biostudies-literatureUnknown84Morsli M<h4>Background</h4>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.<h4>Methods</h4>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.<h4>Findings</h4>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.<h4>Interpretation</h4>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.<h4>Funding</h4>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.EBioMedicine104247https://www.ebi.ac.uk/biostudies/studies/S-EPMC9463524biostudies-literatureReal-time metagenomics-based diagnosis of community-acquired meningitis: A prospective series, southern France.PMC9463524Morsli MSalipante FDrancourt MKerharo QDunyach-Remy CLavigne JPBoudet AStephan RFournier PEHouhamdi LfalseReal-time metagenomics-based diagnosis of community-acquired meningitis: A prospective series, southern France.<h4>Background</h4>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.<h4>Methods</h4>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.<h4>Findings</h4>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.<h4>Interpretation</h4>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.<h4>Funding</h4>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-03.2022-01-01T00:00:00Z2022 Oct2026-05-28T03:18:42.17Z2025-04-07T08:29:42.724ZS-EPMC94635243608752410.1016/j.ebiom.2022.104247