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

项目总结 漫长的周转时间和对传统培养分枝杆菌专业实验室的需求 结核病药敏试验(DST)定期将耐药结核病(DR- 结核病)到数月的潜在无效治疗。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。 从理论上讲，下一代测序(NGS)可以全面识别耐药性图谱，但目前 对于在高负担、低资源环境下的常规使用来说，费用高得令人望而却步，而且还需要了解耐药性 突变仍然不完整。为了解决这一障碍，将开发一种基于荧光的表型分析 在临床上鉴定对所谓的“BPaLM”方案中所含药物具有高特异性抗药性 标本。目前K43应用的总体目标是提出一种新的表型解决方案来转换 高负荷环境下耐药结核病的临床处理。K43提案的目标是：1)提供 关键的职业发展培训和2)推进和评估快速的表现型工作流程，需要最低限度的 在高吞吐量结核病实验室中的实际操作时间。现有的样本收集和技术人员 NIH资助的R01(R01AI153213，PI：John Metcalfe，这项提案的美国主要导师)随机分组 评估与台式NGS测序相关的程序性结果的对照试验(“TS Eliot 研究“)将被杠杆化。Rob Warren博士，StellenBosch大学和该股特聘教授 南非医学研究理事会结核病研究中心主任将担任LMIC 门托。梅特卡夫博士和沃伦博士在分子流行病学、耐药结核病、 以及诊断测试的发展。该项目将有以下具体目标：1)推进荧光-- 基于快速、高通量PDST的检测，以及2)执行初步诊断准确性评估 直接检测临床结核标本表型耐药的方法。在这项研究中，快速、全面 将建立DST方法，该方法将继续为检测新的和 改变用途的毒品。在K43资助的指导计划结束时，申请者将能够领导 在南非资源匮乏的环境下独立开展的研究项目。在未来的研究项目中基于 根据当前项目产生的初步数据，她将能够回答与药物相关的关键问题- 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.