Real-time metagenomics-based diagnosis of community-acquired meningitis: A prospective series, southern France.

BACKGROUND: 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. METHODS: 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. FINDINGS: 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. INTERPRETATION: 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. FUNDING: 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.

Dynamics of community-acquired meningitis syndrome outbreaks in southern France.

In southern France, cases of community-acquired meningitis syndrome (CAM) are typically clustered as outbreaks with determinants which remain unknown. This 61-month retrospective investigation in Nîmes and Marseille university hospital laboratories, yielded 2,209/20,779 (10.63%) documented CAM cases caused by 62 different micro-organisms, represented by seasonal viral etiologies (78.8%), including Enterovirus, Herpes Simplex Virus (HSV), and Varicella-Zoster Virus (VZV; 1,620/2,209 = 73.4%). Multi correspondence analysis revealed an association of infection with age and sex, with the risk of infection being relatively higher in young men, as confirmed by Fisher's exact test (p < 10-3). Bacterial meningitis accounted for 20% of cases, mostly caused by Streptococcus pneumoniae (27.4% of cases), Neisseria meningitidis (12.5%), and Haemophilus influenzae (9.5%) with bacteria/virus coinfection (0.9%), and only six cases of documented fungal meningitis. In total, 62.6% of cases, of which 88.7% were undocumented, arose from 10 outbreaks. 33.2% of undocumented cases were aged >60 years compared to 19.2% of documented cases (p < 0.001), and viral infection was more common in the summer (87.5%) compared to other seasons (72.3%; p < 0.001). Outbreaks most often started in Nîmes and moved eastward toward Marseille at a speed of ~9 km/day, and these dynamics significantly correlated with atmospheric temperature, especially during summer outbreaks. In particular, the incidence of Enterovirus-driven outbreaks correlated with temperature, revealing correlation coefficients of 0.64 in Nîmes and 0.72 in Marseille, and its occurrence in Marseille lagged that in Nîmes by 1-2 weeks. Tracing the dynamics of CAM outbreak during this retrospective investigation in southern France yielded a speed of displacement that correlated with the variation in temperature between both cities, and these results provide clues for the next occurrence of undocumented outbreaks.

Haemophilus influenzae Meningitis Direct Diagnosis by Metagenomic Next-Generation Sequencing: A Case Report.

Current routine real-time PCR methods used for the point-of-care diagnosis of infectious meningitis do not allow for one-shot genotyping of the pathogen, as in the case of deadly Haemophilus influenzae meningitis. Real-time PCR diagnosed H. influenzae meningitis in a 22-year-old male patient, during his hospitalisation following a more than six-metre fall. Using an Oxford Nanopore Technologies real-time sequencing run in parallel to real-time PCR, we detected the H. influenzae genome directly from the cerebrospinal fluid sample in six hours. Furthermore, BLAST analysis of the sequence encoding for a partial DUF417 domain-containing protein diagnosed a non-b serotype, non-typeable H.influenzae belonging to lineage H. influenzae 22.1-21. The Oxford Nanopore metagenomic next-generation sequencing approach could be considered for the point-of-care diagnosis of infectious meningitis, by direct identification of pathogenic genomes and their genotypes/serotypes.