Table 1_Development and performance evaluation of a clinical metagenomics approach for identifying pathogens in the whole blood from patients with undifferentiated fever.docx

IntroductionBlood culture is the cornerstone of microbiological diagnostics for patients with acute undifferentiated fever and no obvious localization of infection; however, up to 50% of cases remain undiagnosed. Infections caused by arboviruses, fastidious or even uncultivable bacteria, or parasites often go undiagnosed without the use of target-specific molecular methods. These are typically performed in a stepwise manner, increasing cost and delaying results. Metagenomic next-generation sequencing (mNGS) has recently gained recognition as a potential universal pathogen detection tool for such cases. Our study aimed to develop a streamlined mNGS workflow for simultaneous detection of intracellular and cell-free pathogens within a single sequencing library.
MethodsTotal nucleic acid was isolated separately from 200 EDTA blood samples. The plasma isolate was processed with DNase, followed by the depletion of host ribosomal and messenger RNA, reverse transcription, and sequence-independent single primer amplification (SISPA). The whole blood isolate was only reverse transcribed, with no other pre-processing manipulation. Finally, the two fractions were combined prior to library preparation and sequencing using either Oxford Nanopore Technologies or Illumina. Following established bioinformatics analysis, we developed a mathematical ranking approach (ClinSeq score) that enabled quick identification of relevant pathogens in approximately one hour.
ResultsThe mNGS workflow reached 79.5% (159/200) overall sensitivity. For bacteria the sensitivity was 88.6% (70/79), DNA viruses, 66.7% (10/15) and for RNA viruses 73.8% (76/103). Pathogen detections by individual sequencing methods showed overall sensitivity of Illumina and ONT to be 80.0% (76/95) and 79.1% (83/105) respectively. The ClinSeq score correctly highlighted the pathogen in 126/200 (63.0%) samples effectively with a Cohen’s kappa (κ) agreement of 0.61 with manual analysis.
ConclusionDeveloped comprehensive mNGS workflow detects a wide range of pathogens in patients with acute undifferentiated fever. The unified workflow improves sensitivity for intracellular bacteria and RNA viruses, reduces time, cost and complexity by eliminating the need for separate library preparations, enabling faster turnaround suitable for clinical settings. The ClinSeq score effectively differentiates true pathogen signals from background noise, reducing false positives and manual interpretation time. Overall, the workflow demonstrates flexible, and efficient pathogen detection, supporting its potential for clinical diagnostics and improved patient management.

【引言】血培养是急性不明热且无明确感染定位患者微生物学诊断的基石，但仍有高达50%的病例无法明确诊断。若不使用靶向特异性分子检测方法，虫媒病毒、苛养菌乃至难以培养的细菌或寄生虫引发的感染往往难以确诊。这类检测通常采用分步式开展，既增加了检测成本，又延长了结果回报时间。宏基因组下一代测序（metagenomic next-generation sequencing，mNGS）近年来被认为是此类病例通用病原体检测的潜在手段。本研究旨在开发一套简化的mNGS流程，可在单一测序文库中同时检测胞内病原体与游离病原体。
【方法】本研究从200份乙二胺四乙酸（EDTA）抗凝全血样本中分别提取总核酸。血浆组分经脱氧核糖核酸酶（DNase）处理后，依次进行宿主核糖体RNA与信使RNA去除、逆转录及序列非依赖性单引物扩增（sequence-independent single primer amplification，SISPA）。全血组分仅进行逆转录处理，无需其他预处理操作。最终将两组分混合，随后进行文库构建与测序，测序平台可选用牛津纳米孔科技公司（Oxford Nanopore Technologies）或因美纳（Illumina）平台。在标准生物信息学分析流程之后，本研究开发了一种数学评分方法（ClinSeq评分），可在约1小时内快速识别相关病原体。
【结果】本mNGS流程的总体敏感度为79.5%（159/200）。其中细菌检测敏感度为88.6%（70/79），DNA病毒为66.7%（10/15），RNA病毒为73.8%（76/103）。单独使用两种测序平台的病原体检测总体敏感度分别为：因美纳（Illumina）平台80.0%（76/95），牛津纳米孔（ONT）平台79.1%（83/105）。ClinSeq评分可在126份（63.0%）样本中准确识别病原体，与人工分析的科恩κ（Cohen’s kappa，κ）一致性系数为0.61。
【结论】本研究开发的完整mNGS流程可检测急性不明热患者体内的多种病原体。该统一流程可提升胞内细菌与RNA病毒的检测敏感度，且无需分别构建文库，从而缩短检测时长、降低检测成本与操作复杂度，更适配临床场景的快速检测需求。ClinSeq评分可有效区分真实病原体信号与背景噪音，减少假阳性结果并缩短人工解读时间。总体而言，本流程展现出灵活高效的病原体检测能力，为其在临床诊断及优化患者管理中的应用潜力提供了支持。