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 虽然具有变革性，但全口服治疗方案最近得到了世界卫生组织的认可 (WHO)对于耐药结核病（DR-TB）的治疗，这些新方案的疗效取决于 在治疗开始之前，对关键药物的敏感性特征有先验知识。伴随分子 新的和重新使用的抗结核药物的药物敏感性测试（DST）的诊断是最 可能的路径，以实现实际和及时的DST为这些新的方案。然而，目前没有 可商购的分子诊断以检测赋予贝达喹啉抗性的突变， pretomanid，利奈唑胺或delamanid，也没有一个简单的开发途径，使用现有的核酸 考虑到这些药物的分子靶点的复杂性，使用扩增试验（NAAT）方法是可行的。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，并依赖于高技能的员工来运行它们。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 大大简化，以降低其复杂性和成本。我们的研究目标是 通过简化耐药结核病诊断的NGS工作流程， NGS可以由任何可以运行NAAT的实验室人员进行大部分免提操作， 几种酶促过程。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遗留药物和新/再用途药物的耐药性， 是公认的分子靶标（即，异烟肼，利福平，吡嗪酰胺，阿米卡星，阿氟沙星，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： 目的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 目的2）将现有的Akonni痰液提取/PCR工作站与我们的新的 在Illumina iSeq 100上进行单扩增测序测定和测序;以及目的3）评估 用于检测表型和基因型参比标准品耐药性的ASAS溶液 在印度、南 非洲和摩尔多瓦。该项目的结果将展示ASAS工作流程的主要特征， 将为该工具在临床环境中的应用提供坚实的发展基础， 以及无需培养的综合DST的结果时间。