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（M.tuberculosis）是结核病（TB）的病原体;尽管是可治疗的结核病，但它是结核病的病原体。 结核病仍然是全球发病率和死亡率的主要原因。这在很大程度上是由 抗分枝杆菌药物耐药性的空前增加。约有1000万人患结核病 2019年，新增利福平耐药病例50万例，其中78%的利福平病例为多药耐药病例 耐药;危及最有效的一线药物。根据世卫组织最近的指导意见， 这些患者用包含贝达喹啉和其它新的抗肿瘤药物的全口服短程治疗来治疗， 剂.新出现的对这些新药的耐药性是扩大缩短的，全口服， 耐药结核病治疗方案。目前的诊断测定不包括对这些新的耐药性检测。 药物，主要是由于我们不完全了解潜在的遗传机制介导 阻力因此，需要结合基因型和表型技术来监测 对这些新药的抵抗力。报告分枝杆菌噬菌体测定代表了一种有前途的方法， 在不依赖复制的系统中导出表型数据，保持高灵敏度，同时减少 与基于增长的方法相伴的结果时间。我们创造了一个TM 4噬菌体载体， 纳米荧光素酶（Nluc）报告酶的盒。在一定范围的细胞上测试TM 4-nluc噬菌体。 营养缺陷型和毒性临床M.结核菌株，评估细胞检测限和相容性 对多种抗分枝杆菌药物进行药敏试验。我们发现， 在预培养阶段，我们可以确定与WHO认可的治疗一致的药物敏感性 一系列一线和二线药物以及新型和再用途药物的浓度 （贝达喹啉、pretomanid和利奈唑胺）。本研究的总体目标是验证 纳米荧光素酶噬菌体用于检测新的抗分枝杆菌药物的耐药性，将在 两个目标。目的1将建立该检测方法在少菌条件下和混合感染中的实用性。AIM2 将推导出临界浓度附近已确定的最小抑制值之间的相关性， 纳米荧光素酶荧光强度测量，并验证这些临界点，在一个既定的前景 耐多药结核病治疗的队列研究。探索性子目标将试验最低限度处理的痰液，而不是 MTB培养用于MTB检测和药物敏感性测试。拟议的工作将提供额外的 在TM 4-nluc噬菌体系统上进行验证，并提供定量表型参考方法， 补充了用于诊断和抗生素敏感性测试的基因型方法。

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.