An RNA Nanosensor for the Diagnosis of Antibiotic Resistance in M. Tuberculosis

用于诊断结核分枝杆菌抗生素耐药性的 RNA 纳米传感器

• 批准号: 10670613
• 负责人: Maha Farhat
• 金额: $ 77.06万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-13 至 2028-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10670613
• 关键词: Address; Antibiotic Resistance; Antibiotic susceptibility; Antibiotics; Antitubercular Agents; Antitubercular Antibiotics; Archives; Benchmarking; Biohazardous Substance; Bioinformatics; Biological Assay; COVID-19 diagnosis; Cells; Cellular Stress; Ciprofloxacin; Clinic; Clinical; Country; Culture Media; DNA; Data; Detection; Development; Diagnosis; Diagnostic; Diagnostic Sensitivity; Diagnostic Specificity; Diagnostic tests; Dose; Drug Exposure; Drug resistance; Evaluation; Evolution; Experimental Designs; Exposure to; Fluoroquinolones; Foundations; Genes; Genetic; Genetic Transcription; Genome; Geography; Growth; Heat-Shock Response; Hospitals; Hour; In Vitro; Incubated; Knowledge; Linezolid; Measurement; Messenger RNA; Methods; Molecular Biology; Monitor; Moxifloxacin; Multidrug-Resistant Tuberculosis; Mutation; Mycobacterium tuberculosis; Newly Diagnosed; Participant; Patients; Performance; Pharmaceutical Preparations; Phase; Phenotype; Positioning Attribute; Predisposition; Public Health; Publishing; Pyrazinamide; RNA; RNA Probes; Rapid diagnostics; Regimen; Research; Resistance; Resistance profile; Ribosomal RNA; Rifampicin resistance; Rifampin; Risk; Sampling; Sensitivity and Specificity; Signal Transduction; Site; South Africa; South African; Specimen; Sputum; Streptomycin; Technology; Testing; Time; Transcript; Treatment outcome; Tuberculosis; Untranslated RNA; Work; accurate diagnosis; accurate diagnostics; bactericide; biobank; cost; design; detection limit; diagnostic strategy; drug mechanism; fluoroquinolone resistance; genetic resistance; genome sequencing; innovation; isoniazid; mycobacterial; nano-string; nanosensors; novel; novel strategies; pathogen; point of care; predictive signature; pressure; prospective; prototype; rapid test; recruit; resistance mechanism; resistance mutation; response; success; targeted sequencing; transcriptome sequencing; transcriptomics; tuberculosis drugs; tuberculosis treatment; whole genome

Project Summary: Mycobacterium tuberculosis (Mtb) has among the highest resistance rates of any pathogen globally, but Mtb resistance remains the most challenging to diagnose due to its very slow growth in in vitro culture and culture’s inordinate cost and biohazard. Rapid and accurate alternative diagnostic approaches are urgently needed. DNA based tests are available for detecting resistance to a few drugs but are limited by their need for extensive knowledge of DNA resistance determinants for interpretation, and concerns about limit of detection at the point- of-care. For novel antibiotics entering clinical use, genetic resistance determinants are particularly poorly understood and may be too dispersed across the genome to lend themselves to targeted sequencing and delays in understanding mechanisms may miss an opportunity to avoid more widespread resistance. A work around is the development of a functional or phenotypic assay that circumvents the need to target specific genetic mechanisms, however there is no such rapid assay currently commercially available largely because these assays have traditionally relied on observed bacterial growth in vitro under antibiotic pressure. Leveraging considerable preliminary data from our group and others, we propose the development of an innovative ‘functional’ RNA-based assay for resistance diagnosis in Mtb. We anticipate increasing sensitivity of resistance detection, expanding the numbers of drugs to which resistance can be detected, while significantly shortening time-to-result. Specifically, in this early phase development proposal we will identify key assay parameters that maximize the Mtb RNA susceptibility signal to five key agents in multi-drug resistant tuberculosis treatment (bedaquiline, pretomanid, linezolid, moxifloxacin and pyrazinamide) using RNA sequencing in a factorial design of experiments assessing drug exposure dose and exposure duration. In addition, we will study the effect of key clinical variables including genetic resistance mechanism, background lineage (Aim 1). In Aim 2 we will work with NanoString technologies to develop hybridization probe sets (that we call nanosensors for short) to target antibiotic responsive and control genes in two iterative phases. This will be followed by assay performance assessment in vitro, and on sputum from participants newly diagnosed with rifampicin resistant TB before and after incubation in culture media. This work will build a strong foundation for an innovative resistance diagnostic that addresses an unmet need.

项目摘要： 结核分枝杆菌（MTB）在全球任何病原体中具有最高的抗药性率，但MTB 由于其体外培养和文化的增长非常缓慢，抵抗仍然是诊断的最大挑战 过分的成本和生物危害。迫切需要快速准确的替代诊断方法。脱氧核糖核酸 可用于检测几种药物的抗性，但受到广泛需求的限制 了解DNA耐药性决定者的解释，并担心该点检测极限 保养。对于进入临床用途的新型抗生素，遗传性抗性确定剂特别差 理解并可能在基因组中分散，以至于将自己放在有针对性的测序和延迟中 在理解机制时，可能会错过避免更多宽度阻力的机会。周围的工作是 开发功能或表型测定法，该测定法规定了针对特定通用的需求 但是，机制，但是目前没有这样的快速组装在很大程度上可用 传统上，测定在抗生素压力下观察到的细菌在体外观察到的生长。利用 我们小组和其他人的大量初步数据，我们提出了创新的开发 基于“功能”的RNA基于MTB的耐药性诊断。我们预计阻力的敏感性会提高 检测，扩大可以检测到抗性的药物的数量，同时显着缩短 时间到回顾。特别是，在这个早期开发建议中，我们将确定关键的测定参数 在多药耐药性结核病治疗中，最大化MTB RNA易感性信号对五个关键药物 （Bedaquiline，Pipomanid，LineZolid，Moxifloxacin和吡嗪酰胺）在阶乘设计中使用RNA测序 评估药物暴露剂量和暴露时间的实验。此外，我们将研究关键的效果 临床变量，包括遗传抗性机制，背景谱系（AIM 1）。在目标2中，我们将工作 借助纳米串技术来开发杂交探针集（我们称为纳米传感器简称）以靶向 在两个迭代阶段中的抗生素反应和控制基因。随后将进行测定性能 在体外评估，以及来自新诊断为利福平抗性结核病的参与者的痰液 在培养基中孵化后。这项工作将为创新的阻力诊断奠定坚实的基础 这解决了未满足的需求。