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.

项目概要： 结核分枝杆菌（Mtb）是全球所有病原体中耐药率最高的，但Mtb 耐药性仍然是最具挑战性的诊断，因为它在体外培养中生长非常缓慢， 成本过高和生物危害。迫切需要快速准确的替代诊断方法。DNA 基础测试可用于检测对一些药物的耐药性，但受限于它们需要广泛的 了解用于解释的DNA耐药决定因素，并关注此时的检测限- 护理。对于进入临床使用的新型抗生素， 这些基因可能过于分散，无法进行有针对性的测序， 在了解机制可能会错过一个机会，以避免更广泛的阻力。一种变通办法是 开发一种功能或表型测定法， 然而，目前还没有这种快速的商业上可获得的测定，这主要是因为这些 传统上，测定依赖于在抗生素压力下观察到的体外细菌生长。利用 根据我们小组和其他人提供的大量初步数据，我们建议开发一种创新的 用于结核分枝杆菌耐药性诊断的基于“功能性”RNA的测定我们预计耐药性的敏感性会增加 检测，扩大可以检测到耐药性的药物数量，同时大大缩短 时间到结果。具体来说，在这个早期开发提案中，我们将确定关键的检测参数， 最大化Mtb RNA敏感性信号，用于耐多药结核病治疗中的五种关键药物 （贝达喹啉、pretomanid、利奈唑胺、阿氟沙星和吡嗪酰胺），采用析因设计中的RNA测序 评估药物暴露剂量和暴露时间的实验。此外，我们将研究关键的影响， 临床变量包括遗传耐药机制、背景谱系（Aim 1）。在目标2中，我们将努力 利用NanoString技术开发杂交探针组（我们简称为纳米传感器）， 抗生素反应基因和控制基因在两个迭代阶段中。随后将进行测定性能 体外评估，并对新诊断为利福平耐药结核病的参与者的痰液进行评估， 在培养基中孵育后。这项工作将为创新的耐药性诊断奠定坚实的基础 来解决未满足的需求。