Development of a Next-generation Rapid Phenotypic Assay for Drug-Resistant M. tuberculosis

项目摘要 常规培养型分枝杆菌的周转时间长，需要专门的实验室 结核病药物敏感性测试（DST）定期提交耐药结核病（DR- 结核病）到几个月的潜在无效治疗。目前的商业分子结核病（TB）测试可以 快速识别耐药结核病，但仅限于测试对少数药物的耐药性，不包括任何新药。 理论上，下一代测序（NGS）可以全面识别耐药谱，但目前 在高负担、低资源的环境中常规使用，以及对耐药性的了解， 突变还不完全。为了解决这一障碍，将开发一种基于荧光的表型测定法 以高特异性鉴定对所谓的“BPaLM”方案中所含药物的耐药性， 标本目前K43应用的总体目标是推进一种新的表型解决方案， 高负担环境下耐药结核病的临床管理。K43提案的目标是：1）提供 关键的职业发展培训和2）推进和评估快速表型工作流程， 在高通量结核病实验室中的实践时间。样本收集和技术人员从现有的 NIH资助的R 01（R 01 AI 153213，PI：John Metcalfe，该提案的美国主要导师）随机分组 对照试验评价与台式NGS测序相关的程序性结果（“TS艾略特 研究”）。斯泰伦博斯大学和该股的杰出教授罗布·沃伦博士 南非医学研究理事会结核病研究中心主任将担任LMIC 导师梅特卡夫博士和沃伦博士在分子流行病学、耐药结核病、 和诊断测试的发展。该项目将有以下具体目标：1）推进荧光- 作为一种快速、高通量pDST，2）进行初步诊断准确性评估， 直接检测临床TB标本中的表型耐药的测定。本研究快速、全面 将建立DST方法，该方法将继续为检测新的和 重新利用的毒品在这个K43资助的辅导计划结束时，申请人预计将能够领导 在南非的低资源环境中独立开展研究项目。在未来的研究项目中， 根据目前项目产生的初步数据，她将能够回答与药物有关的关键问题， 耐药结核病。

PROJECT SUMMARY The long turnaround time and need for specialised laboratories for conventional culture-based Mycobacterium tuberculosis drug-susceptibility testing (DST) regularly commits patients with drug-resistant tuberculosis (DR- TB) to months of potentially ineffective treatments. Current commercial molecular tuberculosis (TB) tests can rapidly identify DR-TB but are limited to testing resistance to a small number of drugs, excluding any new drugs. In theory, next-generation sequencing (NGS) could comprehensively identify resistance profiles, but is currently prohibitively expensive for routine use in high-burden, low-resource settings, and knowledge on drug-resistance mutations is still incomplete. To address this barrier, a fluorescence-based phenotypic assay will be developed to identify with high specificity resistance to drugs contained in the so-called “BPaLM” regimen, in clinical specimens. The overall goal of the current K43 application is to advance a novel phenotypic solution to transform the clinical management of DR-TB in high burden settings. The objectives of the K43 proposal are to 1) provide critical career advancing training and 2) advance and evaluate a rapid phenotypic workflow requiring minimal hands-on time within a high throughput TB laboratory. The sample collections and technical staff from an existing NIH-funded R01 (R01AI153213, PI: John Metcalfe, US primary mentor on this proposal) cluster randomized controlled trial evaluating the programmatic outcomes associated with benchtop NGS sequencing (the “TS Eliot study”) will be leveraged. Dr. Rob Warren, Distinguished Professor at Stellenbosch University and the Unit Director of the South African Medical Research Council Centre for Tuberculosis Research will serve as the LMIC mentor. Both Dr. Metcalfe and Dr. Warren have vast experience in molecular epidemiology, drug-resistant TB, and development of diagnostic tests. The project will have the following specific aims: 1) Advance a fluorescence- based assay as a rapid, high-throughput pDST, and 2) Perform preliminary diagnostic accuracy evaluation of the assay to detect phenotypic resistance directly in clinical TB specimens. In this study rapid, comprehensive DST methods will be established, which will continue to inform molecular assays for testing of new and repurposed drugs. At the end of this K43-funded mentored program, the applicant is expected to be able to lead a research project independently in the low-resource setting of South Africa. In future research projects based on the preliminary data generated in the current project, she will be able to answer key questions related to drug- resistant TB.