杂核酸酶活性可检测超低浓度的 Mtb 无细胞 DNA (cfDNA) 在血液中循环。初步分析研究表明，Mtb cfDNA 的检测限<1fg/μL 血液中，与非结核分枝杆菌 (NTM) DNA 没有交叉反应。初步临床研究 包括疑似患有结核病的成人和儿童，以及来自具有诊断挑战性的队列的样本 对感染艾滋病毒的有症状的免疫功能低下儿童进行的研究显示出高诊断敏感性 成人和儿童合并组的特异性（>90%），儿童的诊断敏感性高（85%）。我们 将通过以下具体目标实现我们提出的目标： 目标 1：验证侧向流动版本 用于低资源环境的 CRISPR-TB 检测。我们目前的工作分析需要 PCR 步骤 荧光阅读器。初步研究表明，我们可以使用等温显着简化此工作流程 扩增和用于检测 Mtb cfDNA 的新型侧流 (LF) 条带。我们将扩大试点研究 使用人工样本和临床样本验证现有的 LF 测定。目标 2：定义 Mtb 的动态 用于治疗监测的 cfDNA 信号。初步研究表明 Mtb cfDNA 信号降低至 治疗开始后消退，但可能具有非线性动力学。我们将定义变异性和 34 名接受耐药结核病治疗的患者的生物库系列样本中 Mtb cfDNA 信号的轨迹 一年多以来，前瞻性收集了 50 名药物敏感结核病患者，接受了六个月以上的治疗。目标 3： 确定 CRISPR-TB 测定在现场环境中的临床敏感性和特异性。我们将 前瞻性地收集并检测结核病流行地区 450 名有结核病风险的患者的血液样本 高（南非）、中（巴基斯坦）和低（墨西哥）艾滋病毒负担。