Rapid phenotypic detection of complex and emergent TB drug-resistance using a next-generation nanoluciferase reporter phage

项目摘要 结核分枝杆菌(结核分支杆菌)是结核病(TB)的病原体；尽管是一种可以治疗的 结核病仍然是全球发病率和死亡率的主要原因。这大体上是由 无与伦比的抗分枝杆菌耐药性增加。大约有1000万人患上了结核病 2019年新增50万例利福平耐药病例，78%为多药耐药病例 耐药；危及最有效的一线药物。根据世卫组织最近的指导方针，大多数 这些患者接受全口服短程治疗，联合贝达奎兰和其他新型药物。 探员们。对这些新药新出现的耐药性是对缩短的、全口服的、 耐药结核病的治疗方案。目前的诊断分析没有将耐药性检测纳入到这些新的 药物，很大程度上是由于我们对潜在的遗传机制的不完全了解 抵抗。因此，需要结合基因分型和表型技术来监测 对这些新药的抗药性。报道：分支杆菌噬菌体分析代表了一种很有希望的方法 在独立于复制的系统中派生表型数据，在保持高敏感度的同时减少 是时候用基于增长的方法来实现结果了。我们创造了一个TM4噬菌体载体，它能传递一个基因 纳米纤维素酶(Nluc)报告酶的盒。TM4-nluc噬菌体在一系列 营养缺乏性和强毒的临床结核分枝杆菌菌株，评估细胞检测极限和配伍 对多种抗分枝杆菌药物进行药敏试验。我们发现在以下情况下 培养前阶段，我们可以确定与世卫组织认可的治疗相一致的药物敏感性 集中到一系列一线和二线药物以及新的和重新定位的药物 (贝达奎林、前胡萝卜素和利奈唑胺)。这项研究的总体目标是验证 用于检测新型抗真菌药物耐药性的纳米纤维素酶噬菌体将于#年完成 两个目标。目的1建立该检测方法在少菌型和混合感染条件下的实用性。AIM2 将推导出临界浓度附近已建立的最小抑制值与 纳米荧光素酶荧光强度测量，并在已建立的预期中验证这些切入点 耐多药结核病治疗的队列研究。一个探索性的子目标将试验最低限度的痰处理，而不是 结核分枝杆菌培养用于结核分枝杆菌检测和药敏试验。拟议的工作将提供额外的 对TM4-nluc噬菌体系统的验证，并提供了一种定量的表型参考方法和 补充用于诊断和药敏试验的基因分型方法。

Project Summary Mycobacterium tuberculosis (M.tuberculosis) is the causative agent of tuberculosis (TB); although a treatable disease TB remains a leading cause of morbidity and mortality globally. This in large is driven by the unparalleled increase in anti- mycobacterial drug resistance. Approximately 10 million people fell ill with TB in 2019, where 500 000 were new rifampicin resistant cases and 78% of rifampicin cases were multi-drug resistant; compromising the most effective first-line drugs. In line with recent WHO guidance, the majority of these patients are treated with all-oral short-course treatment incorporating bedaquiline and other novel agents. Emerging drug resistance to these novel drugs is a critical threat to expansion of shortened, all-oral, DR-TB treatment regimens. Current diagnostic assays do not incorporate resistance detection to these new drugs, largely as a result of our incomplete understanding of the underlying genetic mechanisms mediating resistance. As a result, a combination of genotypic and phenotypic techniques is required to monitor resistance to these new drugs. Reporter mycobacteriophage assays represent a promising approach for deriving phenotypic data in a replication-independent system, maintaining high sensitivity while reducing the time to result attendant with growth-based methods. We created a TM4 phage vector that delivers a gene cassette of the nanoluciferase (Nluc) reporter enzyme. The TM4-nluc phage was tested on a range of auxotrophic and virulent clinical M. tuberculosis strains, assessing cellular limit of detection and compatibility with drug susceptibility testing on a wide range of antimycobacterial drugs. We found that following a preculture period, we could identify drug susceptibility consistent with WHO-endorsed treatment concentrations to a suite of first-line and second-line drugs as well as novel and repurposed drugs (bedaquiline, pretomanid, and linezolid). The overall objective of this research is to validate the nanoluciferase phage for detection of resistance to novel anti-mycobaterial agents, will be accomplished in two Aims. Aim 1 will establish the utility of the assay in paucibacillary conditions and mixed infection. Aim2 will derive correlations between established minimum inhibitory values around critical concentrations and nanoluciferase fluorescence intensity measures, and validate these cut points in a established prospective cohort study of MDR-TB treatment. An exploratory sub Aim will trial minimally processed sputum rather than MTB culture for MTB detection and drug susceptibility testing. The proposed work will provide additional validation on the TM4-nluc phage system and offer a quantitative phenotypic reference method and complement to genotypic methods for diagnosis and antibiotic susceptibility testing.