Redefining Tuberculosis Meningitis with Metagenomics and Host Transciptomics

利用宏基因组学和宿主转录组学重新定义结核性脑膜炎

• 批准号: 10359029
• 负责人: Michael R Wilson
• 金额: $ 67.85万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-11 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10359029
• 关键词: Address; Adult; Affect; Africa South of the Sahara; Aftercare; Antibiotics; Antimicrobial Resistance; Antitubercular Agents; Autopsy; Bioinformatics; Biological Assay; Biological Markers; CRISPR/Cas technology; California; Central Nervous System Infections; Cerebrospinal Fluid; Cessation of life; Clinical; Complication; Coupled; Critical Pathways; DNA; Data; Detection; Diagnosis; Diagnostic; Diagnostic Specificity; Drug resistance in tuberculosis; Early treatment; Enrollment; Fright; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Gene Targeting; Genes; Genetic Transcription; Genome; Genomics; HIV; Infection; Inflammation; Meningeal Tuberculosis; Meningitis; Metagenomics; Methods; Molecular; Multidrug-Resistant Tuberculosis; Mycobacterium tuberculosis; Nature; Neurologic; Nucleic Acid Amplification Tests; Outcome; Pathogenesis; Patients; Pattern; Performance; Persons; Positioning Attribute; Predisposition; Prospective cohort; RNA; Recommendation; Resistance; Resistance profile; Rifampicin resistance; Sampling; San Francisco; Technology; Testing; Time; Tuberculosis; Tuberculosis diagnosis; Uganda; Universities; base; clinical phenotype; cohort; diagnostic assay; disability; experimental study; improved; microbial; mortality; next generation; next generation sequencing; novel; pathogen; point-of-care diagnostics; prognostic; resistance gene; resistance mutation; response; tool; transcriptome sequencing; treatment response; tuberculosis diagnostics; tuberculosis treatment

PROPOSAL SUMMARY/ABSTRACT Mycobacterium tuberculosis (TB) affects >10 million people worldwide and carries devastating consequences with ~1.3 million deaths in 2016. Meningitis is the most feared complication of TB with 50-60% mortality in HIV+ persons. Unfortunately, current diagnostic assays are insensitive, and the ability to rapidly characterize the antimicrobial resistance (AMR) profile is severely limited, resulting in only 1 in 5 of the 480,000 multi-drug resistant TB patients receiving appropriate therapy in 2015. To address this critical gap, we will engage a novel combination of metagenomic next generation sequencing (mNGS) coupled with a CRISPR/Cas9- based targeted enrichment method to detect TB and AMR genes and longitudinal host transcriptional profiling to better understand TB meningitis pathogenesis. The most recent advance in TB diagnostics is the Xpert MTB/RIF Ultra, an automated nucleic acid amplification test. However, Ultra is only ~70% sensitive for probable/definite TB meningitis, and detects only one resistance target, highlighting the need for new and better diagnostics. We have pioneered cerebrospinal fluid (CSF) mNGS at the University of California San Francisco which is an unbiased tool that, in a single assay, can detect the whole range of neurologic infections. Furthermore, we incorporate a novel CRISPR/Cas9-based gene targeting strategy called FLASH (Finding Low Abundance Sequences by Hybridization) to enhance detection of low abundance TB and AMR gene targets by over 104-fold. Thus, we are well positioned to critically test this proposal's central hypotheses which are that CSF mNGS coupled with FLASH is 1) more sensitive and specific than conventional CSF TB diagnostics, 2) enables simultaneous and comprehensive detection of TB AMR genes, and 3) can be used to identify host gene expression biomarkers that discriminate between patients with variable responses to TB meningitis therapy. We will enroll a prospective cohort of over ~1,300 adults presenting with suspected CNS infection in Kampala and Mbarara, Uganda and test >600 subjects with suspected TBM. The deep clinical phenotyping of this cohort will critically inform the hypotheses being tested herein. These experiments will generate the most comprehensive molecular assessment of TB meningitis in sub- Saharan Africa to date and specifically inform 1) revisions of the TB meningitis uniform case definition, 2) detection of non-TB neurologic infections masquerading as suspected TB meningitis, 3) empiric antibiotic recommendations based on AMR patterns, 4) targets for the next-generation point-of-care diagnostic assays for TB meningitis, and 5) candidate prognostic CSF transcriptional biomarkers for treatment response.

提案摘要/摘要 结核分枝杆菌（TB）在全球范围内影响> 1000万人，并带来毁灭性后果 2016年约有130万人死亡。脑膜炎是结核病最担心的并发症，死亡率为50-60％ 艾滋病毒+人。不幸的是，当前的诊断测定不敏感，并且能够快速表征的能力 抗菌素耐药性（AMR）剖面受到严重限制，在480,000个多毒品中只有5个 抗性结核病患者在2015年接受适当的治疗。为了解决这一关键差距，我们将参与 元基因组下一代测序（MNG）的新型组合与CRISPR/CAS9- 基于靶向富集方法检测TB和AMR基因以及纵向宿主转录 分析以更好地了解结核病脑膜炎发病机理。 结核病诊断的最新进展是XPERT MTB/RIF ULTRA，一种自动核酸 放大测试。但是，对可能/明确的TB脑膜炎仅敏感到70％，仅检测 一个阻力目标，强调了对新诊断的需求。我们有开创性的脑脊 加利福尼亚大学旧金山分校的流体（CSF）MNG是一种公正的工具 测定可以检测整个神经系统感染的范围。此外，我们纳入了一本小说 基于CRISPR/CAS9的基因靶向策略称为Flash（通过 杂交）以增强低丰度TB和AMR基因靶标的检测104倍以上。 因此，我们在批判性地测试该提案的中心假设是CSF MNGS 与传统的CSF结核病诊断相比，闪光与闪光灯相结合，更敏感和特异性，2）启用 同时且全面检测结核病基因，3）可用于识别宿主基因 表达生物标志物，可区分对TB脑膜炎治疗的反应可变的患者。 我们将招募一个超过1300名成年人的前瞻性队列 穆巴拉（Mbarara），乌干达和测试> 600名怀疑TBM的受试者。深层临床表型 队列将严格告知本文所述的假设。 这些实验将产生对亚脑膜炎的最全面的分子评估 迄今为止，撒哈拉非洲的非洲，特别是告知1）TB脑膜炎统一病例定义的修订，2） 检测伪装成可疑的TB脑膜炎的非TB神经系统感染，3）经验性抗生素 基于AMR模式的建议，4）下一代护理诊断测定的目标 对于结核病脑膜炎和5）候选预后CSF转录生物标志物用于治疗反应。