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An observational study to examine Streptococcus pneumoniae carriage in Norwegian children was initiated after two cases of pneumococcal meningitis, caused by the England(14)-9 clone, occurred in one day-care centre in Oslo. All children recruited from the day-care centre where the cases occurred were vaccinated with a seven-valent pneumococcal conjugate vaccine; the other participants who attended three other day-care centres nearby were not. The children were followed for 9 months, and three samplings took place. At the first visit, 45.7% of the children were colonised by pneumococci in the nasopharynx. The children harboured a variety of serotypes, with serotypes 6A, 23F, 6B and 19F being the most frequent. The numbers of children carrying vaccine serotypes decreased in both the vaccinated and the non-vaccinated groups. Thus, no significant effect of vaccine on carriage was detected in this relatively small study.

The antimicrobial resistance of 809 Salmonella Typhimurium isolates collected from humans in Norway between 1975 and 1998 was studied. The material was subdivided into domestic and foreign isolates according to whether the patient had recently travelled abroad or not. In imported isolates the largest increase in resistance was in 1996 when 35% of the isolates were multi-resistant. The first multi-resistant isolate acquired in Norway appeared in 1994, but already in 1998 23% of the isolates domestically acquired were multi-resistant, and a majority were S. Typhimurium DT104. We found no ciprofloxacin resistance in domestically acquired isolates. Amplified fragment length polymorphism analysis was performed on selected multi-resistant isolates. The method discriminated well between different multi-resistant isolates, but not between DT104 isolates. Resistant and multi-resistant S. Typhimurium were until 1998 essentially recovered from patients who had travelled abroad, but multi-resistant isolates, mainly DT104, are now also being transmitted within the country.

A new sulphonamide resistant (SR) C[ratio ]15[ratio ]P1.7,16 meningococcal strain, a variant of the ET-5 clone, dominated in an outbreak of 22 cases in western Norway commencing in 1995. The first eight patients were 15–21 years old from the Nordhordland area, initiating a carrier study in the local high schools. Carriage of SR serogroup C meningococci was detected by routine methods and treated with a single dose of ofloxacin 400 mg. Of 20 treated carriers, 14 harboured the outbreak strain C[ratio ]15[ratio ]P1.7,16. Vaccination of 4000 children, adolescents and close contacts of patients was also performed. After the intervention, 14 additional cases of meningococcal disease occurred, 8 due to the outbreak strain. However, incidence rates dropped from 180 to 30 per 100000 per year in the student population, but increased from 0 to 13 in the rest of the population in Nordhordland. Carriage eradication is not generally recommended in Norway. However, tracing and treating meningococcal carriage may have reduced transmission and disease in this outbreak situation.

The aim of this study was to investigate the antibiotic resistance rates of major bacterial pathogens causing bloodstream infections in two very different types of hospital in Norway. We examined all Escherichia coli and staphylococci (330 isolates) causing bloodstream infections from one general county hospital and one specialist national cancer hospital during the periods 1991–92 and 1995–96. Minimal inhibitory concentrations (MICs) were determined using the E-test. E. coli and staphylococci constituted 46.7% of all isolates from bloodstream infections in the two hospitals. Overall, E. coli isolates were resistant to amoxicillin (21%), trimethoprim (21%), doxycycline (20%) and trimethoprim-sulphamethoxazole (17%), while Staphylococcus aureus strains were resistant to benzylpenicillin (66%). No methicillin-resistant S. aureus was detected. Coagulase-negative staphylococci were often multires-istant, but remained fully sensitive to vancomycin. For a few antibiotics, significantly more resistance was found in the specialist hospital. In our material we found no significant increase in resistance between 1991–92 and 1995–96. In conclusion, antimicrobial resistance still remains low in important bacterial pathogens causing bloodstream infections in Norway.

The clustering of four cases of meningococcal disease during a 3-month period in a small community with 2233 inhabitants prompted an interventional carrier survey in persons < 19 years old and in family members of the patients. The aims of the survey were to identify the nasopharyngeal carriers and the carriage rate of the outbreak strain, to offer chemoprophylaxis to those carrying the outbreak strain, and to study the discriminatory power of phenotypic methods versus pulsed-field gel electrophoresis (PFGE) on carrier isolates during an outbreak. A high percentage of the population in the age group 0-19 years (73.7%) participated in the study. Among the 469 samples collected in this age group, meningococci were grown from 43 (9.2%). The highest carriage rates were in the age group 18-19 years (36.4%). With a provisional definition of the outbreak strain (group B or non-groupable Neisseria meningitidis with reduced sulphonamide sensitivity), six carriers were identified. All were treated with a single dose of ofloxacin. Four of these persons (0.76% of all tested) were later shown to have harboured the outbreak strain when analysed by PFGE. Three of them were epidemiologically closely related to one of the index cases. Serogrouping alone is not sufficient for the identification of an epidemic strain of N. meningitidis. Complete concordance of type and subtype antigens correctly identified the outbreak strain in this study. PFGE is well suited for the identification of an outbreak strain of N. meningitidis versus non-epidemic strains in tonsillo-pharyngeal specimens.

An international study supported by the World Health Organization comparing monoclonal antibodies for serotyping and serosubtyping of Neisseria meningitidis strains was performed and the results were assessed in 1992. A collection of 6 serotype-specific (1, 2a, 2b, 4, 14, and 15) and 12 serosubtype-specific (P1.1, P1.2, P1.4, P1.5, P1.6, P1.7, P1.9, P1.10, P1.12, P1.14, P1.15, and P1.16) monoclonal antibodies was provided to 11 participating laboratories throughout the world. Monoclonal antibodies were tested on 85 Neisseria meningitidis strains with known reference results. Whole-cell enzyme-linked immunosorbent assay was used for analysis in 10 of 11 laboratories. The sensitivities and specificities of individual serotype- and subtype-specific monoclonal antibodies were evaluated. Differences in individual laboratories and with individual monoclonal antibodies were assessed. Relatively large differences in sensitivities were achieved in individual laboratories. On the contrary, the specificities remained at high levels in all laboratories. The sensitivities of serotype-specific monoclonal antibodies ranged from 72.0 to 100%. Individual serosubtype-specific monoclonal antibodies showed sensitivities ranging from 64.1 to 98.1%. The most frequent reason for the incorrect results obtained with the monoclonal antibodies were false-negative results. The collaborative study demonstrated that some monoclonal antibodies are not very sensitive. Another study to define the most suitable monoclonal antibodies is planned.

Accurate differentiation between pneumococci and other viridans streptococci is essential given their differences in clinical significance. However, classical phenotypic tests are often inconclusive, and many examples of atypical reactions have been reported. In this study, we applied various phenotypic and genotypic methods to discriminate between a collection of 12 streptococci isolated from the upper respiratory tract of HIV-seropositive individuals in 1998 and 1999. Conventional phenotypic characterization initially classified these streptococci as Streptococcus pneumoniae, as they were all sensitive to optochin and were all bile soluble. However, they did not agglutinate with anti-pneumococcal capsular antibodies and were also far more resistant to antimicrobial agents than typeable pneumococci isolated in the same period. Genotypic characterization of these isolates and control isolates by both multilocus sequence analysis (MLSA) and comparative genomic hybridization (CGH) showed that only a single isolate was genetically considered to be a true S. pneumoniae isolate, and that the remaining 11 non-typable isolates were indeed distinct from true pneumococci. Of these, 10 most closely resembled a subgroup of Streptococcus mitis isolates genetically, while one strain was identified as a Streptococcus pseudopneumoniae isolate. CGH also showed that a considerable part of the proposed pneumococcal core genome, including many of the known pneumococcal virulence factors, was conserved in the non-typable isolates. Sequencing of part of the 16S rRNA gene and investigation for the presence of ply by PCR corroborated these results. In conclusion, our findings confirm the close relationship between streptococci of the Mitis group, and show that both MLSA and CGH enable pneumococci to be distinguished from other Mitis group streptococci.

These data contributed to a 2007 WHO recommendation for a fractional dosing strategy in the context of severe vaccine shortages during a meningitis epidemic., BACKGROUND: Neisseria meningitidis serogroup A is the main causative pathogen of meningitis epidemics in sub-Saharan Africa. In recent years, serogroup W135 has also been the cause of epidemics. Mass vaccination campaigns with polysaccharide vaccines are key elements in controlling these epidemics. Facing global vaccine shortage, we explored the use of fractional doses of a licensed A/C/Y/W135 polysaccharide meningococcal vaccine. METHODS AND FINDINGS: We conducted a randomized, non-inferiority trial in 750 healthy volunteers 2-19 years old in Mbarara, Uganda, to compare the immune response of the full dose of the vaccine versus fractional doses (1/5 or 1/10). Safety and tolerability data were collected for all subjects during the 4 weeks following the injection. Pre- and post-vaccination sera were analyzed by measuring serum bactericidal activity (SBA) with baby rabbit complement. A responder was defined as a subject with a >/=4-fold increase in SBA against a target strain from each serogroup and SBA titer >/=128. For serogroup W135, 94% and 97% of the vaccinees in the 1/5- and 1/10-dose arms, respectively, were responders, versus 94% in the full-dose arm; for serogroup A, 92% and 88% were responders, respectively, versus 95%. Non-inferiority was demonstrated between the full dose and both fractional doses in SBA seroresponse against serogroups W135 and Y, in total population analysis. Non-inferiority was shown between the full and 1/5 doses for serogroup A in the population non-immune prior to vaccination. Non-inferiority was not shown for any of the fractionate doses for serogroup C. Safety and tolerability data were favourable, as observed in other studies. CONCLUSIONS: While the advent of conjugate A vaccine is anticipated to largely contribute to control serogroup A outbreaks in Africa, the scale-up of its production will not cover the entire "Meningitis Belt" target population for at least the next 3 to 5 years. In view of the current shortage of meningococcal vaccines for Africa, the use of 1/5 fractional doses should be considered as an alternative in mass vaccination campaigns. TRIAL REGISTRATION: ClinicalTrials.gov NCT00271479.

Typing of meningococci with a panel of serotype and subtype specific monoclonal antibodies (MAbs) was compared in co-agglutination, dot-blotting and ELISA tests. Twenty reference strains, 50 case isolates and 133 throat isolates from healthy carriers were studied. The typing results with dot-blotting and ELISA were identical, whereas co-agglutination gave different results for three case and 24 carrier strains. The distribution of serotypes and subtypes among the strains is reported. The combination of the subtypes P1.1 and P1.15 in a serotype 15 patient strain was observed. With one case strain and 15 carrier strains, neither serotype nor subtype could be determined. Non-typable and non-subtypable isolates were further characterised by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Co-agglutination is useful for typing small numbers of strains with a few MAbs, but less suitable for large-scale typing than the other two methods. Dot-blotting needs less expensive equipment, smaller volumes of antibodies and fewer manipulations than ELISA.

In September 1988, an epidemic of meningococcal disease started in Ethiopia. 21 Neisseria meningitidis isolates recovered from patients in Addis Ababa and towns 200 km south of the capital were characterized by serogrouping, serotyping, testing of susceptibility to antibiotics, restriction endonuclease fingerprinting, and multilocus enzyme electrophoresis. The 21 isolates were essentially homogeneous for all properties tested and belonged to clone III-1 of serogroup A N. meningitidis, which was also responsible for the epidemics in Saudi Arabia in 1987 and in Sudan and Chad in 1988.

Two new methods for serogrouping of meningococci, whole-cell ELISA and dot-blotting, with monoclonal antibodies against serogroups A, B, C, Y and W135 were compared with slide-agglutination applying polyclonal sera. In addition to a panel of strains with previously determined serogroups by slide-agglutination, two strain collections of meningococci were studied: 1) 50 strains isolated from patients with systemic meningococcal disease in Norway during the winter 1987-1988; 2) 133 throat strains isolated from asymptomatic carriers over the same period. For the disease strains all three methods gave identical results, whereas some carrier strains which were non-agglutinable or polyagglutinable by slide-agglutination were serogroupable by the two other methods. All the systemic strains and about half of the carrier strains were serogroupable. We find that whole-cell ELISA and dot-blotting are specific, easy to read and more sensitive compared to slide-agglutination, but the former methods are at present limited by the availability of monoclonal antibodies against only serogroups A, B, C, Y and W135.

A total of 4624 pneumococcal isolates from episodes of systemic pneumococcal disease were received at the Norwegian Institute of Public Health during the period 1995–2001. All isolates were serotyped and tested for susceptibility to benzylpenicillin, lincomycin, erythromycin, tetracycline and trimethroprim–sulphamethoxazole. The proportion of strains resistant to these antimicrobial agents remained stable at a low level, ranging from 0·1% for benzylpenicillin to 2·5% for erythromycin. The distribution of serotypes was also stable over the 7 years: serotypes 1, 4, 9, 14, 7, 6 and 23 were the most frequent, representing 70·5% of isolates. Overall, 95·8% of the isolates were of serotypes/groups included in the current 23-valent polysaccharide vaccine, 52·2% were of serotypes/groups included in the 7-valent conjugated vaccine and 85·5% were of serotypes/groups included in the 11-valent conjugated vaccine.

The aim of the present study was to investigate whether HIV-infected patients, a group that is supposedly at risk for infection with antibiotic-resistant microbes, really does so, and to assess possible risk factors for acquiring these organisms. During the period from January 1998 to July 1999, samples of normal flora were obtained from 107 HIV-infected patients attending an outpatient clinic in Oslo, Norway. The samples were cultured for Streptococcus pneumoniae, Haemophilus influenzae, Escherichia coli, coagulase-negative staphylococci and Candida spp., and the resulting isolates were tested for antimicrobial susceptibility. The patients studied represented all stages of HIV infection, from recently infected to severely immunocompromised. Samples were taken at one, two or three time-points to determine whether antimicrobial resistance in colonising microorganisms increases over time. Antimicrobial resistance was linked primarily to antimicrobial prophylaxis, but it did not increase during the observation period. The level of a patient's immunodeficiency and the consequently intensified medical care was also of some importance. Even though about 50% of the patients were receiving antimicrobial agents at the time of sampling, the level of resistance found in these patients was very similar to that found in other patient groups in Norway; except for Candida albicans isolates, which were less susceptible to fluconazole. Overall, antimicrobial resistance was uncommon in the HIV-seropositive patients studied, a finding that is probably related to the overall low prevalence of antimicrobial resistance in the general population in Norway.

Three different formulations of an outer membrane vesicle (OMV) vaccine against group B meningococcal disease have been prepared and tested for immunogenicity and reactogenicity in adult volunteers. The vaccines were prepared with or without aluminium hydroxide and serogroup C-polysaccharide (C-ps). Doses from 12.5 to 100 micrograms protein were given twice at a six weeks' interval. All three formulations were well tolerated and highly immunogenic, inducing bactericidal and opsonizing antibodies in humans. Adsorption of OMVs to aluminium hydroxide reduced the pyrogenicity in rabbits. The differences in immunogenicity between the formulations were relatively small, but after the second dose a stronger booster response was observed when the vaccines were adsorbed. Thus, a formulation with OMVs and C-ps represents a safe and highly immunogenic vaccine, even without aluminium hydroxide.

To improve the diagnosis of systemic meningococcal disease we used a nested polymerase chain reaction (nPCR) test to detect meningococcal DNA in cerebrospinal fluid (CSF) samples from patients. The nPCR test was based on the gene coding for the PorA outer membrane protein, a major antigen of Neisseria meningitidis, which is the basis for determining the subtype of the strains. The method was tested on CSF samples from 87 patients with various disease aetiology, including 37 patients with systemic meningococcal disease. It was also applied to all 67 CSF samples from culture-negative patients with suspected meningococcal disease which were collected in the course of the Norwegian serogroup B vaccination trials, between 1987 and 1993. Of the 67 culture-negative CSF samples, 10 were nPCR positive. Two of the 67 CSF were positive with the antigen test, and both of these were nPCR positive. Serum pairs from 46 of the 67 culture-negative patients were analysed for serological serogroup response in an enzyme-linked immunosorbent assay (ELISA) against meningococcal capsular polysaccharides. Nine of the 10 nPCR-positive CSF samples belonged to patients with serological response judged as either typical of, or compatible with systemic meningococcal disease. Sequence analysis of the nPCR products was performed to determine the subtype of the infecting meningococcal strains. Four of the 10 positive CSF samples were infected with a strain of subtype P1.7,16, which is similar to the epidemic strain used to produce the vaccine. Inclusion of nPCR for diagnosis of meningococcal meningitis did not significantly alter the results of the vaccination trial, but added important information about the strain causing disease in 5 of 13 serogroup B cases without preserved isolates.

We surveyed 472 cases of culture-confirmed systemic pneumococcal disease that were reported to the Norwegian Notification System for Infectious Diseases during a 12-month period in 1992-93. The clinicians in charge of the patients filled in a questionnaire providing information on underlying disease and outcome for 461 (98%) of the patients. Eight of these patients were splenectomized; all of them more than ten years before. Four died, two survived but had serious sequelae, and two survived without obvious sequelae upon discharge from hospital. Using a rough estimate of the prevalence of unvaccinated splenectomized persons in Norway, we estimate that this group, compared to the normal population, has a relative risk of 25 of developing systemic pneumococcal disease and a relative risk of 75 of dying from pneumococcal disease. The serotype of the pneumococcal strain that caused the disease was determined for seven of the eight patients. All serotypes were represented in the 23-valent pneumococcal polysaccharide vaccine. We strongly recommend that doctors trace and vaccinate splenectomized individuals.

Splenectomized individuals run increased risk of developing overwhelming septicemia from encapsulated bacteria, especially Streptococcus pneumoniae. Polyvalent pneumococcal polysaccharide vaccine should be given to all splenectomized individuals above two years of age. Antipneumococcal antibody levels should be measured three to five years after the first vaccination, and persons with low antibody levels should be revaccinated. Splenectomized persons may be given a supply of penicillin V to enable them to start therapy while avaiting medical attention.

To estimate the extent of meningococcal carriage in the Norwegian population and to investigate the relationship of several characteristics of the population to the carrier state, 1,500 individuals living in rural and small-town areas near Oslo were selected at random from the Norwegian National Population Registry. These persons were asked to complete a questionnaire and to volunteer for a bacteriological tonsillopharyngeal swab sampling. Sixty-three percent of the selected persons participated in the survey. Ninety-one (9.6%) of the volunteers harbored Neisseria meningitidis. The isolates were serogrouped, serotyped, tested for antibiotic resistance, and analyzed by multilocus enzyme electrophoresis. Eight (8.8%) of the 91 isolates represented clones of the two clone complexes that have been responsible for most of the systemic meningococal disease in Norway in the 1980s. Age between 15 and 24, male sex, and active and passive smoking were found to be independently associated with meningococcal carriage in logistic regression analyses. Working outside the home and having an occupation in transportation or industry also increased the risk for meningococcal carriage in individuals older than 17, when corrections for gender and smoking were made. Assuming that our sample is representative of the Norwegian population, we estimated that about 40,000 individuals in Norway are asymptomatic carriers of isolates with epidemic potential. Thus, carriage eradication among close contacts of persons with systemic disease is unlikely to have a significant impact on the overall epidemiological situation.

Based on differences in reaction pattern with monoclonal antibodies against the P1.16 epitope, a new variant of the class 1 protein in Neisseria meningitidis serogroup B was identified in Norway. A single amino acid deletion was revealed when the part of the gene region encoding the second variable region of the protein was sequenced. This new variant was designated P1.16c. About 5% of the B:15:P1.7,16 strains in Norway from the time period 1987-1991 were P1.16c. In a localized area in Southern Norway, 5/8 (62%) of the P1.7,16 strains were P1.16c. The P1.16b mutant, recently described in England, was not found among the Norwegian meningococcal isolates. Strains carrying the P1.16c mutation showed increased resistance to bactericidal killing, not only by P1.16-specific monoclonal antibodies, but also by the sera from individuals immunized with a vaccine based on outer membranes from a B:15:P1.7,16 strain.

Summary Dot-blot analysis of whole-cell suspensions of meningococci showed that 81% of B:15:P1.16 strains from patients reacted with a monoclonal antibody (MAb) against subtype P1.7. The remaining strains, which did not react on dot-blots or in ELISA, demonstrated the P1.7 subtype epitope on immunoblots after denaturation of the cells with sodium dodecyl sulphate. The monomeric class 1 proteins of the two P1.16 subtype variants had slightly different mol. wts, but bound the P1.7 antibody equally well. These results were explained by a deletion of three codons in the gene encoding the first variable region of the P1.16 class 1 protein. The deletion accounted for the non-exposure of the P1.7 epitope on native cells. Other patient strains, with subtypes P1.3, P1.9 or without any known subtype, also showed a binding site for the P1.7 MAb, which became available only after denaturation. Demonstration of inaccessible epitopes may have consequences for subtype designations and vaccine development.

SUMMARYFollowing the occurrence of a case of systemic meningococcal disease in a military camp in Norway, throat cultures and blood samples were collected from 33 healthy individuals belonging to the same troop as the patient (troop A) and from 29 individuals from a different troop (troop B) in the same camp. Serological studies showed that 91% of the recruits had bactericidal antibodies against the disease-causing strain. The isolates ofNeisseria meningitidisrecovered from the throat cultures were serogrouped, serotyped, and assigned to a clone on the basis of an analysis of the electrophoretic mobilities of 14 metabolic enzymes. None of the 23 carriers in troop A harboured the clone responsible for the case of disease, but 6 carried isolates of the same electrophoretic type, ET-7, which was not identified in any of the 19 carriers of troop B. Individuals in troop A were resampled 2 and 17 weeks after the meningococcal disease episode. Five of the carriers had acquired different clones and one of them changed clone twice in that period. Four of the six newly acquired clones had previously been identified in other carriers of troop A, demonstrating transmission of clones among individuals living and working in close proximity.

The chemiluminescence (CL) was examined when peripheral blood monocytes were incubated with opsonized Neisseria meningitidis, serogroup B, serotype 15:P1.16 or serotype 2a:P1.2. The monocytes were separated from a mononuclear cell suspension by an immunomagnetic negative selection technique using magnetic polystyrene microspheres coated with monoclonal antibodies specific for T and B lymphocytes. More than 90% of the lymphocytes were removed, yielding a suspension containing 93% monocytes. Optimal sensitivity for phagocytosis was obtained using 1% serum (10 microliters), 72 bacteria per monocyte cell, and 7.5 min opsonization and incubation time during continuous agitation at 37 degrees C. The CL was amplified by lucigenin. Preliminary experiments suggest that convalescent sera from patients with group B meningococcal disease induced increased CL responses compared to acute sera. Sera from volunteers immunized with an outer membrane complex vaccine from serogroup B, serotype 15:P1.16 or 2a:P1.2 meningococci also induced increased CL activity compared to preimmune sera. No such response was shown when a group B capsular polysaccharide vaccine was given. This response pattern was also demonstrated by a flow cytometric phagocytosis technique (FCM). Internalization of meningococci by monocytes was demonstrated by a FCM quenching technique and by transmission electron microscopy. CL and FCM represent rapid and reproducible methods for the measurement of opsonophagocytosis of meningococci by monocytes and may be performed with minute amounts of sera.

Results from the serum bactericidal assay (BA) after immunization of human volunteers with the Norwegian serogroup B meningococcal outer membrane vesicle vaccine are surveyed. In the phase II trials with adults we found very high seroconversion rates (greater than 98%) against the vaccine strain in the BA. Details in the antigenic composition of the inoculum used in the BA seem very important as shown here by finding lower bactericidal titres with teenager sera when tested with a variant of the standard inoculum. The present preliminary report corresponds to the presentation given at the Report Meeting on the Norwegian Meningococcal Vaccine Trial, Oslo, 12 September, 1991.

A survey is given of the efforts made to inform the general public, the potential vaccinees and their parents, and the health care personnel about meningococcal disease in general and the vaccination trial in particular, as a preparation for the meningococcal outer membrane vesicle serogroup B vaccine (MenB-vaccine "Folkehelsa") trials in secondary school students and military conscripts in Norway. Our case reporting system, supplementing the official notification, concerning even vaguely suspected cases in the age cohorts involved, is described. The efforts made to collect clinical material as well as laboratory and clinical data from 221 registered suspected cases are delineated. We also briefly summarize our cerebrospinal fluid antigen detection methods and diagnostic meningococcal serology work on these suspected cases. The compiled information on findings done at the admitting hospital of the possible cases and the additional diagnostic data provided at the National Institute of Public Health were put at the disposal of the independent Diagnosis Review Committee (DRC) as a basis for their diagnostic decisions before code opening for the meningococcal serogroup B outer membrane vesicle vaccine protection trial 3 June 1991.

An outer membrane vesicle vaccine against acute, systemic disease caused by meningococci of serogroup B has been developed. The vaccine has been tested consecutively in phase I and phase II clinical trials including more than 5000 volunteers. These trials provided data on safety, immunogenicity and reactogenicity and possible effect on carriage of meningococci in the throat, and consequently formed the basis for two major protection trials; one in secondary school students and one among military recruits. The aims, design and major results of phase I and phase II studies are described as well as the design and organization of the protection trials.

For more than 15 years, Norway has had the highest incidence of meningococcal disease in northern Europe, with 80% of cases being due to serogroup B meningococci. The case-fatality has remained high, at about 10%. In this study, an outer membrane vaccine, which had previously been shown to induce an increase in bactericidal antibodies to the parent strain, was assessed in a large-scale, randomised, double-blind trial. From October, 1988, 171,800 students in secondary schools volunteered to take part in a double-blind, placebo-controlled, efficacy trial with school as the randomisation unit. Hospitals and clinics that routinely receive patients with infectious disease were asked to report urgently all cases of suspected meningitis and/or septicaemia in 13-21-year-old students in Norway. These cases were registered and further investigated according to a detailed protocol. 89 out of the 221 cases investigated by June 3, 1991, were shown to be severe systemic disease due to group B meningococci. 36 cases in 35 schools took part in the trial (11 schools with vaccinated students and 24 with students given placebo). The calculated rate of protection was thus 57.2% (p = 0.012, one-sided test). The findings suggest that, although the vaccine conferred protection against group B meningococcal disease, the effect was insufficient to justify a public vaccination programme.

The reactions of serogroup A strains of Neisseria meningitidis with one monoclonal antibody specific for serotype 21 and three different monoclonal antibodies specific for serotype 4 were compared with those of serogroup B strains previously assigned to serotype 4. Antibody binding was studied by enzyme-linked immunosorbent assay (ELISA), dot blotting, and immunoblotting. Characterization of the isolates by the electrophoretic mobilities of 14 metabolic enzymes showed 50 multilocus enzyme genotypes. All except two genotypes fell into three distinct clusters: I, IIa and IIb. The enzyme genotypes of serogroup B strains were mainly in cluster I, and 88% of the serogroup A strains had genotypes in clusters IIa and IIb. Serogroup B strains generally reacted with all three serotype 4 monoclonal antibodies in ELISA and dot blotting but with only two in immunoblots. Serogroup A strains showed two different reactions in the blotting methods: either binding of the serotype 21 antibody only or binding of this and two of the three serotype 4 monoclonal antibodies. Strains of the first pattern were in clusters I and IIa, whereas all but two strains in cluster IIb were of the second pattern. In ELISA, an additional reaction of two of the serotype 4 monoclonal antibodies with serogroup A isolates was observed. The different binding of these two monoclonal antibodies in ELISA and the blotting methods appeared to result from heat inactivation of the meningococcal cells and use of detergent-containing reagents in ELISA. The results show that the serotype of serogroup A strains is distinct from serotype 4 of serogroup B strains.

Two new methods for serogrouping of meningococci, whole-cell ELISA and dot-blotting, with monoclonal antibodies against serogroups A, B, C, Y and W135 were compared with slide-agglutination applying polyclonal sera. In addition to a panel of strains with previously determined serogroups by slide-agglutination, two strain collections of meningococci were studied: 1) 50 strains isolated from patients with systemic meningococcal disease in Norway during the winter 1987-1988; 2) 133 throat strains isolated from asymptomatic carriers over the same period. For the disease strains all three methods gave identical results, whereas some carrier strains which were non-agglutinable or polyagglutinable by slide-agglutination were serogroupable by the two other methods. All the systemic strains and about half of the carrier strains were serogroupable. We find that whole-cell ELISA and dot-blotting are specific, easy to read and more sensitive compared to slide-agglutination, but the former methods are at present limited by the availability of monoclonal antibodies against only serogroups A, B, C, Y and W135.

A clone of serogroup A Neisseria meningitidis recognized by multilocus enzyme electrophoresis and designated as clone III-1, has caused major epidemics of meningococcal disease in various parts of the world since the 1970s. In Norway, serogroup B meningococci have been responsible for an epidemic since the mid-1970s. We have studied a sample of 53 meningococci isolated from patients in western Norway prior to the serogroup B epidemic. 22/35 meningococcal isolates collected 1969-73 represented clone III-1, whereas this clone was not found in the 18 isolates from 1962-68. It has been speculated that the epidemic of meningococcal disease in Finland caused by clone III-1 in 1973-75 had spread from an epidemic in USSR that began in 1969. Our findings demonstrate, however, that the clone III-1 was present in Scandinavia in 1969.

Several different meningococcal outer membrane protein vaccines have been prepared and used in human safety and immunogenicity studies. The results of these studies have led to some general conclusions regarding the human antibody response to these vaccines. A review of these conclusions, however, indicates that a number of important questions and problems still need to be addressed. Two of these are the determination of the protective level of bactericidal antibody in human serum and the impact of phase variation of surface antigens on vaccine strategy. Bactericidal assays using intrinsic complement and high concentrations of serum suggest that the level of natural immunity to group B meningococci is quite high, but is increased by vaccination with outer membrane protein vaccine. Phase variation in meningococcal surface antigens including capsule, class 1 protein, class 5 protein, and lipopolysaccharide was demonstrated using colony blotting with monoclonal antibodies. Phase variation resulted in differences in susceptibility to the bactericidal activity of human sera.

SUMMARY: Variation in nine enzymes in 152 isolates of Neisseria meningitidis from Norway (118 from blood or cerebrospinal fluid of patients with systemic disease and 34 from the pharynx of healthy carriers) was analysed by starch-gel electrophoresis. All nine enzymes were polymorphic and the number of allozymes (electromorphs) identified per locus ranged from 3 to 12, with a mean of 6.1. Among the 152 isolates, 55 unique combinations of electromorphs (electrophoretic types, ETs) were distinguished. Twenty ETs were represented among the carrier isolates and 37 among the systemic isolates; hence, only two ETs were found in both groups of isolates. ET-5 was identified 67 times among the 118 systemic isolates (58%), indicating an association of this ET with invasiveness; ET-5 was also the most common type among the carrier isolates (18%). Genetic similarity between ETs was analysed by pairwise comparison of all 55 ETs with respect to the number of electromorphs by which they differed. No evidence of a general genetic difference between carrier and case isolates was found. Two well-defined clusters of ETs were observed, each including one of the two most common ETs identified among the systemic isolates (ET-5 and ET-37), together with isolates differing from them only at one or two loci. All isolates of ET-5 and ET-37, as well as their closely related variants defined by the similarity matrix, were resistant to sulphonamide, independent of their antigenic characteristics and isolation site. The extensive allozyme variation among isolates of the same serogroup demonstrated the limited value of serogrouping as an epidemiological tool. All but one isolate of serotype 15:P1.16 were electrophoretically similar, as were all the 2a:P1.2 isolates. The 15:P1.15 isolates, however, were genetically heterogeneous. The distribution of alleles in genotypes identified among the systemic isolates indicated that genetic recombination may occur in natural populations of N. meningitidis.

The antibody response of 30 volunteers vaccinated with a complex of group B polysaccharide and outer membrane vesicles (OMV) from serotype 2a Neisseria meningitidis and of 3 individuals who received a placebo vaccine was determined by immunoblotting. OMV were separated by sodium dodecyl sulfate-gel electrophoresis and electrotransferred to nitrocellulose filters. Binding of immunoglobulin G (IgG), IgA, and IgM antibodies in the human sera to OMV components was detected with class-specific peroxidase-conjugated antibodies. The immunoblotting results were also related to the bactericidal activity of the sera and the meningococcal carrier status of the volunteers. Before vaccination weakly reactive bands in the molecular weight range of 140,000 to 10,000 were observed on the blots. Sera from carriers showed more marked bands. Individual patterns of increased reactivity were seen 6 weeks after vaccination. The main immunoreactive components of OMV corresponded to a molecular weight of 43,000 (class 1 protein), 30,000 (class 5 proteins), and 22,000. IgG antibodies in postvaccination sera of high bactericidal titers showed distinct binding to the 43,000-molecular-weight antigen. Meningococcal carriers had antibodies against an antigen of 22,000 molecular weight; in polyacrylamide gels this component did not stain with Coomassie brilliant blue or silver. The marked binding of IgG antibodies to the class 5 proteins decreased considerably between weeks 6 and 25 after vaccination. Periodate oxidation of OMV abolished the binding of IgG antibodies to the class 5 proteins, whereas the antigenicity of the 43,000-molecular-weight (class 1 protein) and 22,000-molecular-weight antigens was unaffected.

Antibody responses of adult volunteers given a vaccine containing meningococcal capsular polysaccharides (serogroups A, C, Y, and W-135) noncovalently complexed with serotype 2b:P1.2 and 15:P1.16 outer membrane proteins have been studied. Sera were analyzed by enzyme-linked immunosorbent assay methods for immunoglobulin G (IgG), IgM, and IgA antibodies and for bactericidal activities against the homologous strains. The vaccination was performed as a double-blind experiment with 47 volunteers, of whom 23 received the protein-polysaccharide vaccine and 24 received the control preparation containing the polysaccharides only. Ten additional persons volunteered for the protein-polysaccharide vaccine. Before vaccination, carriers of meningococci had significantly higher levels of specific IgG and IgA and also higher bactericidal activities than noncarriers. At 2 weeks postvaccination we found significant IgG and bactericidal antibody responses against both the 2b:P1.2 and 15:P1.16 strains in about 70% of the protein-polysaccharide vaccinees. The immune response induced by disease was compared with that induced by vaccination by analyzing paired sera from 13 survivors of serogroup B serotype 15 meningococcal disease. We found that the mean specific IgG level in acute-phase sera was lower than average in prevaccination sera from the vaccinees but similar to that of healthy noncarriers before vaccination. The convalescent-phase sera showed IgG responses similar to those of the vaccinees, but the IgM response to disease was significantly higher than after vaccination. The immune response to disease caused by serogroup B serotype 15 meningococci was found by enzyme-linked immunosorbent assay analysis to be about the same with outer-membrane antigens from a serotype 2b strain as it was with antigens from a serotype 15 strain.

To elucidate pathogenic aspects and serodiagnostic possibilities for meningococcal disease, we investigated levels of specific antimeningococcal immunoglobulin G (IgG), IgA, and IgM in serum by using an enzyme-linked immunosorbent assay with outer membrane antigen prepared from a Neisseria meningitidis B:15:P1.16 strain. Serum samples were drawn on hospital admission as well as during convalescence from patients suspected of purulent meningitis or meningococcal septicemia, and single samples were drawn from population controls. A total of 637 samples were examined blindly. On admission, the average antimeningococcal immunoglobulin levels were about the same in the meningococcal disease patients as in the population controls. Septicemic patients, however, had significantly lower values. During one week the mean specific immunoglobulin levels in meningococcal-disease patients increased 6 times for IgG, 14 times for IgA, and 5 times for IgM. Children younger than 1 year showed a modest and more slowly developing antibody response. There were no statistically significant differences in average antibody responses among patients infected with meningococci of different serotypes. At 100% specificity, the increase in IgG, IgA, and IgM yielded diagnostic sensitivities for meningococcal disease of 84, 52, and 66%, respectively. One of seven serum pairs from the patient control group with unknown etiology was positive for meningococcal disease in this assay. The patients with meningococcal disease originally diagnosed only by clinical signs and symptoms showed a slightly lower rate of seroconversion than the patients in whom the diagnosis was supported by test results showing a systemic Neisseria meningitidis isolate.

Endotoxin liberation was studied in a blinded material of 121 Neisseria meningitidis isolates; from nasopharynx of 58 carriers and from cerebrospinal fluid or blood of 63 cases with meningococcal disease. Endotoxin activity in culture filtrates was determined by a Limulus lysate test. Meningococci isolated from clinical cases were significantly more frequently endotoxin-liberating (E+) (84.1%) than in carriers (25.9%); p less than 0.001. Serogroupable carrier isolates had a significantly higher frequency of E+ meningococci (61.9%) than non-groupable ones (5.4%); p less than 0.002. Serogroup B case isolates, which generally had a larger amount of capsular polysaccharide than B meningococci from carriers, had a significantly higher proportion of E+ meningococci than group B from carriers; p = 0.007. All 7 serogroup C isolates were E+ (5 cases and 2 carriers). No correlation was found between endotoxin liberation and the serotype: subtype 15:P1.16, tested by a selection of monoclonal antibodies, or between endotoxin liberation and sulfonamide resistance, when carrier and case isolates were studied separately. Meningococci isolated from cases had the following mean endotoxin titres: 320.5 in the meningitis group, 408.2 in the septicaemic group, 462.1 in the septicaemic and meningitis group, and 123.7 in the group with other systemic disease. E+ meningococci were isolated from 5/6 fatal cases. Thus, endotoxin liberation from meningococci is strongly, but not completely associated with establishment of meningococcal disease and with the presence of capsular polysaccharide.

The genetic structure of populations of Neisseria meningitidis was examined by an analysis of electrophoretically demonstrable allelic variation at 15 structural genes encoding enzymes in 688 isolates. Variation among strains in serogroup and serotype has little relationship to the complex structure of populations revealed by enzyme electrophoresis, which involves 14 major lineages of clones diverging from one another at more than half their genetic loci. Clones of one of these lineages, the ET-5 complex, have been identified as the causative agent of recent outbreaks and epidemics of meningococcal disease in Europe, South Africa, Latin America, and the United States. There is evidence that organisms of the ET-5 complex reached Florida via human immigrants from Cuba.

Examination of the trends of meningococcal infection in Norway, Iceland, Faroe Islands, Denmark, England and Wales, and the Netherlands, has shown that Neisseria meningitidis B:2b:P1.2 and/or B:2a:P1.2 phenotypes were associated with peaks of infection in the Netherlands in 1966, in Iceland 1976-77, and in England and Wales in 1973-75. These strains were present in all six countries in the decade 1970-80 but their prevalence is now practically negligible. In contrast the prevalence of the B:15:P1.16 phenotype has risen. In the Faroe Islands and northern Norway this change in serotype prevalence has been followed by rises in incidence of meningococcal disease; the same is happening in England and Wales but not yet in the other countries.

Two groups of volunteers were immunized with either a serogroup A plus C meningococcal polysaccharide vaccine or a combined serogroup B polysaccharide-serotype 2 protein vaccine. Serum opsonin responses were measured by chemiluminescence of polymorphonuclear leukocytes exposed to opsonized live meningococci. Two of the six volunteers immunized with the A plus C vaccine had an increase in serum opsonins to group A meningococci, four responded to group C meningococci, and none to group B meningococci. Five other volunteers who were immunized with the combined group B polysaccharide-serotype 2 protein vaccine responded with an increase in serum opsonins to group B meningococci of two different protein serotypes, as well as to a group C-serotype 2 meningococcal strain. Although no booster effect was observed after a second dose of the combined vaccine, both the polysaccharide and the protein components appear to be able to stimulate an opsonin response.

serogroup B,serotype 15meningococci. Throat specimenswerecollected from552healthy contacts, including allchildren below age17andtheir parents. Among the36carrier isolates (carrier rate, 6.5%)13showed DNAfingerprints identical, oralmost identical, tothe index pattern. Allofthese 13isolates weresulfonamide resistant, 12wereofserotype 15,and8wereof polysaccharide serogroup B (5werenongroupable). Theseclosely related isolates werealmost exclusively recovered fromschoolchildren of2of15small villages, oneofwhich included thehomesoftwoofthepatients. Theremaining 23carrier isolates werenonresistant, non-type 15meningococci ofwidely differing DNA restriction patterns. Ourresults confirm that DNAfingerprinting haspotential asanefficient tool inpractical meningococcal epidemiology. Tounderstand theepidemiology ofNeisseria meningitidis itismandatory tostudy theinterplay ofneisserial strains in thepopulation. Little isknownaboutthedynamics and epidemiological consequences ofthis interplay, partly becauseofproblems ofexactdifferentiation between similar strains. Intheearly 1970s, theprevailing serogroup A andC meningococci almost disappeared fromNorwayasthenew serogroup B,type15,sulfonamide-resistant meningococci became predominant (4). Thus, mostsystemic meningococcalisolates appeared identical. However, following adaptation oftheDNA fingerprinting technique toanalysis ofthe meningococcal genome, werecently foundthatthefingerprint ofB15meningococci mayshowa widerangeof different patterns (13), although thefingerprints ofepidemiologically closely linked isolates maybeidentical (15). Thepresent investigation wasundertaken tocharacterize isolates ofmeningococci andstudy their distribution inthe healthy population during anoutbreak ofmeningococcal disease inasmall community innorthern Norway. Forthis purpose DNA fingerprinting wasapplied incombination with serotyping bymonoclonal antibodies, serogrouping, and sulfonamide susceptibility testing.

A 20 fold purification of the gramicidin S synthesizing activity in a 11,000 ×g supernatant from Bacillus brevis has been achieved. The purification procedure involves precipitation of undesired material with streptomycin sulphate, followed by precipitation of gramicidin S synthesizing activity at 40% (NH4)2SO4 saturation and fractionation on DEAE-Sephadex. All gramicidin S synthesizing activity appeared in one peak. It incorporated labelled phenylalanine, proline, valine, ornithine and leucine to the same extent into gramicidin S. The gramicidin S synthesizing activity was found to be stable in a 0.05 M potassium phosphate buffer (pH 7.1) containing glutathione (0.01 M) and glycerol (20%, v/v) for at least 3 days at + 4° and four weeks at −20°.

1. An improved 11000g cell-free system for the incorporation of [(14)C]valine into gramicidin S has been obtained. The cell-free extract used was the supernatant obtained by treating Bacillus brevis with ultrasonics for 1min. followed by centrifugation at 11000g. The optimum pH for the incorporation was 8.2-8.4 and the optimum Mg(2+) concentration 0.05m. The presence of ammonium sulphate (0.1m) and K(+) (0.01m) increased the incorporation. 2. Cell-free extracts prepared from cells harvested in the early phase of growth (extinction value 0.1) incorporated negligible amounts of [(14)C]valine into gramicidin S compared with that incorporated by cell-free extracts prepared from cells harvested in the late phase of growth (extinction value 0.5). This was not due to the presence of inhibitors in the cell-free extracts prepared from cells harvested early, since there was no marked decrease in gramicidin S synthesis in a mixture of extracts prepared from cells harvested early and late in the growth phase. 3. The small incorporation of [(14)C]valine into protein, which took place in cell-free extracts from cells harvested in the late growth phase, was not inhibited by puromycin, chloramphenicol and ribonuclease. However, the substantial incorporation that took place in cell-free extracts prepared from cells harvested in the early phase of growth was completely inhibited by puromycin, chloramphenicol and ribonuclease. On mixing cell-free extracts prepared from cells harvested early and late in the growth phase, it appeared that the small incorporation that occurs in extracts from cells harvested in the late phase of growth was not due to cellular inhibitors.

1. A cell-free system prepared from Bacillus brevis cells, harvested in the late phase of growth and consisting of the 11000g supernatant, has been shown to incorporate into gramicidin S the five constituent amino acids added in labelled form. The results are consistent with complete synthesis and not merely a completion of pre-existing intermediate peptides. 2. The incorporation of 14C-labelled amino acids by the 11000g supernatant into gramicidin S requires an energy source. Omission of phosphoenolpyruvate and pyruvate kinase from the incubation mixture prevents incorporation into gramicidin S. The cell-free system incorporates [14C]-leucine, -proline and -phenylalanine over a period of 4hr. With [14C]leucine, incorporation into gramicidin S takes place in the range pH6–9 with maximum incorporation at pH7·0. High concentrations of chloramphenicol or puromycin decreased the incorporation into gramicidin S by only about 20%. 3. The 50000g supernatant exhibited no decrease in ability of incorporating [14C]valine into gramicidin S as compared with the 11000g supernatant. About 40% of the incorporating ability remained in the 105000g supernatant after 3hr. centrifugation. When recombining the 105000g sediment with the 105000g supernatant, some increase in incorporation over that obtained with the supernatant alone was obtained. The findings tend to support the view that gramicidin S is synthesized in a different manner from that of proteins.