Detecting heteroresistant M. tuberculosis infections using SuperSelective PCR

使用超选择性 PCR 检测异质耐药结核分枝杆菌感染

• 批准号: 9808584
• 负责人: Salvatore A.E. Marras
• 金额: $ 23.41万
• 依托单位: RBHS-NEW JERSEY MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2021-07-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9808584
• 关键词: Agar; Antitubercular Agents; Bacillus (bacterium); Botswana; Cell division; Cessation of life; Clinical; Clinical Research; Collaborations; DNA; Detection; Development; Directly Observed Therapy; Disease Resistance; Drug Kinetics; Drug resistance; Drug resistance in tuberculosis; Environment; Ethambutol; Future; Genes; Genetic; Goals; Growth; Individual; Institutes; Intermediate resistance; Intervention; Isoniazid resistance; Lead; Lung; Methodology; Methods; Molecular; Monitor; Multidrug-Resistant Tuberculosis; Mutation; Mycobacterium tuberculosis; Organizational Objectives; Oryctolagus cuniculus; Pathway interactions; Patients; Pharmaceutical Preparations; Phenotype; Polymerase Chain Reaction; Population; Predisposition; Preparation; Prevention; Pyrazinamide; Research; Resistance; Rifampicin resistance; Rifampin; Sampling; Source; Sputum; Standardization; Tanzania; Testing; Tissues; Treatment Protocols; Treatment outcome; Tuberculosis; Universities; Validation; Vertebral column; Work; World Health Organization; acquired drug resistance; base; digital; drug development; innovation; isoniazid; molecular diagnostics; mutant; novel; novel strategies; pressure; prevent; programs; tool; tuberculosis drugs; tuberculosis treatment

PROJECT SUMMARY A primary objective of tuberculosis (TB) treatment is to prevent the emergence of drug-resistant disease. Standardized treatment regimens that include isoniazid, rifampin, pyrazinamide, and ethambutol are the backbone of the directly observed therapy-short course strategy for global TB control. The emergence of drug resistance during anti-TB therapy presents a major challenge to these efforts. Treatment of multidrug resistant TB requires the use of more toxic and less effective second-line drugs, and thus the prevention of emergence of drug resistance is a key aspect of TB elimination efforts. Our ability to study the development of drug resistance during M. tuberculosis infection is severely limited by insensitive tools to detect rare drug-resistant mutants in mixed populations. Conventional drug susceptibility testing using the agar proportion method defines phenotypic drug resistance at a threshold of 1% colony growth on drug-containing media. Traditionally, heteroresistant TB has been defined as between 1-99% colony growth on drug-containing media, with 100% defined as full resistance. Importantly, M. tuberculosis isolates display molecular heteroresistance when mutant M. tuberculosis DNA- conferring drug resistance- is simultaneously detected alongside wild-type (i.e. drug-susceptible) DNA. However, a major limitation of conventional polymerase chain reaction (PCR) methodology is the inability to amplify rare mutant sequences in a background of abundant wild-type DNA. Our long-term goal is to develop a new tool (“SuperSelective” PCR methods) to detect and monitor heteroresistant M. tuberculosis infections during the course of treatment. The objectives of this application are to develop and test SuperSelective PCR methods for the detection of rare mutations in genes encoding isoniazid or rifampin resistance, despite an abundant background of wild-type M. tuberculosis DNA. This work will directly lead to clinical studies of patient factors that contribute to the emergence of rare drug-resistant mutants during anti-TB treatment. With this new understanding, we can direct interventions towards preventing anti-TB drug resistance.

项目摘要 结核病（TB）治疗的主要目的是防止耐药性疾病的出现。 标准化治疗方案包括异烟肼、利福平、吡嗪酰胺和乙胺丁醇， 直接观察治疗的骨干--全球结核病控制的短期战略。药物的出现 抗结核治疗期间的耐药性对这些努力提出了重大挑战。多药耐药的治疗 结核病需要使用毒性更大、效果更差的二线药物，因此， 耐药性是消除结核病努力的一个关键方面。我们研究抗药性发展的能力 在M.结核病感染严重受限于检测罕见耐药突变体的不敏感工具， 混合人口。使用琼脂比例法的常规药物敏感性试验定义了表型 在含药培养基上1%菌落生长阈值下的耐药性。传统上，异源耐药结核病 定义为含药培养基上菌落生长在1-99%之间，100%定义为完全生长 阻力重要的是，M。当突变的M.结核 赋予耐药性的DNA与野生型（即药物敏感）DNA同时检测。 然而，常规聚合酶链式反应（PCR）方法的主要限制是不能 在大量野生型DNA的背景下扩增罕见的突变序列。我们的长期目标是发展一个 新的工具（“SuperSelective”PCR方法）检测和监测异源耐药M。结核病感染期间 治疗过程本申请的目的是开发和测试SuperSelective PCR方法 用于检测编码异烟肼或利福平耐药的基因中的罕见突变， 野生型M.结核病DNA这项工作将直接导致对患者因素的临床研究， 在抗结核治疗过程中，导致罕见的耐药突变体的出现。有了这个新 了解这些情况后，我们可以将干预措施导向预防抗结核药物耐药性。