MBL2 deficiency is associated with higher genomic bacterial loads during meningococcemia in young children.

Mannose binding lectin (MBL2) is a soluble pattern recognition receptor that is key to generating innate immune responses to invasive infection, including against the cardinal Gram-negative bacterium Neisseria meningitidis. Individuals homozygous or heterozygous for any of three variant alleles of MBL2 (O/O or A/O genotypes) have deficient concentrations of MBL2 in circulating blood, but previous studies linking MBL deficiency to susceptibility to meningococcal disease have not revealed a consistent association. We genotyped 741 patients with microbiologically-proven meningococcal disease and correlated MBL2 genotype with plasma bacterial load of N. meningitidis with blood samples taken during hospital admission. We show that individuals with genotypes compatible with MBL2 deficiency have higher measurable levels of bacterial plasma genomic load with the greatest effect seen in children <2 years of age. However, the overall impact of this is minor, because there was no evidence that such genotypes are more common in children with meningococcal disease compared with uninfected cohorts. The findings suggest that MBL2 supports innate immune defence against meningococcal disease in the early months of life, before acquired immunity is sufficiently robust for effective natural protection.

Usefulness of porA sequencing in distinguishing sporadic and linked cases of serogroup B invasive meningococcal disease in Suffolk, United Kingdom, December 2009 to January 2010.

A cluster of three fatal cases of invasive meningococcal disease due to Neisseria meningitidis serogroup Bin a town in Suffolk, United Kingdom, during December 2009 to January 2010 was reported to the local Health Protection Unit. This paper describes the investigation undertaken to identify any potential epidemiological links among the cases, to determine if this was an outbreak and to consider whether to implement community-wide interventions and control measures. Case epidemiological information in addition to serogroup and genosubtyping (porA gene sequencing) data of the infecting organism was gathered on all cases in this reported cluster. Genosubtyping was also retrospectively requested for all serogroup B cases confirmed in Suffolk during 2009. Extensive investigation failed to establish an epidemiological link among the cluster of fatal cases of serogroup B invasive meningococcal disease in Suffolk. By demonstrating a number of distinct strains, the genosubtyping of isolates proved to be useful in the public health management of this incident by serving to exclude a community outbreak and preventing unnecessary mass chemoprophylaxis.

Clusters of meningococcal disease in school and preschool settings in England and Wales: what is the risk?

AIMS: To assess the risk of further cases in educational settings in order to inform policy on managing cases and clusters of meningococcal disease. METHODS: Between 1 April 1995 and 31 March 2001, surveillance in preschool and school settings in England and Wales identified 114 clusters of meningococcal disease. Twenty clusters were reported in preschool settings, 43 in primary, 46 in secondary, and five in independent schools. Seventy three clusters (64%) consisted of two or more confirmed cases, of which 30 had two or more serogroup C cases. Following the introduction of the national meningococcal serogroup C vaccination programme in 1999, no serogroup C clusters were observed between April 2000 and March 2001. RESULTS: The relative risk of further cases in the four weeks after a single case compared with the background rate was raised in all settings, ranging from RR 27.6 (95% CI 15.2 to 39.9) in preschool settings to RR 3.6 (95% CI 2.5 to 4.6) in secondary schools. Absolute risk estimates ranged from 70/100 000 in preschool settings to 3.0/100 000 in secondary schools. The relative risk of clustering was similar for serogroup B and C strains. Most (68%) second cases occurred within seven days of the first case. CONCLUSIONS: Although there was a higher risk of further cases of meningococcal disease in schools and especially in preschool settings, it is not known whether widespread antibiotic use after single cases reduces risk of further cases and if there is a real risk of harm. Evidence of risk reduction is needed to inform public health policy.

Measurement and interpretation of pneumococcal IgG levels for clinical management.

The detection of pneumococcal IgG antibodies is helpful for the evaluation of response to pneumococcal vaccination and need for revaccination. Results generated by the clinical assay which is currently used, in which the 23 valent polysaccharide vaccine is the antigen, were compared to those obtained by a capsular polysaccharide serotype-specific assay that measures IgG antibodies to 9 common serotypes causing invasive disease. Discrepancies in 21/47 (45%) of the results were observed in a direct comparison between the two assays. In each case a positive titre was obtained on the clinical assay but IgG levels on the serotype-specific assay were below the putative protective level of 0.2 micro g/ml for at least one of the 9 serotypes assayed. The generation of false positives by the current clinical assay is due to its lack of specificity. Antibodies to C-polysaccharide and all of the 23 serotypes included in the pneumococcal polysaccharide vaccine are incorporated into the final titre whereas the serotype-specific assay adsorbs out noncapsular polysaccharide antibodies. The discrepancies between the two assays highlight the importance of standardized assays that measure putative correlates of protection and demonstrate the need to re-evaluate the current clinical assay. A tool that allows the interpretation of the results of the serotype-specific assay is provided and its potential for assessing individual susceptibility levels to vaccine preventable pneumococcal infection is discussed.

Enhanced surveillance scheme for suspected meningococcal disease in five regional health authorities in England: 1998.

Enhanced surveillance of meningococcal disease (ESMD) began in five English regions on 1st January 1998. The aims of the scheme were to obtain accurate incidence data and develop a robust surveillance system with which to monitor the impact of a new meningococcal serogroup C conjugate vaccine. During 1998, 2,314 suspected cases of meningococcal disease were identified. The majority (84%) was classified as invasive meningococcal disease, with infection of N. meningitidis confirmed in 66%. Sixteen per cent of suspected cases were subsequently given an alternative diagnosis. Age differences between those classified as meningococcal disease and those not, implied a higher index of suspicion of meningococcal disease in younger children. Regions with high rates of meningococcal disease were due to a higher rate of serogroup C. ESMD increased ascertainment of meningococcal disease and deaths. Cases were 34% greater than identified through statutory notifications, an additional 6.8% confirmed infections were identified than were reported to the PHLS Meningococcal Reference Unit (MRU) and deaths were 24% greater than death registrations. These data were used to inform the national meningococcal serogroup C conjugate vaccination programme in England and Wales. In 1999 ESMD was extended to all regions of England, Wales and Northern Ireland.

Estimating the burden of serogroup C meningococcal disease in England and Wales.

In 1999 a new conjugate vaccine for serogroup C meningococcal disease was licensed for use in the UK. In order for an appropriate vaccination strategy to be developed the burden of serogroup C disease in England and Wales needed to be established. This was done using data from an enhanced surveillance scheme alongside routine laboratory reports and a total of 5,052 cases of serogroup C disease in England and Wales between 1993 and 1998 were estimated. Among these, an estimated 398 died and 1,767 were admitted to intensive care units (ITUs). The greatest burden of disease was in young children and teenagers. The current literature identified four studies reporting sequelae following serogroup C meningococcal disease. These provided estimates of sequelae in the range of 6.5% and 45% and presented some evidence of higher levels than occur following serogroup B meningococcal disease. This information was provided to the Joint Committee on Vaccination and Immunisation to inform policy to implement a serogroup C conjugate vaccination programme in the UK. The vaccination programme has since been justified by the dramatic reduction in serogroup C meningococcal cases.

Meningococcal serogroup C conjugate vaccination in England and Wales: coverage and initial impact of the campaign.

The UK was the first country to introduce meningococcal serogroup C conjugate vaccination. The vaccine was incorporated into the routine infant immunisation schedule and was offered to all under 18 year olds in a catch-up campaign. The vaccine has been well accepted in infants receiving routine vaccination, with coverage around 89%. Coverage in older children targeted in the catch-up campaign was above 85% up to the age of 14, and was lowest (43%) in 15-17 year olds not in education. The winter of 2000-01 was the first meningococcal season following the offer of the vaccination to all children and adolescents. The incidence of serogroup C disease in the targeted age groups fell by 80%, and the number of deaths in laboratory confirmed cases in 0-19 year olds decreased from 78 to 8 between 1998-99 and 2000-01. The incidence of serogroup B disease in all age groups was slightly higher in 2000-01 than previous years, and there was an increase in the incidence of serogroup C disease in those aged over 20 during the study period, leading to the extension of the vaccination campaign to 20-24 year olds.

Improved case confirmation in meningococcal disease with whole blood Taqman PCR.

BACKGROUND: The clinical diagnosis of meningococcal disease (MCD) can be difficult. Non-culture methods like the previous ELISA meningococcal PCR improved case confirmation rates, but were not ideal. A Taqman meningococcal PCR, using DNA extracted from serum (S-Taqman), which has an improved sensitivity compared to the ELISA method in vitro, was introduced into clinical practice in July 1997. A new whole blood DNA extraction method for Taqman (WB-Taqman) was introduced in September 1999. AIMS: To determine the degree of improvement in the confirmation rate in clinically diagnosed MCD, following the introduction of WB-Taqman. METHODS: A total of 192 patients (WB-Taqman) with possible or probable MCD, including those admitted to our paediatric intensive care unit, were studied. Admission EDTA samples obtained were sent for bacterial DNA detection at the Meningococcal Reference Unit (MRU), Manchester. These patients were compared to 319 patients with possible and probable MCD, seen at the same hospital prior to the introduction of WB-Taqman. RESULTS: Following the introduction of WB-Taqman, 82 of the 95 probable cases (88%) had a positive meningococcal PCR result. This gives a diagnostic sensitivity and specificity for WB-Taqman of 87% and 100% respectively. Following WB-Taqman all blood culture positive patients were also PCR positive. Confirmation of cases by PCR rose from 47% (S-Taqman, n = 166) to 88% (WB-Taqman). When all confirmatory tests were included, case confirmation increased from 72% (S-Taqman) to 94% (WB-Taqman). CONCLUSION: The sensitivity of PCR in confirming clinical MCD has improved significantly with this new method. The gold standard for confirming cases of MCD is now the WB-Taqman PCR.

Ultrasound enhanced detection of individual meningococcal serogroups by latex immunoassay.

AIMS: To examine A, C, Y, and W135 Neisseria meningitidis serogroup characterisation by ultrasonic standing wave enhanced latex agglutination tests (USELATs) of clinical samples. In addition, to determine USELAT enhancement of detection sensitivity for the individual antigens compared with conventional card latex agglutination tests (LATs). METHODS: Wellcogen (Abbott Murex), Slidex meningite kit 5 (bioMerieux), and Pastorex (Sanofi) kits and beads coated in house with antibodies to Y and to W135 alone were tested. Positive control antigens consisted of A and C polysaccharide preparations and the Pastorex Y/W135 kit sample. The limiting concentrations of antigen detection were determined by USELAT and by LAT. Thirty five clinical samples (plasma), previously characterised by the polymerase chain reaction (PCR) and culture, were tested by USELAT and, when sample volume allowed, by LAT. RESULTS: USELAT enhancement of control antigen detection ranged from 16 to 128 fold for the different latex systems. Enhancements for the different control antigens were comparable between kits. USELAT tests of clinical (A/C/Y/W135) samples (n = 15) with the Wellcogen (A/C/Y/W135) and Slidex meningite (A/C/Y/W135) kits showed comparable specificities. A set (n = 22) of Y and W135 samples gave 18, 19, and 17 positive results for Wellcogen (A/C/Y/W135), Pastorex (A/C/Y/W135), and in house beads (Y/W135), respectively. Positive USELAT PCR and culture results were concordant. A typical sensitivity for the commercial kits was 80% (Wellcogen). CONCLUSIONS: USELAT identified serogroups for 80% of samples, whereas LATs identified only 40%. The USELAT detection of the A, C, Y, and W135 antigen serogroups showed comparable enhancement for the kits tested. The commercial availability of latex beads coated with antibody to the Y and W135 serogroups would expedite their identification.

Meningococcal bacterial DNA load at presentation correlates with disease severity.

AIMS: To determine bacterial loads in meningococcal disease (MCD), their relation with disease severity, and the factors which determine bacterial load. METHODS: Meningococcal DNA quantification was performed by the Taqman PCR method on admission and sequential blood samples from patients with MCD. Disease severity was assessed using the Glasgow Septicaemia Prognostic Score (GMSPS, range 0-15, severe disease > or =8). RESULTS: Median admission bacterial load was 1.6 x 10(6) DNA copies/ml of blood (range 2.2 x 10(4) to 1.6 x 10(8)). Bacterial load was significantly higher in patients with severe (8.4 x 10(6)) compared to milder disease (1.1 x 10(6), p = 0.018). This difference was greater in septicaemic patients (median 1.6 x 10(7) versus 9.2 x 10(5), p < 0.001). Bacterial loads were significantly higher in patients that died (p = 0.017). Admission bacterial load was independent of the duration of clinical symptoms prior to admission, with no difference between the duration of symptoms in mild or severe cases (median, 10.5 and 11 hours respectively). Bacterial loads were independent of DNA elimination rates following treatment. CONCLUSION: Patients with MCD have higher bacterial loads than previously determined with quantitative culture methods. Admission bacterial load is significantly higher in patients with severe disease (GMSPS > or =8) and maximum load is highest in those who die. Bacterial load is independent of the duration of clinical symptoms or the decline in DNA load.

Risk of laboratory-acquired meningococcal disease.

Five probable secondary cases of meningococcal disease were identified in microbiology laboratory workers in England and Wales during a 15-year period. All cases had prepared suspensions of Neisseria meningitidis outside a safety cabinet upto seven days before onset of illness. Relative risk in laboratory workers compared with the background adult population was 184 (95% CI 60-431). In view of the potentially serious outcome from this infection, a safety cabinet should always be used when preparing or working with suspensions of meningococci. Vaccination policy for microbiology laboratory workers should be reviewed.

Preventing secondary meningococcal disease in health care workers: recommendations of a working group of the PHLS meningococcus forum.

Based on new data on the risk of secondary meningococcal disease in health care workers, a review of published cases and an assessment of the available evidence, a change to the recommendations for giving chemoprophylaxis to health care workers in England and Wales is proposed. Previous guidance recommended prophylaxis only for those who had given mouth to mouth resuscitation. Chemoprophylaxis is now recommended for health care workers whose mouth or nose has been directly and heavily exposed to respiratory droplets/secretions from a case of meningococcal disease around the time of hospital admission. Wearing surgical face masks is encouraged to reduce risk of exposure.

Prevention of meningococcal infection in laboratory workers--an audit of practice in England and Wales.

A survey of clinical microbiology laboratories in England and Wales found that in 12% of laboratories a class 1 microbiological safety cabinet was not always used for manipulating suspensions of live meningococci. The recommendation that a safety cabinet should always be used for such purposes needs reiteration and implementation.

A functional polymorphism of toll-like receptor 4 is not associated with likelihood or severity of meningococcal disease.

Human Toll-like receptor 4 (TLR4) transduces proinflammatory cytokine release by human cells in response to lipopolysaccharide (LPS). This study tested the hypothesis that, if TLR4 is rate limiting for a successful response to bacterial LPS in humans, a human gene polymorphism that results in the amino acid substitution Asp299Gly and causes reduced expression and function of TLR4 should influence susceptibility to or severity of natural gram-negative infection. The allele frequency of the Asp299Gly polymorphism was 5.9% among 879 blood donors, 6.5% among 1047 patients with microbiologically proven meningococcal disease, and 4.1% among 86 patients who died of meningococcal disease. No significant differences were observed, including those analyzed after stratification of the infected population by age and by meningococcal serogroup. Therefore, this functional TLR4 polymorphism does not influence susceptibility to or severity of meningococcal disease.

Simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in suspected cases of meningitis and septicemia using real-time PCR.

A single-tube 5' nuclease multiplex PCR assay was developed on the ABI 7700 Sequence Detection System (TaqMan) for the detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae from clinical samples of cerebrospinal fluid (CSF), plasma, serum, and whole blood. Capsular transport (ctrA), capsulation (bexA), and pneumolysin (ply) gene targets specific for N. meningitidis, H. influenzae, and S. pneumoniae, respectively, were selected. Using sequence-specific fluorescent-dye-labeled probes and continuous real-time monitoring, accumulation of amplified product was measured. Sensitivity was assessed using clinical samples (CSF, serum, plasma, and whole blood) from culture-confirmed cases for the three organisms. The respective sensitivities (as percentages) for N. meningitidis, H. influenzae, and S. pneumoniae were 88.4, 100, and 91.8. The primer sets were 100% specific for the selected culture isolates. The ctrA primers amplified meningococcal serogroups A, B, C, 29E, W135, X, Y, and Z; the ply primers amplified pneumococcal serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10A, 11A, 12, 14, 15B, 17F, 18C, 19, 20, 22, 23, 24, 31, and 33; and the bexA primers amplified H. influenzae types b and c. Coamplification of two target genes without a loss of sensitivity was demonstrated. The multiplex assay was then used to test a large number (n = 4,113) of culture-negative samples for the three pathogens. Cases of meningococcal, H. influenzae, and pneumococcal disease that had not previously been confirmed by culture were identified with this assay. The ctrA primer set used in the multiplex PCR was found to be more sensitive (P < 0.0001) than the ctrA primers that had been used for meningococcal PCR testing at that time.

Efficacy of meningococcal serogroup C conjugate vaccine in teenagers and toddlers in England.

The UK was the first country to use meningococcal serogroup C conjugate (MCC) vaccines, which were licensed on the basis of immunogenicity and safety data but without a formal efficacy study. Increased surveillance during the first 9 months since introduction has shown that short-term efficacy of the MCC vaccine in England was 97% (95% CI 77-99) for teenagers and 92% (65-98) for toddlers. These early results confirm the superiority of MCC over plain C polysaccharide vaccines, which are ineffective in young children.

Meningococcal serology.

Meningococcal serology has been mainly used over the last 20 years in the field of vaccinology, to evaluate candidate vaccines and quantify individuals' immune responses. With the increasing usage of pre-admission antibiotic treatment (1), nonculture diagnostic methods such as polymerase chain reaction (PCR) ( Chapter 3 ), antigen detection ( Chapter 4 ), and serology have become important tools. Nonculture diagnosis of meningococcal disease is rapidly becoming of equal importance for the confirmation of meningococcal infection as the isolation of Neisseria meningitidis organisms (2). This has occurred at a time when accurate case ascertainment of meningococcal disease has become a crucial aspect of assessing effectiveness of the recently introduced serogroup C polysaccharide-protein conjugate vaccine in the UK (3). N. meningitidis polysaccharide vaccines have been available for over 20 years (4) and evaluation of candidate vaccines and assessment of levels of antibody requires accurate and reproducible assays.

Improved methods of detection of meningococcal DNA from oropharyngeal swabs from cases and contacts of meningococcal disease.

In the UK the increasing use of pre-admission parenteral antibiotic therapy in meningococcal disease has lessened the value of routine cultures as a tool to confirm diagnosis, and laboratory confirmation of invasive meningococcal infection is achieved increasingly by non-culture, nucleic acid amplification methods. The purpose of this study was to evaluate a DNA extraction and meningococcal-specific DNA amplification methodology for detection of meningococci from oropharyngeal swabs. One hundred and six swabs from suspected or confirmed cases of meningococcal disease, and 94 swabs from contacts of meningococcal disease cases were examined. Of laboratory-confirmed cases, 38/65 (58.5%) yielded a positive oropharyngeal swab PCR result and 5/24 (20.8%) swabs from suspected but laboratory-unconfirmed cases were PCR positive. No significant differences in PCR positivity rates were found between the types of swab transport systems utilized, but transport time to the testing laboratory was found to affect PCR positivity (P < 0.05). Application of meningococcus-specific PCR to oropharyngeal swabs, in addition to routine culture of swabs, can provide valuable epidemiological information as well as case confirmation for contact management. PCR amplification of meningococcal PCR from oropharyngeal swabs will also increase the ascertainment in swabbing surveys carried out as part of meningococcal disease outbreak investigation and management.

Carriage of Neisseria meningitidis in residents and staff at a residential home for elderly people following a case of invasive disease.

A study of the carriage of Neisseria meningitidis among staff and residents of a 'closed community' (a residential home for elderly people) was conducted after a resident developed invasive meningococcal disease. All 39 other residents and 49 staff who worked at the home during the previous seven days were offered a throat swab and 38 residents and 49 staff consented. Two residents (none in the index case's social group) and one staff member were found to be carrying N. meningitidis, all phenotypically distinct from the infecting isolate. Thus, all four individuals carried different organisms and the index case was sporadic. Our findings suggest that residents or staff in long stay residential settings where a sporadic case occurs need not be offered meningococcal prophylaxis unless they fulfil the existing definition of 'close personal contacts' of the case.

An interleukin-1 genotype is associated with fatal outcome of meningococcal disease.

To determine whether known variants of the interleukin-1 (IL-1) and tumor necrosis factor (TNF) gene families are associated with severe manifestations of meningococcal disease, 276 white patients 4-70 years of age (median, 17 years) were genotyped. All patients had microbiologically proven Neisseria meningitidis infection; 39 died and 237 survived. A significant association (P<.001) was found between fatal outcome and genotype at IL1B (nucleotide position -511). Homozygous individuals, both for the common (1/1) and the rare (2/2) alleles, had increased odds ratios (ORs) for death, compared with heterozygous individuals (1/2): ORs (95% confidence intervals [CIs]) were 3.39 (1.39-8.29) and 7.35 (2.51-21.45), respectively. The mortality rates according to genotype at IL1B (-511) were 18.0% (1/1), 6.1% (1/2), and 32.3% (2/2), compared with 14.2% overall. The composite genotype, consisting of heterozygosity of IL1B (-511) together with homozygosity of the common allele of the IL-1 receptor antagonist gene (IL1RN) at +2018, was significantly associated with survival (P=.018; OR, 7.78 [95% CI, 1. 05-59.05]). There was no association between TNF genotype and fatal outcome. These data suggest that IL-1 genotype influences the severity of meningococcal disease.