Individual responsiveness of macrophage migration inhibitory factor predicts long-term cognitive impairment after bacterial meningitis.

BACKGROUND: Patients with pneumococcal meningitis are at risk for death and neurological sequelae including cognitive impairment. Functional genetic polymorphisms of macrophage migration inhibitory factor (MIF) alleles have shown to predict mortality of pneumococcal meningitis. METHODS: We investigated whether MIF concentrations during the acute phase of disease were predictive for death in a nationwide prospective cohort study. Subsequently, we studied whether individual ex vivo MIF response years after meningitis was associated with the development of cognitive impairment. RESULTS: We found that in the acute illness of pneumococcal meningitis, higher plasma MIF concentrations were predictive for mortality (p = 0.009). Cognitive impairment, examined 1-5 years after meningitis, was present in 11 of 79 patients after pneumococcal meningitis (14%), as compared to 1 of 63 (2%) in controls, and was consistently associated with individual variability in MIF production by peripheral blood mononuclear cells after ex vivo stimulation with various infectious stimuli. CONCLUSIONS: Our study confirms the role of MIF in poor disease outcome of pneumococcal meningitis. Inter-individual differences in MIF production were associated with long-term cognitive impairment years after pneumococcal meningitis. The present study provides evidence that MIF mediates long-term cognitive impairment in bacterial meningitis survivors and suggests a potential role for MIF as a target of immune-modulating adjunctive therapy.

Community-acquired Bacterial Meningitis in Adults With Cerebrospinal Fluid Leakage.

BACKGROUND: Cerebrospinal fluid (CSF) leakage is a risk factor for developing bacterial meningitis. METHODS: We analyzed episodes of community-acquired bacterial meningitis associated with CSF leakage from a prospective nationwide cohort study. RESULTS: CSF leakage was identified in 65 episodes of 2022 episodes (3%) in 53 patients. The cause of CSF leakage was identified in 49 of 65 episodes (75%), which most commonly consisted of ear-nose-throat surgery (19 of 49 episodes [29%]) and remote head trauma (15 of 49 episodes [23%]). The episode was a recurrent meningitis episode in 38 patients (59%). Of the recurrent episodes, 27 had known CSF leakage (71%) of whom 20 (53%) had previous surgery aiming to close the leak. Nine patients (38%) with known CSF leakage had been vaccinated (23-valent pneumococcal vaccine in 9 patients, meningococcal serogroup C vaccine in 2, meningococcal serogroup A and Haemophilus influenzae type b vaccine each in 1 patient). Streptococcus pneumoniae was cultured in 33 episodes (51%) and H. influenzae in 11 episodes (17%). The most common pneumococcal serotypes were 3 (4 episodes), 35B, 9N, 38, and 15C (each 2 episodes). Haemophilus influenzae was unencapsulated in all 10 episodes with known capsule type. The outcome was unfavorable in 8 episodes (12%) and no patient died. CONCLUSIONS: Bacterial meningitis in patients with CSF leakage has a high recurrence rate, despite surgical repair or vaccination, and outcome is generally favorable. CSF leakage should be suspected in patients with bacterial meningitis presenting with liquorrhea, recurrent meningitis, or with disease caused by H. influenzae.

Incidence and Risk Factors for Invasive Pneumococcal Disease and Community-acquired Pneumonia in Human Immunodeficiency Virus-Infected Individuals in a High-income Setting.

BACKGROUND: Although people living with human immunodeficiency virus (PLWH) are at increased risk of invasive pneumococcal disease (IPD) and community-acquired pneumonia (CAP), it is unclear whether this remains the case in the setting of early initiation of combination antiretroviral therapy (cART), at high CD4 cell counts. This is important, as pneumococcal vaccination coverage in PLWH is low in Europe and the United States, despite longstanding international recommendations. METHODS: We identified all CAP and IPD cases between 2008 and 2017 in a cohort of PLWH in a Dutch HIV referral center. We calculated incidence rates stratified by CD4 count and cART status and conducted a case-control study to identify risk factors for CAP in PLWH receiving cART. RESULTS: Incidence rates of IPD and CAP in PLWH were 111 and 1529 per 100 000 patient-years of follow-up (PYFU). Although IPD and CAP occurred more frequently in patients with CD4 counts <500 cells/µL (incidence rate ratio [IRR], 6.1 [95% confidence interval, 2.2-17] and IRR, 2.4 [95% confidence interval, 1.9-3.0]), the incidence rate in patients with CD4 counts >500 cells/µL remained higher compared with the general population (946 vs 188 per 100 000 PYFU). All IPD isolates were vaccine serotypes. Risk factors for CAP were older age, CD4 counts <500 cells/µL, smoking, drug use, and chronic obstructive pulmonary disease. CONCLUSIONS: The incidence of IPD and CAP among PLWH remains higher compared with the general population, even in those who are virally suppressed and have high CD4 counts. With all serotyped IPD isolates covered by pneumococcal vaccines, our study provides additional argumentation against the poor current adherence to international recommendations to vaccinate PLWH.

[Cranial computed tomography, lumbar puncture and clinical deterioration in bacterial meningitis: a nationwide cohort study]. / CT-hersenen, lumbaalpunctie en klinische achteruitgang bij bacteriële meningitis.

BACKGROUND: Investigate how often cerebral herniation occurs following lumbar puncture (LP) in patients with bacterial meningitis, and whether cranial computed tomography (CT) can be used to identify patients at a higher risk of cerebral herniation. STUDY DESIGN: Prospective, nationwide cohort study covering the period March 2006 - November 2014. METHOD: We identified patients with community-acquired bacterial meningitis who showed signs of clinical deterioration, possibly caused by LP. For systematic evaluation of contraindications for LP on cranial CT, the included patients were matched to bacterial meningitis patients without deterioration. Four experts, blinded for patient outcome, scored cranial CT scan imaging for the cases as well as control patients in relation to contraindications for LP. Inter-assessor reliability was determined with Fleiss' generalized κ. RESULTS: Of the 1533 bacterial meningitis patients included, 47 (3.1%) exhibited clinical deterioration possibly caused by LP. Two patients deteriorated within 1 hour after LP (0.1%). In 43 of 47 patients that showed signs of clinical deterioration, cranial CT was performed prior to LP. The inter-rater reliability of assessment of contraindications for LP on cranial CT was moderate (Fleiss' generalized κ = 0.47). A contraindication for LP was reported by all four raters in 6 patients with clinical deterioration (14%) and in 5 patients without clinical deterioration (11%). CONCLUSION: LP can be performed safely in the large majority of patients with bacterial meningitis, as it only very rarely results in cerebral herniation. Cranial CT can be considered a screening method to identify patients who are at a higher risk of cerebral herniation, but the inter-rater reliability of the CT scan assessment for contraindications of LP is moderate.

pIgR and PECAM-1 bind to pneumococcal adhesins RrgA and PspC mediating bacterial brain invasion.

Streptococcus pneumoniae is the main cause of bacterial meningitis, a life-threating disease with a high case fatality rate despite treatment with antibiotics. Pneumococci cause meningitis by invading the blood and penetrating the blood-brain barrier (BBB). Using stimulated emission depletion (STED) super-resolution microscopy of brain biopsies from patients who died of pneumococcal meningitis, we observe that pneumococci colocalize with the two BBB endothelial receptors: polymeric immunoglobulin receptor (pIgR) and platelet endothelial cell adhesion molecule (PECAM-1). We show that the major adhesin of the pneumococcal pilus-1, RrgA, binds both receptors, whereas the choline binding protein PspC binds, but to a lower extent, only pIgR. Using a bacteremia-derived meningitis model and mutant mice, as well as antibodies against the two receptors, we prevent pneumococcal entry into the brain and meningitis development. By adding antibodies to antibiotic (ceftriaxone)-treated mice, we further reduce the bacterial burden in the brain. Our data suggest that inhibition of pIgR and PECAM-1 has the potential to prevent pneumococcal meningitis.

Long-term mortality after IPD and bacteremic versus non-bacteremic pneumococcal pneumonia.

BACKGROUND: Short-term mortality after invasive pneumococcal disease (IPD) and pneumococcal pneumonia is high but data on long-term mortality (including the comparison between bacteremic and non-invasive/non-bacteremic pneumococcal pneumonia) within the first years after diagnosis are scarce. METHODS: Adult patients with 'non-pneumonia' IPD and 'invasive pneumonia' (from 2004 to 2012) and with 'bacteremic' vs 'non-invasive/non-bacteremic (NI/NB)' pneumococcal pneumonia (from 2008 to 2012) diagnosed by negative blood culture but a positive urinary antigen test (UAT) were identified in a Dutch hospital. Mortality of patients up to 10years after diagnosis was compared with age- and sex-matched life-expectancy data of the general population. Multivariable Cox regression analysis was used to study predictors for mortality in invasive pneumonia patients and to adjust for confounders comparing mortality between bacteremic and NI/NB/UAT-positive pneumonia patients. RESULTS: Of 228 invasive pneumonia patients 17% died within 30days and in 30-day survivors cumulative long-term mortality at 1 and 5years were 16% and 39% as compared with 3% and 15% in age- and sex-matched persons. High mortality was largely dependent on pre-existent comorbidities. In invasive pneumonia patients who survived the first 30days, age, male gender, chronic cardiovascular disease, malignancy and PCV7 serotype disease were independent predictors for higher long-term mortality. For bacteremic pneumonia patients (n=128) 30-day mortality was 16% and almost similar to 14% in NI/NB/UAT-positive pneumococcal pneumonia patients (n=170). In 30-day survivors of bacteremic pneumonia (n=108, median age 66years), cumulative mortality at 1 and 3years were 13% and 29% as compared with 18% and 40% in NI/NB/UAT-positive pneumococcal pneumonia patients (n=146, median age 67years) without a significant difference in mortality. CONCLUSIONS: Approximately 40% of all patients, who survived the first 30days after presentation with non-pneumonia IPD and pneumococcal pneumonia died within the following 5years. High long-term mortality was largely dependent on pre-existent comorbidity.

Expression of the Gene for Autotransporter AutB of Neisseria meningitidis Affects Biofilm Formation and Epithelial Transmigration.

Neisseria meningitidis is a Gram-negative bacterium that resides as a commensal in the upper respiratory tract of humans, but occasionally, it invades the host and causes sepsis and/or meningitis. The bacterium can produce eight autotransporters, seven of which have been studied to some detail. The remaining one, AutB, has not been characterized yet. Here, we show that the autB gene is broadly distributed among pathogenic Neisseria spp. The gene is intact in most meningococcal strains. However, its expression is prone to phase variation due to slipped-strand mispairing at AAGC repeats located within the DNA encoding the signal sequence and is switched off in the vast majority of these strains. Moreover, various genetic disruptions prevent autB expression in most of the strains in which the gene is in phase indicating a strong selection against AutB synthesis. We observed that autB is expressed in two of the strains examined and that AutB is secreted and exposed at the cell surface. Functionality assays revealed that AutB synthesis promotes biofilm formation and delays the passage of epithelial cell layers in vitro. We hypothesize that this autotransporter is produced during the colonization process only in specific niches to facilitate microcolony formation, but its synthesis is switched off probably to evade the immune system and facilitate human tissue invasion.

Toll-like receptor 9 enhances bacterial clearance and limits lung consolidation in murine pneumonia caused by methicillin resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen in pneumonia, associated with severe lung damage. Tissue injury causes release of Damage Associated Molecular Patterns (DAMPs), which may perpetuate inflammation. DNA has been implicated as a DAMP that activates inflammation through Toll-like receptor (TLR)9. The aim of this study was to evaluate the role of TLR9 in MRSA pneumonia. Wild-type (Wt) and TLR9 knockout (tlr9-/-) mice were infected intranasally with MRSA USA300 (BK 11540) (5E7CFU) and euthanized at 6,24,48 or 72 hours for analyses. MRSA pneumonia was associated with profound release of cell-free host DNA in the airways, as reflected by increases in nucleosome and DNA levels in bronchoalveolar lavage fluid (BALF), accompanied by transient detection of pathogen DNA in MRSA-free BALF supernatants. In BALF, as compared to Wt -mice tlr9-/- mice showed reduced TNFα and IL-6 levels at 6 hours and reduced bacterial clearance at 6 and 24 hours post infection. Furthermore, tlr9-/- mice exhibited a greater influx of neutrophils in BALF and increased lung consolidation at 24 and 48 hours. This study demonstrates the release of host- and pathogen-derived TLR9 ligands (DNA) into the alveolar space after infection with MRSA via the airways and suggests that TLR9 has pro-inflammatory effects during MRSA pneumonia associated with enhanced bacterial clearance and limitation of lung consolidation.

V-akt murine thymoma viral oncogene homolog 3 (AKT3) contributes to poor disease outcome in humans and mice with pneumococcal meningitis.

Pneumococcal meningitis is the most common and severe form of bacterial meningitis. Fatality rates are substantial, and long-term sequelae develop in about half of survivors. Here, we have performed a prospective nationwide genetic association study using the Human Exome BeadChip and identified gene variants in encoding dynactin 4 (DCTN4), retinoic acid early transcript 1E (RAET1E), and V-akt murine thymoma viral oncogene homolog 3 (AKT3) to be associated with unfavourable outcome in patients with pneumococcal meningitis. No clinical replication cohort is available, so we validated the role of one of these targets, AKT3, in a pneumococcal meningitis mouse model. Akt3 deficient mice had worse survival and increased histopathology scores for parenchymal damage (infiltration) and vascular infiltration (large meningeal artery inflammation) but similar bacterial loads, cytokine responses, compared to wild-type mice. We found no differences in cerebrospinal fluid cytokine levels between patients with risk or non-risk alleles. Patients with the risk genotype (rs10157763, AA) presented with low scores on the Glasgow Coma Scale and high rate of epileptic seizures. Thus, our results show that AKT3 influences outcome of pneumococcal meningitis.

Community-acquired bacterial meningitis in adults with cancer or a history of cancer.

OBJECTIVES: To study the incidence, clinical presentation, causative bacteria, and outcome of community-acquired bacterial meningitis in adults with cancer. METHODS: We evaluated incidence and characteristics of patients with cancer included in a nationwide prospective cohort study of adults with community-acquired meningitis performed in the Netherlands from March 1, 2006, to September 31, 2014. All patients underwent a neurologic examination at hospital discharge, and outcome was graded using the Glasgow Outcome Scale. RESULTS: Active cancer was identified in 68 of 1,351 episodes (5%) and a history of cancer in 87 (6%). The annual incidence of community-acquired bacterial meningitis was 2.71-fold (95% confidence interval [CI] 1.68-4.36, p < 0.001) increased for patients with cancer compared to patients without cancer in 2010, and 3.52-fold (95% CI 2.16-5.73, p < 0.001) in 2013. The clinical presentation of bacterial meningitis in patients with cancer compared to patients without cancer was similar. Patients with active cancer presented with lower leukocyte count in blood (12.1 × 10(9) cells/L vs 17.3 × 10(9) cells/L, p < 0.001) and CSF (670 cells/mm(3) vs 2,567 cells/mm(3), p < 0.001) and were more likely to be infected with Listeria monocytogenes (21% vs 5%, p < 0.001) than patients without cancer. Active cancer was identified as an independent risk factor for unfavorable outcome in bacterial meningitis (odds ratio 1.85, 95% CI 1.09-3.13). CONCLUSIONS: One of 8 patients with community-bacterial meningitis was identified to have a history of cancer and cancer was considered active in half of these patients. Patients with active cancer present with lower CSF leukocyte counts, are more likely to be infected with L monocytogenes, and are at high risk of unfavorable outcome.

Clinical, environmental, and serologic surveillance studies of melioidosis in Gabon, 2012-2013.

Burkholderia pseudomallei, an environmental gram-negative bacillus, is the causative agent of melioidosis and a bio-threat agent. Reports of B. pseudomallei isolation from soil and animals in East and West Africa suggest that melioidosis might be more widely distributed than previously thought. Because it has been found in equatorial areas with tropical climates, we hypothesized that B. pseudomallei could exist in Gabon. During 2012-2013, we conducted a seroprevalance study in which we set up microbiology facilities at a large clinical referral center and prospectively screened all febrile patients by conducting blood cultures and testing for B. pseudomallei and related species; we also determined whether B. pseudomallei could be isolated from soil. We discovered a novel B. pseudomallei sequence type that caused lethal septic shock and identified B. pseudomallei and B. thailandensis in the environment. Our data suggest that melioidosis is emerging in Central Africa but is unrecognized because of the lack of diagnostic microbiology facilities.

Host-pathogen Interaction at the Intestinal Mucosa Correlates With Zoonotic Potential of Streptococcus suis.

BACKGROUND: Streptococcus suis has emerged as an important cause of bacterial meningitis in adults. The ingestion of undercooked pork is a risk factor for human S. suis serotype 2 (SS2) infection. Here we provide experimental evidence indicating that the gastrointestinal tract is an entry site of SS2 infection. METHODS: We developed a noninvasive in vivo model to study oral SS2 infection in piglets. We compared in vitro interaction of S. suis with human and porcine intestinal epithelial cells (IEC). RESULTS: Two out of 15 piglets showed clinical symptoms compatible with S. suis infection 24-48 hours after ingestion of SS2. SS2 was detected in mesenteric lymph nodes of 40% of challenged piglets. SS2 strains isolated from patients showed significantly higher adhesion to human IEC compared to invasive strains isolated from pigs. In contrast, invasive SS9 strains showed significantly higher adhesion to porcine IEC. Translocation across human IEC, which occurred predominately via a paracellular route, was significantly associated with clonal complex 1, the predominant zoonotic genotype. Adhesion and translocation were dependent on capsular polysaccharide production. CONCLUSIONS: SS2 should be considered a food-borne pathogen. S. suis interaction with human and pig IEC correlates with S. suis serotype and genotype, which can explain the zoonotic potential of SS2.

Epidemiology of invasive meningococcal disease in the Netherlands, 1960-2012: an analysis of national surveillance data.

BACKGROUND: Epidemiological data for invasive meningococcal disease is essential for public health policy and vaccine development. We analysed national surveillance data from the Netherlands for PorA coverage of two PorA-based meningococcal serogroup B vaccines to describe the epidemiology of invasive meningococcal disease. METHODS: We examined national surveillance data from the Netherlands Reference Laboratory of Bacterial Meningitis (NRLBM) for cases of culture-positive invasive meningococcal disease from Jan 1, 1960, to Jan 1, 2013. We included cases with a meningococcal isolate cultured from cerebrospinal fluid, blood, skin biopsy, or all, and cases with a positive cerebrospinal fluid latex agglutination test, counter current electrophoresis test, or positive cerebrospinal fluid PCR for Neisseria meningitidis. To test for completeness of case ascertainment, we compared data of the NRLBM with those of the Dutch National institute for public health and the environment (RIVM) to which notification was compulsory. We did serogrouping by ouchterlony gel diffusion. We tested susceptibility of meningococcal strains by agar dilution and E test. We tested differences between proportions with the Pearson χ(2) test or Fisher's exact test, and differences in frequencies between time periods with the Mann-Whitney U test. We defined penicillin resistance as MIC of 1·0 µg/mL or higher. FINDINGS: Annual incidence rates of invasive meningococcal disease per 100 000 population increased from 0·5 in 1960, to 4·5 in 2001, and subsequently decreased to 0·6 in 2012. Median age increased from 1·8 years in 1960, to 6·1 years in 2012 for all serogroups. The proportion of blood culture positive cases increased from 4% in 1960, to 60% in 2012 (p<0·0001). Serogroup B was the most common serogroup over time, 64% of isolates were from ST-41/44 complex. We established PorA finetype of 4133 isolates, 19 of 252 variable regions covered 99% of sequenced serogroup B cases. Coverage of the 4CMenB PorA component was 4% in 1960-65, and 36% in 2006-12. In response to a serogroup C epidemic (1999-2001), serogroup C conjugate vaccine was introduced, which reduced serogroup C disease by 95%. Since 2003, serogroup Y disease emerged and serogroup A disease disappeared. We identified evidence of capsular switching, but not of serogroup replacement. The rate of reduced penicillin susceptibility increased to 37% from 1993 to 2012, but penicillin resistance was not recorded. INTERPRETATION: Incidence of invasive meningococcal disease has decreased, but decreasing rates of penicillin susceptibility and the possible resurgence of this devastating disease remain a threat to public health. Our long-term serosubtyping and porA sequencing data is valuable for the assessment of vaccine coverage and future serogroup B vaccine development. FUNDING: National Institute of Public Health and the Environment, the European Union's seventh Framework programme, Netherlands Organization for Health Research and Development, Academic Medical Center, and the European Research Council.

Meningitis caused by a lipopolysaccharide deficient Neisseria meningitidis.

OBJECTIVE: Lipopolysaccharide (LPS) is a major component of the Neisseria meningitidis outer membrane. Here we report a patient with meningococcal meningitis of which the causative isolate lacked LPS. Thus far, no naturally occurring LPS-deficient meningococcal isolate has been known to cause clinical disease. METHODS: We used SDS-PAGE, silver staining and LPS-specific antibodies in whole cell ELISA to determine LPS presence in the causative isolate. Meningococcal whole genome sequencing was performed using Roche 454-sequencing. The N. meningitidis strain MC58 was used to compare all LPS biosynthesis associated genes. We compared growth characteristics of Escherichia coli transformed with a plasmid containing 2 lpxH types. RESULTS: The patient presented with isolated thunderclap headache. Analysis of the causative N. meningitidis showed no LPS. Whole genome sequencing revealed a mutation located in lpxH explaining LPS-deficiency. Expression of this lpxH variant in E. coli resulted in growth impairment compared to E. coli expressing the meningococcal wild type lpxH variant. In addition, inactivating lpxH in N. meningitidis H44/76 by insertional inactivation with a kanamycin cassette resulted in a LPS-deficient phenotype. CONCLUSIONS: We describe invasive meningococcal disease caused by a naturally occurring LPS-deficient meningococcal isolate.

Leukocyte attraction by CCL20 and its receptor CCR6 in humans and mice with pneumococcal meningitis.

We previously identified CCL20 as an early chemokine in the cerebrospinal fluid (CSF) of patients with pneumococcal meningitis but its functional relevance was unknown. Here we studied the role of CCL20 and its receptor CCR6 in pneumococcal meningitis. In a prospective nationwide study, CCL20 levels were significantly elevated in the CSF of patients with pneumococcal meningitis and correlated with CSF leukocyte counts. CCR6-deficient mice with pneumococcal meningitis and WT mice with pneumococcal meningitis treated with anti-CCL20 antibodies both had reduced CSF white blood cell counts. The reduction in CSF pleocytosis was also accompanied by an increase in brain bacterial titers. Additional in vitro experiments showed direct chemoattractant activity of CCL20 for granulocytes. In summary, our results identify the CCL20-CCR6 axis as an essential component of the innate immune defense against pneumococcal meningitis, controlling granulocyte recruitment.

Association between population prevalence of smoking and incidence of meningococcal disease in Norway, Sweden, Denmark and the Netherlands between 1975 and 2009: a population-based time series analysis.

OBJECTIVE: To investigate the relationship between the prevalence of smoking in the population and incidence of invasive meningococcal disease (IMD) among children under 5 years of age. DESIGN: Retrospective, longitudinal, observational study. Poisson regression controlled for confounding factors. SETTING: Norway, Sweden, Denmark and the Netherlands between 1975 and 2009. POPULATION: Total population of approximately 35 million people in these four countries. DATA SOURCES: Data were collected from the Ministries of Health, National Statistics Bureaus and other relevant national institutes. RESULTS: In Norway, there was a significant positive relationship between the annual prevalence of daily smokers among individuals aged 25-49 years and the incidence of IMD in children under 5 years of age, unadjusted (RR=1.04-1.06, 95% CI 1.02 to 1.07, p<0.001) and after adjustment for time of year (quarter), incidence of influenza-like illness and household crowding (RR=1.05-1.07, 95% CI 1.03 to 1.09, p<0.001). Depending on age group, the risk of IMD increased by 5.2-6.9% per 1% increase in smoking prevalence among individuals aged 25-49 years in adjusted analyses. Using limited datasets from the three other countries, unadjusted analysis showed positive associations between IMD in children related to older smokers in Sweden and the Netherlands and negative associations related to younger smokers in Sweden. However, there were no demonstrable associations between incidence of IMD and prevalence of smoking, after adjustment for the same confounding variables. CONCLUSIONS: The reduced incidence of IMD in Norway between 1975 and 2009 may partly be explained by the reduced prevalence of smoking during this period. High-quality surveillance data are required to confirm this in other countries. Strong efforts to reduce smoking in the whole population including targeted campaigns to reduce smoking among adults may have a role to play in the prevention of IMD in children.

Meningococcal serogroup Y lpxL1 variants from South Africa are associated with clonal complex 23 among young adults.

OBJECTIVES: To determine the genotypes of serogroup Y meningococcus (MenY), and to determine the prevalence of and identify factors associated with MenY lpxL1 variants. METHODS: Isolates, collected from 2003 to 2007 through national surveillance for invasive meningococcal disease, were characterized by multilocus sequence typing and screened for interleukin-6 induction. LpxL1 genes were sequenced from low IL-6 inducers. RESULTS: MenY represented 13% (n = 219/1702) of meningococcal disease. Clonal complex (cc) 175, ST-23/Cluster A3 (cc23), cc11 and cc167 accounted for 82% (176/214), 11% (24/214), 3% (6/214) and 3% (7/214) respectively. Low cytokine induction was evident in 15% (32/218). Cc23 isolates (24/24) had an lpxL1 mutation, while among the remaining isolates the proportion of lpxL1 variants was 4% (8/189, p < 0.001), and these were all cc175. Compared to wild type isolates, lpxL1 variants were associated with patients aged 5-14 years [unadjusted OR (95% CI): 4.3 (1.5-12)] or 15-24 years [unadjusted OR (95% CI): 9.1 (2.8-29)] compared to children <5 years; and were more likely have been isolated from CSF than blood [unadjusted OR (95% CI): 3.5 (1-11.9)]. On multivariable analysis, age remained significant [adjusted OR (95% CI), 5-14 years: 4.2 (1.5-12); 15-24 years: 8.9 (2.7-29)]. CONCLUSION: LpxL1 variants were associated with cc23 among young adults.

Genetic variation in inflammasome genes is associated with outcome in bacterial meningitis.

Bacterial meningitis is a severe and deadly disease, most commonly caused by Streptococcus pneumoniae. Disease outcome has been related to severity of the inflammatory response in the subarachnoid space. Inflammasomes are intracellular signaling complexes contributing to this inflammatory response. The role of genetic variation in inflammasome genes in bacterial meningitis is largely unknown. In a prospective nationwide cohort of patients with pneumococcal meningitis, we performed a genetic association study and found that single-nucleotide polymorphisms in the inflammasome genes CARD8 (rs2043211) and NLRP1 (rs11621270) are associated with poor disease outcome. Levels of the inflammasome associated cytokines interleukin (IL)-1ß and IL-18 in cerebrospinal fluid also correlated with clinical outcome, but were not associated with the CARD8 and NLRP1 polymorphisms. Our results implicate an important role of genetic variation in inflammasome genes in the regulation of inflammatory response and clinical outcome in patients with bacterial meningitis.

Association of chronic meningococcemia with infection by meningococci with underacylated lipopolysaccharide.

OBJECTIVES: Chronic meningococcemia is an uncommon manifestation of meningococcal disease. Our objective was to asses whether a bacterial factor, a mutation in the lpxL1 gene resulting in underacylated lipopolysaccharide, might be important in chronic meningococcemia. METHODS: We identified 15 patients with chronic meningococcemia over a 50-year period. Chronic meningococcemia episodes were defined by a febrile episode of at least one week and presence of meningococci in blood and/or cerebrospinal fluid (CSF). Meningococcal isolates from these patients were characterised by serogrouping, multi-locus sequence typing, and in vitro interleukin 6 inducing capacity. lpxL1 gene mutations were determined by direct sequencing. RESULTS: The median age was 21 years (range, 2-62) and median duration of symptoms before diagnosis was four weeks (range, 1-12). Of the 15 isolates, seven (47%) strains had a reduced interleukin 6 inducing capacity and were found to have a mutation in lpxL1 resulting in penta-acylated lipid A. This frequency is higher than previously reported among adult patients with meningococcal meningitis (7%; p < 0.0001) and invasive meningococcal disease (9%; p = 0.001). CONCLUSIONS: We conclude that chronic meningococcemia patients are often infected with meningococci with a mutation in lpxL1 resulting in underacylated lipid A. The lpxL1 mutations may well explain the protracted and benign clinical course in these patients.

Complement factor 7 gene mutations in relation to meningococcal infection and clinical recurrence of meningococcal disease.

Meningococcal disease is caused by Neisseria meningitidis which is associated with high morbidity and mortality. Recurrences of meningococcal infection have been observed in patients with terminal complement component defects, because of the inefficient formation of the lytic membrane attack complex (MAC), C5b-9. Complement component C7 is one of the five plasma proteins to form the MAC. The gene C7 may carry mutations that cause functional abnormalities or the mere absence of the C7 protein. More than 200 patients were screened for aberrant C7 protein by isoelectric focusing (C7 IEF). These were compared with patients in whom recurrent meningococcal infection had resulted in the diagnosis of complete C7 absence (C7Q0). A higher proportion of C7 IEF variants were found in meningitis cases compared to controls (p=0.03). In contrast to C7Q0 patients, recurrent meningococcal infection was never observed in C7 IEF cases. Whereas C7Q0 sera were defective in meningococcal serogroup B and W135 killing assays, the sera of patients with C7 IEF variants were only defective in complement-mediated killing when classical pathway activation by (endogenous) anti-meningococcal antibodies was blocked. Upon sequence analysis we characterized the genetic background of the C7*6 and C7*8 IEF pattern and identified three novel C7 gene mutations in 13 C7Q0 patients. In conclusion, C7 IEF variants can determine meningococcal killing in the early stage of infection when antibody-independent killing prevails. The results endorse the lack of clinical recurrences once antibodies are present, whereas in C7Q0 patients the anti-meningococcal antibodies may not suffice to protect from recurrent meningococcal infection.