Redefining Tuberculosis Meningitis with Metagenomics and Host Transciptomics

提案摘要/摘要 结核分枝杆菌（TB）影响全世界超过1000万人，并带来毁灭性的后果 2016年死亡人数约为130万。脑膜炎是结核病最可怕的并发症， 艾滋病毒阳性者。不幸的是，目前的诊断测定是不敏感的，并且快速表征的能力是有限的。 抗生素耐药性（AMR）严重受限，导致480，000种多药耐药中只有五分之一 2015年接受适当治疗的耐药结核病患者。为了解决这一关键差距，我们将采取 宏基因组下一代测序（mNGS）与CRISPR/Cas9- 基于靶向富集的方法检测TB和AMR基因和纵向宿主转录 分析以更好地了解结核性脑膜炎的发病机制。 结核病诊断的最新进展是Xpert MTB/RIF Ultra，一种自动核酸检测系统， 放大试验然而，Ultra对可能/确定的结核性脑膜炎的敏感性仅为~70%， 一个耐药性目标，突出了对新的和更好的诊断的需要。我们开创了脑脊髓 在加州大学旧金山弗朗西斯科，这是一个公正的工具，在一个单一的 检测，可以检测到整个范围的神经系统感染。此外，我们还将一部小说 基于CRISPR/Cas9的基因靶向策略称为FLASH（通过寻找低异常序列 杂交）以将低丰度TB和AMR基因靶标的检测增强超过104倍。 因此，我们处于有利地位，以严格测试这一建议的中心假设，即CSF mNGS 与闪存相结合，1）比传统CSF TB诊断更灵敏和特异，2） 可同时、全面检测结核菌AMR基因，3）可用于鉴定宿主基因 表达生物标志物，区分对结核性脑膜炎治疗具有不同反应的患者。 我们将在坎帕拉招募超过1,300名疑似中枢神经系统感染的成年人 和乌干达的姆巴拉拉，并测试> 600名患有疑似TBM的受试者。这种疾病的深层临床表型 队列将批判性地告知本文所测试的假设。 这些实验将产生最全面的分子评估结核性脑膜炎在亚- 撒哈拉非洲迄今为止，特别通知1）修订结核性脑膜炎统一病例定义，2） 检测伪装成疑似结核性脑膜炎的非结核性神经系统感染，3）经验性抗生素 基于AMR模式的建议，4）下一代床旁诊断检测的目标 用于TB脑膜炎，和5）用于治疗反应的候选预后CSF转录生物标志物。

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.