Standardization and a multilaboratory comparison of Neisseria meningitidis serogroup A and C serum bactericidal assays. The Multilaboratory Study Group.

A standardized serum bactericidal assay (SBA) is required to evaluate the functional activity of antibody produced in response to Neisseria meningitidis serogroup A and C vaccines. We evaluated assay parameters (assay buffer, target strains, growth of target cells, target cell number, complement source and concentration, and methods for growth of surviving bacteria) which may affect the reproducibility of SBA titers. The various assay parameters and specificity of anticapsular antibody to five serogroup A strains (A1, ATCC 13077, F8238, F9205, and F7485) and four serogroup C strains (C11, G7880, G8050, and 1002-90) were evaluated with Centers for Disease Control and Prevention meningococcal quality control sera. The critical assay parameters for the reproducible measurement of SBA titers were found to include the target strain, assay incubation time, and complement. The resulting standardized SBA was used by 10 laboratories to measure functional anticapsular antibody against serogroup A strains F8238 and serogroup C strain C11. In the multilaboratory study, SBA titers were measured in duplicate for 14 pairs of sera (seven adults and seven children) before and after immunization with a quadrivalent polysaccharide (A, C, Y, and W-135) vaccine. The standardized SBA was reliable in all laboratories regardless of experience in performing SBAs. For most sera, intralaboratory reproducibility was +/- 1 dilution; interlaboratory reproducibility was +/- 2 dilutions. The correlation between median titers (interlaboratory) and enzyme-linked immunosorbent assay total antibody concentrations was high for both serogroup A (r = 0.86; P < 0.001; slope = 0.5) and serogroup C (n = 0.86; P < 0.001; slope = 0.7). The specified assay, which includes the critical parameters of target strain, incubation time, and complement source, will facilitate interlaboratory comparisons of the functional antibody produced in response to current or developing serogroup A and C meningococcal vaccines.

Septicaemia caused by phenotypic variants of group A streptococci.

Clinical isolates (115) of Streptococcus pyogenes responsible for septicaemia were investigated to determine whether invasiveness may be correlated with a large variety of strains, or concerns only particular phenotypes. Irrespective of their clinical origin, the strains studied displayed seven of the ten biotypes described within this species. As already observed in a series of strains isolated from pharyngitis, a restricted association between M-types and biotypes was demonstrated; each M-type corresponded to a sole biotype. However, the proportion of biotypes 3, 5 and 7 was higher in the septicaemia series than in the pharyngitis series, with a larger variety of M-types and with more non-typable strains. Despite these differences, the restricted associations between these characters were concordant in both series. These results demonstrate that streptococcal septicaemias appear to be caused by a wide variety of strains, suggesting that multiple factors may be involved in the invasiveness of the bloodstream during streptococcal infections.

[Post-streptococcal reactive arthritis: diagnostic problems]. / Poststreptokokni reaktivni artritis: dijagnosticke teskoce.

The authors present clinical, microbiological and scintigraphic findings in eight children with poststreptococcal reactive arthritis, hospitalized in the Zadar General Hospital from 1990 to 1992. In all patients an antecedent throat infection was noticed, 7-10 days before the first symptoms and signs of arthritis developed. Antistreptolysin O microtitration and/or throat culture revealed streptococcal infection in all patients. Scintigraphic examination with Te-99m yielded four positive (50%) results. In the follow-up one of the positive scans was later suggestive of tumorous process. The authors conclude that accurate diagnosis of poststreptococcal reactive arthritis depends on microbiological identification of streptococcal infection and longitudinal clinical follow-up in which scintigraphy may be of differential diagnostic benefit.

Serotypes and subtypes of Neisseria meningitidis: results of an international study comparing sensitivities and specificities of monoclonal antibodies.

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.

Restricted association between biotypes and serotypes within group A streptococci.

Investigating individual variations between different isolates of group A streptococci, we observed a close correlation between biotypes and serotypes in 46 strains from pharyngitis patients. Biotyping, carried out with a commercially available rapid identification gallery, delineated 10 different associations of characteristics, designated biotypes 1 to 10, observed both in the manufacturer's (127 strains) and our personal (98 strains) collections of group A strains. Only the most frequent biotypes (biotypes 1 to 6) were observed in the pharyngitis cohort, but the overall frequencies of the biotypes did not display striking differences compared with the control collections. Serotyping of the pharyngitis strains showed that each M type was restricted to a sole biotype. For example, M types 1, 4, and 28 were found only in biotype 1 and M type 6 was found only in biotype 6 strains. This association was not due to an epidemiologic bias, since it was also observed in a control series consisting of reference strains and isolates from distant countries (the United States and Czech Republic versus France). An exception was for M type 78, which exhibited biotype 3 or biotype 4. Investigation of the heterogeneity of the strains at the DNA level showed no significant variations of the ribotype patterns between strains of different biotypes, confirming that group A streptococci belong to a unique and homogeneous species. This previously undescribed association between serotypes and biotypes is of interest for a rapid and preliminary characterization of strains isolated in individual patients or during an outbreak. A possible pathogenic association of some biotypic characteristics with specific M proteins is envisaged.

[Increased incidence of invasive meningococcal disease caused by C:2a:P1.2 meningococci in certain regions of the Czech Republic]. / Zvýsený výskyt invazívního meningokokového onemocnení v nekterých oblastech Ceské republiky zpusobený meningokokem C:2a:P1.2.

In the first half of 1993 an increased incidence of invasive meningococcal disease was recorded in the Czech Republic. In the CR a total of 59 cases were revealed, incl. 10 fatal ones. This high lethality (16.9%) is markedly higher than that from 036 recorded in this country during previous years. The highest incidence and death rate was recorded in the North Moravian region and in the age group from 15-19 years. Based on active surveillance of the invasive meningococcal disease by epidemiologists, microbiologists and infectiologists of the entire Czech Republic, it may be concluded that the sulphonamide sensitive strain Neisseria meningitidis C:2a:P1.2 caused in the North Moravian region a local epidemic of invasive meningococcal disease in the army and civilian population. In other regions of the CR the epidemiological situation in the first half of 1993 did not differ from the previous period: sporadic incidence of meningococcal disease, prevalence of the serological group B, highest incidence in the youngest age groups. The meningococcus C:2a:P1.2 was not detected in the CR before 1993. This uncommon epidemiological situation was resolved by immunization, aimed from the antigenic aspect and with regard to age and locality.

[Genotypes of Neisseria meningitidis strains isolated from patients in the Czech Republic]. / Genotypy kmenu Neisseria meningitidis izolovaných od nemocných v Ceské republice.

A group of 75 strains of Neisseria meningitidis isolated from patients with meningococcal meningitis in the Czech Republic during 1980-1988 was characterized by assessment of serogroups, serotypes, serosubtypes and genotypes. Twelve strains belonged into serogroup A, 27 into serogroup B and 36 into serogroup C. The most frequently found serotype was 4, subserotype P1.2. The authors identified 48 different enzyme genotypes among which four genetically related groups were assessed. Strains of Neisseria meningitidis of the same genotype were found more frequently during the period of 1980-1984, when a rise of meningococcal meningitis in the Czech Republic was recorded. During this period 73% of the strains belonged into three genetically related groups, while during the subsequent period (1985-1988) these genetically related groups comprised only 47% of the strains. Only one strain of Neisseria meningitidis (878/85) represented a genetic clone ET-5 complex responsible for an epidemic of meningococcal meningitis in western European countries from the mid-seventies.

The complex of flavivirus envelope polypeptide with meningococcal proteosomes elicits formation of virus-neutralizing antibodies.

Polypeptide E of tick-borne encephalitis virus was isolated in sucrose density gradient and mixed with equal weight portion of meningococcal proteosomes in the presence of N-dodecyl-N,N-dimethylglycine. Mutual complexing of viral and bacterial molecules occurred after removal of detergent by dialysis. Complexed particles appeared in the electron microscope as 40-50 microns thick short-rod structures covered on their surface with both, delicate poppy-like grains, or envelope subunit-like clustered molecules. Even when applied without adjuvant, the complex of tick-borne encephalitis virus polypeptide E with meningococcal proteosomes elicited in mice a marked antiviral as well as antibacterial humoral response.

[Serotyping and subtyping strains of Neisseria meningitidis isolated from patients in Czechoslovakia 1975-1986]. / Sérotypizace a subtypizace kmenu Neisseria meningitidis izolovaných od nemocných v CSR v letech 1975-1986.

The paper presents results of serotyping and subtyping of 181 strains of N. meningitidis isolated from patients in the CSR during 1975-1988. A new method of whole-cell ELISA with monoclonal antibodies was used. During the investigation period in strains of N. meningitidis serogroup A only serotype 4 was detected (68.4%) and no subtype was found. In N. meningitidis, serogroups B and C, heterogeneity of serotypes and subtypes was found and a relatively high percentage of strains where the type or subtype could not be detected. In N. meningitidis serogroup B serotype 15 (11.0%) and 4 (8.2%) and subtype P1.15 (14.9%) and P1.2 (12.9%) predominated. In N. meningitidis serogroup C serotype 4 (28.8%) and 15 (7.7%) and subtype P1.2 (21.1%) and P1.6 (11.6%) prevailed. This detected heterogeneity of serotypes and subtypes corresponds to the findings of other authors during non-epidemic conditions. The authors present the most frequent combinations of serotypes and subtypes in strains of different serological groups A : 4 : NT, B : NT : P1.2, B : 4 : P1.15, B : 15 : 1.15, B : 15 : NT, C : 4 : NT, C : NT : P1.2. The work confirms the homogeneity of antigenic properties of N. meningitidis strains isolated from one focus.

Meningitis in a child due to Neisseria meningitidis group K.

A four-year-old boy with meningitis due to Neisseria meningitidis group K, serotype 14, serosubtype P1.6, is reported to be, we believe, the first case caused by this strain.

[Specific antibody response in carriers of N. meningitidis]. / Specifická protilátková odpoved na nosicstvo N. meningitidis.

When investigating the specific immune response, the authors found that as many as 97.95% subjects in the investigated community had positive titres of anti A meningococcal antibodies. The values of geometrical means of anti A antibody titres varied between 16.85 and 20.25. Short-time carriers have substantially higher antibody levels than long-term carriers, the difference being statistically significant p less than 0.001. In the course of the investigation antibody levels rose despite the fact that meningococci of group A were not isolated. The position as regards anti C meningococcal antibodies is much worse. There the percentage of positivity is only 24.48% and the values of the geometrical means of titres in positive subjects varied between 7.78 and 8.48. On the whole group of subjects the values were, however, 2.09-2.42. This means they did not by far reach the value of the 1:6 titre, which is considered the first positive titre. Similarly as in anti A antibodies, there is a significant difference between the antibody level in short-term and long-term carriers in favour of the short-term ones.

[Changes in the age distribution of meningococcal diseases in Czechoslovakia]. / Zmena vekové distribuce meningokokového onemocnení v CSR.

Based on an analysis of the specific morbidity by age, we tried to explain the development of the morbidity from meningococcal meningitis in the CRS during the past 35 years. After the epidemic of meningococcal meningitis at the beginning of the fifties, the morbidity had a declining trend to the mid-seventies. During the period between 1975 and 1984 there was a rising trend of the notified morbidity, while in the subsequent years a decline followed. From the analysis of the specific morbidity by age it is obvious that during the period between 1972-1986 a significant rise of the morbidity of the age groups 0-11 months, 15-19 years and 25-29 years occurred. Analysis of the morbidity of five-year cohorts of subjects born in 1947-1971 revealed that after 1977 ex post herd immunization of all cohorts occurred with the exception of the cohort born in 1947-1951 which acquired a maximum immunity state already in the epidemic at the beginning of the fifties. The authors express the hypothesis that these changes are directly associated with the antigenic changes of N. meningitidis.

Meningococcal meningitis in CSR: a preepidemic situation?

Meningococcal meningitis has become a problem in many European countries, with epidemics occurring during the past 10-15 years. There have also been outbreaks in some non-European countries. The latest meningococcal meningitis epidemic recorded in the CSR (Czech Socialist Republic) occurred in 1953, with the morbidity reaching 14.8 per 100 000 population. After the mid 1950's there was a gradual decline in morbidity, the lowest value (0.3 per 100 000 population) being reached in 1974. Since 1975 there has been a continual rise in morbidity, the value notified for 1982 was 1.2 per 100 000 population. An analysis of the epidemiological situation in meningococcal meningitis in CSR for the past 30 years was performed. Different indicators of the onset of epidemics were evaluated. Shift in the age distribution of patients was found to be the most valuable sign of changing epidemiological situtation. An attempt was made to prognosticate mathematically its further development. The analysis of the epidemiological data suggests that CSR is in a preepidemic situation. This finding is all the more important because a change has been found in the serogroup and serotype patterns of Neisseria meningitidis strains isolated from cerebrospinal fluid.

Neisseria meningitidis strains isolated in CSR from cerebrospinal fluid: preepidemic shift in serogroup and serotype distribution?

Antigenic properties of N. meningitidis strains isolated from CSF in CSR were surveyed. Capsular antigens (serogroups) were tested from 1970, noncapsular antigens (serotypes) from 1975. Results up to the end of 1982 are presented. In 1980 shifts were encountered in the distribution of both capsular and noncapsular antigens. Serogroups. Group B predominated throughout the surveillance period, but from 1980 this predominance was significantly lower. Group C was the second most frequent throughout the observed period, but its frequency rose significantly from 1980 and stayed high. Group A was the third in frequency all the time, its relative prevalence increased (but not significantly) from 1980. Serotypes. Serotype 2 predominated during the whole period, but its predominance rose significantly from 1980. Serotype 4 was second in frequency, but from 1980 its frequency also increased significantly. Serotypes other than 2 or 4 decreased significantly in frequency and variability from 1980, also nontypable strains decreased significantly from 1980. Changes in the distribution of the capsular and noncapsular antigens on N. meningitidis strains from CSR are characteristic of epidemic situation. The present finding is all the more serious because it comes in a context of rising meningococcal morbidity and a shift in the morbidity into older age groups.