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

基本信息

  • 批准号:
    10620065
  • 负责人:
  • 金额:
    $ 79.52万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 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

项目摘要

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.
项目摘要 虽然90%的结核病病例是可以治愈的,但治疗完全依赖于基于检测的诊断 患者痰中结核分枝杆菌(Mtb)的存在,尽管痰作为诊断的局限性 样本。痰很难处理,几乎不可能从少杆菌结核病患者那里获得,例如 艾滋病毒携带者(PLWH)和儿童,对肺外结核病患者不能诊断。一个低点- 成本,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, 缩小目前艾滋病毒合并感染患者和未合并感染患者之间的诊断差距。最活跃的结核病 尽管痰涂片镜检的敏感性和特异度较低,但仍可诊断该病。这 缺乏诊断的严谨性导致每年约300万结核病病例持续诊断不足,导致 持续传播和糟糕的患者结局,特别是在死亡率最高的PLWH中。我们的 长期目标是通过血液测试准确区分ATB患者来改变结核病的诊断 不受潜在结核病感染(LTBI)和其他肺部疾病的影响,而不考虑与艾滋病毒的混合感染。这个 这项研究的目的是证明一种新的快速检测方法的分析和临床性能。 利用CRISPR和CRISPR的精确分子亲和力进行ATB的诊断和治疗监测 Cas12a杂合核酸酶活性检测超低浓度结核分枝杆菌游离DNA(CfDNA) 在血液中循环。初步分析研究表明,Mtb cfDNA的检出限为~lt;1fg/µL。 血液和非结核分枝杆菌(NTM)DNA无交叉反应。初步临床研究 包括成人和患有推定结核病的儿童,以及来自具有诊断挑战性的队列的样本 有症状的免疫功能低下的艾滋病毒携带者,表现出高度的诊断敏感性和 在成人和儿童组中的特异性(90%),以及在儿童中的高诊断敏感性(85%)。我们 将通过以下具体目标实现我们提议的目标:目标1:验证横向流动版本 用于低资源环境的CRISPR-TB检测。我们目前的检测需要一个聚合酶链式反应步骤 荧光读取器。初步研究表明,使用等温技术可以大大简化这一工作流程 一种用于检测结核分枝杆菌cfDNA的新型横向流动(LF)条带。我们将扩大我们的试点研究 用人工和临床标本验证现有的LF检测方法。目标2:确定MTB的动态 用于治疗监测的cfDNA信号。初步研究表明,结核分枝杆菌cfdna信号减少到 在治疗开始后消退,但可以有非线性动力学。我们将定义可变性和 34例耐药结核病患者的生物库系列样本中结核分枝杆菌cfDNA信号的轨迹 一年多来,50名预期收集的药物敏感结核病患者接受了六个月的治疗。目标3: 在现场环境下确定CRISPR-TB检测的临床敏感性和特异性。我们会 在结核病流行现场,前瞻性地收集和检测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。

项目成果

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Richard S Garfein其他文献

Richard S Garfein的其他文献

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{{ truncateString('Richard S Garfein', 18)}}的其他基金

A Novel Point-of-Care Assay for Diagnosing TB from Blood using Exosomes
使用外泌体从血液中诊断结核病的新型护理点检测
  • 批准号:
    10596302
  • 财政年份:
    2022
  • 资助金额:
    $ 79.52万
  • 项目类别:
Cell Phone Video Directly Observed Therapy to Monitor Short Course LTBI Treatment
手机视频直接观察治疗监测短期 LTBI 治疗
  • 批准号:
    8848622
  • 财政年份:
    2015
  • 资助金额:
    $ 79.52万
  • 项目类别:
Cell Phone Video Directly Observed Therapy to Monitor Short Course LTBI Treatment
手机视频直接观察治疗监测短期 LTBI 治疗
  • 批准号:
    9221964
  • 财政年份:
    2015
  • 资助金额:
    $ 79.52万
  • 项目类别:
Drug tourism to Mexico: Impact of Mexicos new drug law on HIV-HCV-TB in US IDUs
墨西哥毒品旅游:墨西哥新毒品法对美国注射吸毒者 HIV-HCV-TB 的影响
  • 批准号:
    8452218
  • 财政年份:
    2011
  • 资助金额:
    $ 79.52万
  • 项目类别:
Drug tourism to Mexico: Impact of Mexicos new drug law on HIV-HCV-TB in US IDUs
墨西哥毒品旅游:墨西哥新毒品法对美国注射吸毒者 HIV-HCV-TB 的影响
  • 批准号:
    8477164
  • 财政年份:
    2011
  • 资助金额:
    $ 79.52万
  • 项目类别:
Drug tourism to Mexico: Impact of Mexicos new drug law on HIV-HCV-TB in US IDUs
墨西哥毒品旅游:墨西哥新毒品法对美国注射吸毒者 HIV-HCV-TB 的影响
  • 批准号:
    8662547
  • 财政年份:
    2011
  • 资助金额:
    $ 79.52万
  • 项目类别:
Drug tourism to Mexico: Impact of Mexicos new drug law on HIV-HCV-TB in US IDUs
墨西哥毒品旅游:墨西哥新毒品法对美国注射吸毒者 HIV-HCV-TB 的影响
  • 批准号:
    8304207
  • 财政年份:
    2011
  • 资助金额:
    $ 79.52万
  • 项目类别:
Drug tourism to Mexico: Impact of Mexicos new drug law on HIV-HCV-TB in US IDUs
墨西哥毒品旅游:墨西哥新毒品法对美国注射吸毒者 HIV-HCV-TB 的影响
  • 批准号:
    8141202
  • 财政年份:
    2011
  • 资助金额:
    $ 79.52万
  • 项目类别:
Drug tourism to Mexico: Impact of Mexicos new drug law on HIV-HCV-TB in US IDUs
墨西哥毒品旅游:墨西哥新毒品法对美国注射吸毒者 HIV-HCV-TB 的影响
  • 批准号:
    8685629
  • 财政年份:
    2011
  • 资助金额:
    $ 79.52万
  • 项目类别:
Mobile Phone-Based Video Directly Observed Therapy for Tuberculosis
结核病手机视频直观治疗
  • 批准号:
    7878280
  • 财政年份:
    2010
  • 资助金额:
    $ 79.52万
  • 项目类别:

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