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.

项目摘要 虽然世界卫生组织最近认可了变革性的全口处理方案 （WHO）为了治疗耐药性结核（DR -TB），这些新方案的功效取决于 在治疗开始之前，对关键药物的易感性概况的先验知识。伴侣分子 新的和再利用的抗结核药物的药物敏感性测试（DST）的诊断是最多的 为这些新方案实现实用和及时的DST的途径。但是，目前没有 市售的分子诊断，以检测突变赋予对贝喹的耐药性， pipomanid，linezolid或delamanid，也没有现有核酸的易于发展路径 鉴于这些药物的分子靶标的复杂性，放大测试（NAAT）方法。文化- 免费的下一代测序（NGS）具有提供全面诊断的最大潜力 解决方案，但现有的工作流程非常复杂，昂贵，并且依靠高技能的员工来运行它们。到 通过广泛采用NGS方法，尤其是在低资源设置中，这些工作流程必须是 大大简化以降低其复杂性和成本。我们提出的研究的目标是带来 通过简化NGS工作流以DR-TB诊断，无文化的NG靠近患者护理 NG可以由任何可以运行NAAT并通过删除来降低成本的实验室大部分运行 几个酶促过程。我们的目标是证明自动化的单个扩增测序 （ASA）可用于准确诊断对所有遗产和新的/重新利用的药物的抗性 是公认的分子靶标（即异念珠菌，利福平，吡嗪酰胺，amikacin，moxifloxacin，bedaquiline，bedaquiline， 氯富齐明，linezolid和delamanid）。我们将通过完成以下三个目标来实现这一目标； 目标1）用其他基因靶向现有的单个放大针对NGS分析的基因扩展并描述 测定性能；目标2）将现有的Akonni痰液提取/PCR工作站与我们的小说集成 Illumina ISEQ100上的单个扩增测序测定和序列；目标3）评估的准确性 ASAS检测耐药性对表型和基因型参考标准的耐药性 在印度南部的临床实验室，来自现场条件下有DR-TB风险的患者的痰液样品 非洲和摩尔多瓦。该项目的结果将展示ASAS工作流程的关键特征和 将为在临床环境中应用该工具提供坚实的发展基础，从而降低成本 以及不进行培养的全面DST的时间。