Virology Laboratory, Genomics and Health Area, Center for Public Health Research (FISABIO-Public Health), Generalitat Valenciana, Valencia, Spain; Microbiology & Ecology Department, Medical School, University of Valencia, Spain; and CIBERESP, Instituto de Salud Carlos III, Spain.
Institute of Medical Virology, University of Zurich, Switzerland.
J Clin Virol. 2024 Aug;173:105695. doi: 10.1016/j.jcv.2024.105695. Epub 2024 May 25.
Metagenomics is gradually being implemented for diagnosing infectious diseases. However, in-depth protocol comparisons for viral detection have been limited to individual sets of experimental workflows and laboratories. In this study, we present a benchmark of metagenomics protocols used in clinical diagnostic laboratories initiated by the European Society for Clinical Virology (ESCV) Network on NGS (ENNGS). A mock viral reference panel was designed to mimic low biomass clinical specimens. The panel was used to assess the performance of twelve metagenomic wet lab protocols currently in use in the diagnostic laboratories of participating ENNGS member institutions. Both Illumina and Nanopore, shotgun and targeted capture probe protocols were included. Performance metrics sensitivity, specificity, and quantitative potential were assessed using a central bioinformatics pipeline. Overall, viral pathogens with loads down to 10 copies/ml (corresponding to C values of 31 in our PCR assays) were detected by all the evaluated metagenomic wet lab protocols. In contrast, lower abundant mixed viruses of C values of 35 and higher were detected only by a minority of the protocols. Considering the reference panel as the gold standard, optimal thresholds to define a positive result were determined per protocol, based on the horizontal genome coverage. Implementing these thresholds, sensitivity and specificity of the protocols ranged from 67 to 100 % and 87 to 100 %, respectively. A variety of metagenomic protocols are currently in use in clinical diagnostic laboratories. Detection of low abundant viral pathogens and mixed infections remains a challenge, implying the need for standardization of metagenomic analysis for use in clinical settings.
宏基因组学正逐渐被应用于传染病的诊断。然而，对于病毒检测的深入协议比较仅限于个别实验工作流程和实验室。在本研究中，我们展示了欧洲临床病毒学学会（ESCV）下一代测序（NGS）网络（ENNGS）发起的临床诊断实验室中使用的宏基因组学协议的基准。设计了一个模拟低生物量临床标本的模拟病毒参考面板。该面板用于评估 12 种目前在参与 ENNGS 成员机构的诊断实验室中使用的宏基因组学湿实验室协议的性能。包括 Illumina 和 Nanopore 、 shotgun 和靶向捕获探针协议。使用中央生物信息学管道评估性能指标敏感性、特异性和定量潜力。总体而言，所有评估的宏基因组学湿实验室协议都可以检测到负载量低至 10 拷贝/ml（对应于我们 PCR 检测中的 C 值 31）的病毒病原体。相比之下，只有少数协议可以检测到负载量较低的混合病毒（C 值为 35 及更高）。考虑到参考面板为金标准，根据水平基因组覆盖率，为每个协议确定了定义阳性结果的最佳阈值。根据这些阈值，协议的敏感性和特异性范围分别为 67%至 100%和 87%至 100%。目前在临床诊断实验室中使用了多种宏基因组学协议。检测低丰度病毒病原体和混合感染仍然是一个挑战，这意味着需要对宏基因组分析进行标准化，以用于临床环境。
