Metagenomic next-generation sequencing (mNGS) for pan-pathogen detection has been successfully tested in proof-of-concept case studies in patients with acute illness of unknown etiology but to date has been largely confined to research settings. Here, we developed and validated a clinical mNGS assay for diagnosis of infectious causes of meningitis and encephalitis from cerebrospinal fluid (CSF) in a licensed microbiology laboratory. A customized bioinformatics pipeline, SURPI+, was developed to rapidly analyze mNGS data, generate an automated summary of detected pathogens, and provide a graphical user interface for evaluating and interpreting results. We established quality metrics, threshold values, and limits of detection of 0.2-313 genomic copies or colony forming units per milliliter for each representative organism type. Gross hemolysis and excess host nucleic acid reduced assay sensitivity; however, spiked phages used as internal controls were reliable indicators of sensitivity loss. Diagnostic test accuracy was evaluated by blinded mNGS testing of 95 patient samples, revealing 73% sensitivity and 99% specificity compared to original clinical test results, and 81% positive percent agreement and 99% negative percent agreement after discrepancy analysis. Subsequent mNGS challenge testing of 20 positive CSF samples prospectively collected from a cohort of pediatric patients hospitalized with meningitis, encephalitis, and/or myelitis showed 92% sensitivity and 96% specificity relative to conventional microbiological testing of CSF in identifying the causative pathogen. These results demonstrate the analytic performance of a laboratory-validated mNGS assay for pan-pathogen detection, to be used clinically for diagnosis of neurological infections from CSF.

中文翻译：

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用于脑脊液中病原体检测的临床宏基因组测序测定的实验室验证。

用于泛病原体检测的宏基因组下一代测序（mNGS）已在病因不明的急性疾病患者的概念验证案例研究中成功进行了测试，但迄今为止主要局限于研究环境。在这里，我们开发并验证了一种临床 mNGS 检测方法，用于在获得许可的微生物学实验室中通过脑脊液 (CSF) 诊断脑膜炎和脑炎的感染原因。开发了定制的生物信息学流程 SURPI+，用于快速分析 mNGS 数据，生成检测到的病原体的自动摘要，并提供用于评估和解释结果的图形用户界面。我们为每种代表性生物体类型建立了每毫升 0.2-313 个基因组拷贝或菌落形成单位的质量指标、阈值和检测限。严重溶血和过量宿主核酸会降低检测灵敏度；然而，用作内部对照的尖刺噬菌体是敏感性丧失的可靠指标。通过对 95 名患者样本进行盲法 mNGS 测试来评估诊断测试的准确性，与原始临床测试结果相比，敏感性为 73%，特异性为 99%，差异分析后阳性一致性百分比为 81%，阴性一致性百分比为 99%。随后对从患有脑膜炎、脑炎和/或脊髓炎的住院儿科患者队列中前瞻性收集的 20 个阳性 CSF 样本进行 mNGS 攻击检测，在识别致病病原体方面，相对于传统的 CSF 微生物检测，显示出 92% 的敏感性和 96% 的特异性。这些结果证明了经实验室验证的 mNGS 检测用于泛病原体检测的分析性能，可在临床上用于诊断脑脊液神经系统感染。