IHU Méditerranée Infection, Marseille, France; IRD, MEPHI, IHU Méditerranée Infection, Aix-Marseille-Université, France.
VBIC, INSERM U 1047, Université de Montpellier, France; Service de Microbiologie et Hygiène Hospitalière, CHU Nîmes, Nîmes, France.
EBioMedicine. 2022 Oct;84:104247. doi: 10.1016/j.ebiom.2022.104247. Epub 2022 Sep 7.
Point-Of-Care (POC) diagnosis of life-threatening community-acquired meningitis currently relies on multiplexed RT-PCR assays, that lack genotyping and antibiotic susceptibility profiling. We assessed the usefulness of real-time metagenomics (RTM) directly applied to the cerebrospinal fluid (CSF) for the identification, typing and susceptibility profiling of pathogens responsible for community-acquired meningitis.
A series of 52 CSF samples from patients suspected of having community-acquired meningitis, were investigated at POC by direct RTM in parallel to routine real-time multiplex PCR (RT-PCR) and bacterial culture, for the detection of pathogens. RTM-generated sequences were blasted in real-time against an in-house database incorporating the panel of 12 most prevalent pathogens and against NCBI using EPI2ME online software, for pathogen identification. In-silico antibiogram and genotype prediction were determined using the ResFinder bio-tool and MLST online software.
Over eight months, routine multiplex RT-PCR yielded 49/52 positive CSFs, including 21 Streptococcus pneumoniae, nine Neisseria meningitidis, eight Haemophilus influenzae, three Streptococcus agalactiae, three Herpesvirus-1, two Listeria monocytogenes, and one each of Escherichia coli, Staphylococcus aureus and Varicella-Zoster Virus. Parallel RTM agreed with the results of 47/52 CSFs and revealed two discordant multiplex RT-PCR false positives, one H. influenzae and one S. pneumoniae. Both multiplex RT-PCR and RTM agreed on the negativity of three CSFs. While multiplex RT-PCR routinely took 90 min, RTM took 120 min, although the pipeline analysis detected the pathogen genome after 20 min of sequencing in 33 CSF samples; and after two hours in 14 additional CSFs; yielding > 50% genome coverage in 19 CSFs. RTM identified 14 pathogen genotypes, including a majority of H. influenzae b, N. meningitidis B and S. pneumoniae 11A and 3A. In all 16 susceptible cultured bacteria, the in-silico antibiogram agreed with the in-vitro antibiogram in 10 cases, available within 48 h in routine bacteriology.
In addition to pathogen detection, RTM applied to CSF samples offered supplementary information on bacterial profiling and genotyping. These data provide the proof-of-concept that RTM could be implemented in a POC laboratory for one-shot diagnostic and genomic surveillance of pathogens responsible for life-threatening meningitis.
This work was supported by the French Government under the Investments in the Future programme managed by the National Agency for Research reference: Méditerranée Infection 10-IAHU-03.
目前，危及生命的社区获得性脑膜炎的即时检测诊断依赖于多重实时 RT-PCR 检测，该方法缺乏基因分型和抗生素药敏分析。我们评估了直接应用于脑脊液的实时宏基因组学（RTM）在鉴定、分型和检测社区获得性脑膜炎病原体的药敏分析方面的实用性。
本研究对疑似患有社区获得性脑膜炎的 52 例患者的脑脊液进行了研究，通过直接 RTM 与常规实时多重 PCR（RT-PCR）和细菌培养平行进行，以检测病原体。实时生成的序列实时与包含 12 种最常见病原体的内部数据库和 NCBI 上的 EPI2ME 在线软件进行比对，以进行病原体鉴定。使用 ResFinder 生物工具和 MLST 在线软件进行体内抗生素分析和基因型预测。
在 8 个月的时间里，常规多重 RT-PCR 检测出 52 例 CSF 中有 49 例呈阳性，包括 21 例肺炎链球菌、9 例脑膜炎奈瑟菌、8 例流感嗜血杆菌、3 例无乳链球菌、3 例单纯疱疹病毒 1 型、2 例李斯特菌单核细胞增生症、1 例大肠埃希菌、1 例金黄色葡萄球菌和 1 例水痘带状疱疹病毒。平行 RTM 与 47/52 例 CSF 结果一致，并发现 2 例多重 RT-PCR 假阳性，1 例为流感嗜血杆菌，1 例为肺炎链球菌。多重 RT-PCR 和 RTM 均对 3 例 CSF 呈阴性。虽然多重 RT-PCR 通常需要 90 分钟，但 RTM 需要 120 分钟，尽管在 33 例 CSF 样本中，测序 20 分钟后即可进行管道分析检测病原体基因组；而在另外 14 例 CSF 样本中，需要 2 小时；在 19 例 CSF 样本中，基因组覆盖率超过 50%。RTM 鉴定了 14 种病原体基因型，包括大多数 b 型流感嗜血杆菌、B 型脑膜炎奈瑟菌和 11A 型和 3A 型肺炎链球菌。在所有 16 株可培养的敏感细菌中，10 株的体内抗生素分析与 48 小时内常规细菌学中的体外抗生素分析一致。
除了病原体检测外，直接应用于 CSF 样本的 RTM 还提供了关于细菌分析和基因分型的补充信息。这些数据提供了概念验证，即 RTM 可在即时检测实验室中实施，用于对危及生命的脑膜炎的病原体进行一次性诊断和基因组监测。
本研究由法国政府资助，该研究由国家研究署管理的未来投资计划（Investissements dans l'avenir）提供资金，参考号为 Méditerranée Infection 10-IAHU-03。
