Clinical isolates of Neisseria meningitidis with reduced susceptibility to penicillin G (intermediate isolates, PenI ) harbor alterations in the penA gene encoding the penicillin binding protein 2 (PBP2). A 402-bp DNA fragment in the 3 half of penA was sequenced from a collection of 1,670 meningococcal clinical isolates from 22 countries that spanned 60 years. Phenotyping, genotyping, and the determination of MICs of penicillin G were also performed. A total of 139 different penA alleles were detected with 38 alleles that were highly related, clustered together in maximum-likelihood analysis and corresponded to the penicillin G-susceptible isolates. The remaining 101 penA alleles were highly diverse, corresponded to different genotypes or phenotypes, and accounted for 38% of isolates, but no clonal expansion was detected. Analysis of the altered alleles that were represented by at least five isolates showed high correlation with the Pen I phenotype. The deduced amino acid sequence of the corresponding PBP2 comprised five amino acid residues that were always altered. This correlation was not complete for rare alleles, suggesting that other mechanisms may also be involved in conferring reduced susceptibility to penicillin. Evidence of mosaic structures through events of interspecies recombination was also detected in altered alleles. A new website was created based on the data from this work (http://neisseria.org/nm/typing/penA). These data argue for the use of penA sequencing to identify isolates with reduced susceptibility to penicillin G and as a tool to improve typing of meningococcal isolates, as well as to analyze DNA exchange among Neisseria species.

Rapid, accurate and inexpensive diagnosis of bacterial meningitis is critical for patient management. This study describes the development and evaluation of a multiplex PCR assay for the detection of Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae type b, which globally account for 90% of cases of bacterial meningitis. The single-tube assay, based on the ctrA, ply and bex targets, respectively, enabled detection of 5-10 pg DNA. When the assay was tested with clinical samples (n = 425), its sensitivity for the three targets was 93.9%, 92.3% and 88%, respectively, while the overall specificity and positive predictive value of the assay was 100%. The negative predictive value was 99.1-99.5%. The methodology permits rapid and accurate detection of the three main pathogens that cause bacterial meningitis.

Twenty clinical samples (18 cerebrospinal fluid samples and 2 articular fluid samples) were sent to 11 meningococcus reference centers located in 11 different countries. Ten of these laboratories are participating in the EU-MenNet program (a European Union-funded program) and are members of the European Monitoring Group on Meningococci. The remaining laboratory was located in Burkina Faso. Neisseria meningitidis was sought by detecting several meningococcus-specific genes (crgA, ctrA, 16S rRNA, and porA). The PCRbased nonculture method for the detection of N. meningitidis gave similar results between participants with a mean sensitivity and specificity of 89.7 and 92.7%, respectively. Most of the laboratories also performed genogrouping assays (siaD and mynB/sacC). The performance of genogrouping was more variable between laboratories, with a mean sensitivity of 72.7%. Genogroup B gave the best correlation between participants, as all laboratories routinely perform this PCR. The results for genogroups A and W135 were less similar between the eight participating laboratories that performed these PCRs.

The differential diagnosis of acute community-acquired meningitis is of paramount importance in both therapeutic and healthcare-related economic terms. Despite the routinely used markers, novel, easily calculated, and rapidly available biomarkers are needed particularly in resource-poor settings. A promising, exponentially studied inflammatory marker is the neutrophil-to-lymphocyte ratio (NLR), albeit not assessed in meningitis. The aim of this study was to investigate the utility of the NLR in the differential diagnosis of acute meningitis. Data on cerebrospinal fluid (CSF) and blood leukocyte parameters from more than 4,000 patients diagnosed with either bacterial or viral meningitis in Greece during the period 2006-2013 were retrospectively examined. The diagnostic accuracy of the NLR and neutrophil counts in CSF and blood were evaluated by receiver operating characteristic curves. The discrimination ability of both the NLR and neutrophil counts was significantly higher in CSF than in blood. The optimal cutoff values of the NLR and neutrophil counts were 2 in CSF vs 8 in blood, and 287 cells in CSF vs 12,100 cells in blood, respectively. For these values, sensitivity, negative predictive value, and odds ratio were statistically significantly higher in CSF than blood for both markers. Logistic regression analysis showed that the CSF NLR carries independent and additive information to neutrophil counts in the differential diagnosis of acute meningitis. This study is the first one to assess NLR in acute meningitis, providing promising results for its differential diagnosis.

BackgroundSerogroup B meningococcal (MenB) isolates currently account for approximately 90% of invasive meningococcal disease (IMD) in Greece with ST-162 clonal complex predominating. The potential of a multicomponent meningococcal B vaccine (4CMenB) recently licensed in Europe was investigated in order to find whether the aforementioned vaccine will cover the MenB strains circulating in Greece. A panel of 148 serogroup B invasive meningococcal strains was characterized by multilocus sequence typing (MLST) and PorA subtyping. Vaccine components were typed by sequencing for factor H-binding protein (fHbp), Neisserial Heparin Binding Antigen (NHBA) and Neisseria adhesin A (NadA). Their expression was explored by Meningococcal Antigen Typing System (MATS).ResultsGlobal strain coverage predicted by MATS was 89.2% (95% CI 63.5%-98.6%) with 44.6%, 38.5% and 6.1% of strains covered by one, two and three vaccine antigens respectively. NHBA was the antigen responsible for the highest coverage (78.4%), followed by fHbp (52.7%), PorA (8.1%) and NadA (0.7%). The coverage of the major genotypes did not differ significantly. The most prevalent MLST genotype was the ST-162 clonal complex , accounting for 44.6% of the strains in the panel and with a predicted coverage of 86.4%, mainly due to NHBA and fHbp.Conclusions4CMenB has the potential to protect against a significant proportion of Greek invasive MenB strains.