Automated Sequencing for Culture-free Diagnosis of Drug Resistant TB

用于耐药结核病免培养诊断的自动测序

• 批准号: 10662791
• 负责人: Rebecca Elizabeth Colman
• 金额: $ 76.42万
• 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: 2023-04-17 至 2028-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10662791
• 关键词: Adherence; Adoption; Amikacin; Biological Assay; Bypass; Catalogs; Characteristics; Clinical; Collaborations; Companions; Complex; Development; Diagnosis; Diagnostic; Dimensions; Drug resistance; Drug resistance in tuberculosis; Effectiveness; Ensure; Foundations; Frequencies; Funding; Gene Targeting; Genotype; Goals; Grant; Hand; Health Personnel; Hour; Incidence; India; Intermediate resistance; Investments; Knowledge; Laboratories; Leadership; Linezolid; Moldova; Molecular Diagnostic Testing; Molecular Target; Moxifloxacin; Multi-Drug Resistance; Mutation; Mutation Detection; Mycobacterium tuberculosis; Nucleic Acid Amplification Tests; Oral; Patient Care; Patients; Performance; Pharmaceutical Preparations; Phenotype; Predisposition; Process; Pyrazinamide; Recommendation; Reference Standards; Regimen; Resistance; Resistance profile; Resource-limited setting; Rifampicin resistance; Rifampin; Risk; Running; Sampling; Solid; South Africa; Specificity; Sputum; System; Testing; Time; Translating; Treatment Protocols; Tuberculosis; Tuberculosis diagnosis; United States National Institutes of Health; Update; Work; World Health Organization; accurate diagnosis; blind; cost; detection limit; drug repurposing; drug testing; field study; human error; instrument; isoniazid; molecular diagnostics; molecular drug target; molecular recognition; next generation sequencing; novel; novel therapeutics; point of care; preservation; prevent; profiles in patients; rapid test; skills; solid state; tool; treatment organization; tuberculosis drugs; tuberculosis treatment; uptake

PROJECT SUMMARY While transformative, all-oral treatment regimens have recently been endorsed by World Health Organization (WHO) for the treatment of drug resistant TB (DR -TB), the efficacy of these new regimens is dependent on prior knowledge of the susceptibility profile of key drugs before treatment initiation. Companion molecular diagnostics for drug susceptibility testing (DST) of the new and repurposed anti-tuberculosis drugs is the most likely path to achieving practical and timely DST for these new regimens. However, there are currently no commercially available molecular diagnostics to detect mutations conferring resistance to bedaquiline, pretomanid, linezolid or delamanid, nor is there an easy developmental path forward using existing nucleic acid amplification test (NAAT) approaches given the complexity of the molecular targets for these drugs. Culture- free, next generation sequencing (NGS) has the greatest potential for delivering a comprehensive diagnostic solution, but existing workflows are highly complex, expensive, and rely on highly skilled staff to run them. To achieve broad adoption of NGS approaches, particularly in low resource settings, these workflows must be simplified substantially to reduce both their complexity and cost. Our goals for the proposed study are to bring culture-free NGS closer to patient care by simplifying NGS workflows for DR-TB diagnosis to the point that NGS can be run mostly hands-free by any laboratorian who can run a NAAT and to reduce costs by removing several enzymatic processes. Our objective is to demonstrate that automated, single amplification sequencing (ASAS) can be used to accurately diagnose resistance to all legacy and new/repurposed drugs for which there are recognized molecular targets (i.e., isoniazid, rifampicin, pyrazinamide, amikacin, moxifloxacin, bedaquiline, clofazimine, linezolid, and delamanid). We will achieve this objective by completing the following three aims; Aim 1) Expand with additional gene targets an existing single amplification targeted NGS assay and describe the assay performance; Aim 2) Integrate an existing Akonni sputum extraction/PCR workstation with our novel single amplification sequencing assay and sequence on Illumina iSeq100; and Aim 3) Evaluate the accuracy of the ASAS solution for detection of drug resistance against a phenotypic and genotypic reference standard in sputum samples from patients at risk for DR-TB under field conditions at clinical laboratories in India, South Africa, and Moldova. The results of this project will demonstrate key characteristics of the ASAS workflow and will provide a solid developmental foundation for the application of this tool in clinical settings, reducing cost and time-to-result for comprehensive DST without culturing.

项目概要 虽然具有变革性，但全口服治疗方案最近已获得世界卫生组织的认可 (WHO) 对于治疗耐药结核病 (DR -TB)，这些新疗法的疗效取决于 在开始治疗之前预先了解关键药物的敏感性概况。伴侣分子 新的和重新利用的抗结核药物的药敏试验（DST）的诊断是最重要的 这些新方案实现实用且及时的药敏试验的可能途径。不过，目前还没有 商业上可用的分子诊断来检测赋予贝达喹啉耐药性的突变， pretomanid、利奈唑胺或德拉马尼，使用现有核酸也没有简单的开发路径 考虑到这些药物分子靶点的复杂性，扩增测试（NAAT）方法。文化- 免费的下一代测序 (NGS) 在提供全面诊断方面具有最大潜力 解决方案，但现有的工作流程非常复杂、昂贵，并且需要高技能的员工来运行。到 要实现 NGS 方法的广泛采用，特别是在资源匮乏的环境中，这些工作流程必须 大幅简化以降低其复杂性和成本。我们拟议研究的目标是 通过简化耐药结核病诊断的 NGS 工作流程，使无培养 NGS 更贴近患者护理 NGS 几乎可以由任何能够运行 NAAT 的实验室人员免提运行，并通过消除 几个酶促过程。我们的目标是证明自动化的单扩增测序 (ASAS) 可用于准确诊断对所有传统药物和新药/再利用药物的耐药性 是公认的分子靶标（即异烟肼、利福平、吡嗪酰胺、阿米卡星、莫西沙星、贝达喹啉、 氯法齐明、利奈唑胺和德拉马尼）。我们将通过完成以下三个目标来实现这一目标； 目标 1) 用额外的基因靶标扩展现有的单扩增靶向 NGS 测定并描述 测定性能；目标 2) 将现有的 Akonni 痰液提取/PCR 工作站与我们的新型工作站集成 单扩增测序分析和 Illumina iSeq100 上的测序；和目标 3) 评估准确性 ASAS 解决方案用于检测针对表型和基因型参考标准的耐药性 印度南部临床实验室在现场条件下采集的具有耐药结核病风险的患者的痰样本 非洲、摩尔多瓦。该项目的结果将展示 ASAS 工作流程的关键特征以及 将为该工具在临床环境中的应用提供坚实的发展基础，降低成本 以及无需培养的全面 DST 的出结果时间。