Development and validation of a sample-to-answer metagenomics workflow for comprehensive pathogen detection in central nervous system infections

摘要 这项研究最终将通过改善中枢神经系统疾病的诊断和治疗来促进人类健康 神经系统感染，包括脑膜炎和脑炎，原因不明 在高达50%的案例中。这些感染与严重的发病率和死亡率有关；因此，迅速 而准确的诊断对于适当的患者管理和改善预后至关重要。不幸的是， 由于可导致中枢神经系统感染的微生物种类繁多，目前的护理标准 诊断方法存在周转时间长和假阴性率高的问题。元基因组学下一步- 世代测序(MNGS)是一种很有前途的传染病通用检测方法 疾病，使得能够同时识别和表征病毒、细菌和真菌病原体 直接从临床样本中存在的DNA/RNA中提取。然而，尽管越来越多的人采用分子 临床微生物学实验室中的诊断，由于其成本、周转时间、 以及产生和分析数据所需的技术专长。我们正在开发的产品 解决了所有这些障碍。Galileo One-CSF是一个样本到答案平台，它包括所有 直接检测和报告临床相关病原体所需的试剂、对照、方案和软件 在约24小时内从脑脊液(CSF)样本中提取。该平台结合了几个专有技术 技术，包括减少大量人类序列的文库准备模块和 提高序列变异敏感性的定量生物信息学流水线。此一体式捆绑包 允许实验室在自己的实验室中轻松评估和采用mNGS，而不需要广泛的 基因组学或生物信息学专业知识。在我们的第一阶段-相当于工作期间，我们使用人造样本来 评估使用CSF作为样本类型的可行性；结果表明，我们可以生成高质量的 库，并使用我们的Galileo One工作流检测脑脊液中的一组代表性病原体，这是 最初在等离子体上进行了验证。因此，我们已经准备好进入产品的开发阶段 发展进程。在第二阶段，我们将对伽利略进行分析和初步临床验证 One-CSF平台，完成特定于CSF的试剂盒控制的开发，并对人工设计的 和我们的合作者的临床样本。该产品将是第一个经过验证的部署的mNGS平台 使我们能够利用不断增长的基于NGS的传染病诊断市场， 预计到2027年，这一数字将达到21亿美元。Galileo One-CSF将增强终端用户的能力，其中包括 研究人员、参考实验室和制药/生物技术合作伙伴，采用元基因组学来解决 目前在检测中枢神经系统感染的工具方面存在差距。

ABSTRACT This research will ultimately advance human health by improving the diagnosis and treatment of central nervous system infections including meningitis and encephalitis, where the causative pathogen is not identified in up to 50% of cases. These infections are associated with significant morbidity and mortality; therefore, rapid and accurate diagnosis is crucial for appropriate patient management and to improve outcomes. Unfortunately, due to the variety of organisms that can cause central nervous system infections, current standard of care diagnostic methods suffer from long turnaround times and high false negative rates. Metagenomic next- generation sequencing (mNGS) has emerged as a promising universal detection method for infectious disease, enabling the simultaneous identification and characterization of viral, bacterial, and fungal pathogens directly from the DNA/RNA present in clinical samples. However, despite the increasing adoption of molecular diagnostics in clinical microbiology labs, mNGS has not become widespread due to its cost, turnaround time, and the technical expertise required to produce and analyze the data. The product we are developing addresses all of these obstacles. Galileo ONE – CSF is a sample-to-answer platform that includes all of the reagents, controls, protocols, and software required to detect and report clinically relevant pathogens directly from a cerebrospinal fluid (CSF) sample in ~24 hours. This platform incorporates several proprietary technologies, including a library preparation module that reduces abundant human sequences and a quantitative bioinformatics pipeline that increases sensitivity for sequence variants. This all-in-one bundle allows labs to easily evaluate and adopt mNGS in their own laboratories without the need for extensive genomics or bioinformatics expertise. During our Phase I-equivalent work, we used contrived samples to assess the feasibility of using CSF as a sample type; the results show that we can generate high-quality libraries and detect a representative set of pathogens in CSF using our Galileo ONE workflow, which was initially validated on plasma. Therefore, we are ready to move into the development phase of the product development process. In Phase II, we will perform analytical and preliminary clinical validation of the Galileo ONE – CSF platform, complete development of CSF-specific kit controls, and perform beta testing on contrived and clinical samples with our collaborators. This product will be the first deployed mNGS platform validated for CSF, enabling us to take advantage of the growing market for NGS-based infectious disease diagnostics, which is expected to reach $2.1B by 2027. Galileo ONE – CSF will empower end users, who include researchers, reference laboratories, and pharma/biotech partners, to adopt metagenomics to address the current gap in tools for detecting central nervous system infections.