Production and immunochemical characterization of Neisseria meningitidis group B antiserum for the diagnosis of purulent meningitis.

Unlike Neisseria meningitidis groups A, C, Y and W135, the group B capsular polysaccharide has been shown to be chemically and immunologically identical to the capsular polysaccharide of Escherichia coli K1. Both components are sialic acid homopolymers and are poorly immunogenic. Nevertheless, due to the high incidence of Neisseria meningitidis group B meningitis in the population of the State of São Paulo, preparing antiserum to this serogroup for diagnostic purposes has become a matter of high priority. Of the many immunization schemes proposed, intravenous inoculation of whole bacteria previously inactivated with formaldehyde and simultaneous intradermal inoculation with a mixture of the bacterial polysaccharide fraction and whole bacteria in complete Freund;s adjuvant have produced the best results. The antiserum was treated with immunoadsorbents prepared with aluminum chloride and protein and/or polysaccharide antigens from each of the following heterologous bacteria: Haemophilus influenzae type b, Streptococcus pneumoniae, Escherichia coli other than K1, and Staphylococcus aureus, in order to eliminate cross-reactivity. For quality control analysis, the antiserum was assessed by the immunodiffusion, counterimmunoelectrophoresis, dot-ELISA, and immuno-blot techniques against homologous antigens. Specificity was obtained after treating the antiserum with Haemophilus influenzae type b polysaccharide immunosorbent.

[Differences in the adhesive properties of Staphylococcus saprophyticus to HEp-2 cells and erythrocytes]. / Diferenças nas propriedades adesivas de Staphylococcus saprophyticus a células HEp-2 e eritrócitos.

S. saprophyticus has been frequently isolated from urinary tract infections in young women. In contrast with S. aureus, no defined virulence factors have been recognized for the coagulase negative Staphylococcus species. The objective this study was to analyze the adherence of S. saprophyticus to HEp-2 cells and sheep erythrocytes. The sample were isolated from urine of patients with urinary infection. Hemagglutination, adherence to HEp-2 cells tests and inhibition by specific carbohydrates of the interactions between these cells were analyzed. Most of the strains were hemagglutinating whose properties was inhibited by mannose (100mM). There was a high adherence level to HEp-2 cells. The differences in specificity and attachment level noted in this study suggest that multiple adhesins are involved in the mechanism of cellular interaction.

Listeria monocytogenes in renal transplant recipients.

Five cases of Listeria monocytogenes bacteremia were observed from April to December 1985, among renal transplant recipients from the same hospital in São Paulo, Brazil. The patients were adults (mean age: 40.6 years), and the basic complaint was fever, with no report of meningeal syndrome. Laboratory tests revealed the presence of two serovars, (1/2)a and 4b, which were classified into three lysotypes. The four strains of serovar 4b showed the same antibiotype, with resistance to cefoxitin, clindamycin, oxacillin and penicillin.

[Serotypes of Streptococcus pneumoniae isolated from cerebrospinal fluid in 1977-1988 in São Paulo City, Brazil]. / Sorotipos de Streptococcus pneumoniae isolados de líquido cefalorraquidiano no período de 1977-1988 na cidade de São Paulo, Brasil.

Since 1977, the Instituto Adolfo Lutz (IAL) is having interest in the serotyping of S. pneumoniae or pneumococcus from infections caused by this bacteria. The isolated strains have been sent to the WHO Pneumococcal Reference Center, Pennsylvania, U.S.A.. From 1977 to 1988, 1.000 pneumococcus strains isolated from cerebrospinal fluid were typed, according to Danish nomenclature, and 60 serotypes were identified. The most frequent serotypes were 1, 6B, 18C, 14, 5, 3, 6A, 23F, 19F, and 38. Among different age groups, they showed a variable incidence, with serotype 6B in the ages of zero to almost 2 years old, serotype 1 in the age group of 2 to 50 years old, and serotype 3 in the ages over 50. During the 12 years study, 25 serotypes showed some uniformity in the frequency, the same as with the seasonal fluctuations. Concerning the severity of the pneumococcal infections, chiefly meningitis, and the few information related to pneumococcus serotypes which occur in the area, it was considered of high relevance to have the information of serotypes, once polysaccharide vaccines have been employed with success to prevent these infections.

Genetic relationships among serogroup B: serotype 4 Neisseria meningitidis strains.

We compared the results obtained by serotyping of PorB epitopes using an expanded panel of monoclonal antibodies (mAb) including mAb 7 and mAb 10, with results obtained by RFLP of rRNA genes (ribotyping). The purpose of this study was to assess the correlation between phenotypic- and genotypic- methods for typing N. meningitidis. The ribotypes obtained using ClaI or EcoRV endonucleases grouped the strains in seven and two different patterns, respectively. This additional characterization of PorB epitopes improved the correlation between these two methods of typing N. meningitidis.

Meningococcal disease caused by Neisseria meningitidis serogroup B serotype 4 in São Paulo, Brazil, 1990 to 1996.

A large epidemic of serogroup B meningococcal disease (MD), has been occurring in greater São Paulo, Brazil, since 1988. A Cuban-produced vaccine, based on outer-membrane-protein (OMP) from serogroup B: serotype 4: serosubtype P1.15 (B:4:P1.15) Neisseria meningitidis, was given to about 2.4 million children aged from 3 months to 6 years during 1989 and 1990. The administration of vaccine had little or no measurable effects on this outbreak. In order to detect clonal changes that could explain the continued increase in the incidence of disease after the vaccination, we serotyped isolates recovered between 1990 and 1996 from 834 patients with systemic disease. Strains B:4:P1.15, which was detected in the area as early as 1977, has been the most prevalent phenotype since 1988. These strains are still prevalent in the area and were responsible for about 68% of 834 serogroup B cases in the last 7 years. We analyzed 438 (52%) of these strains by restriction fragment length polymorphism (RFLPs) of rRNA genes (ribotyping). The most frequent pattern obtained was referred to as Rb1 (68%). We concluded that the same clone of B:4:P1.15-Rb1 strains was the most prevalent strain and responsible for the continued increase of incidence of serogroup B MD cases in greater São Paulo during the last 7 years in spite of the vaccination trial.

The use of oligonucleotide probes for meningococcal serotype characterization.

In the present study we examine the potential use of oligonucleotide probes to characterize Neisseria meningitidis serotypes without the use of monoclonal antibodies (MAbs). Antigenic diversity on PorB protein forms the bases of serotyping method. However, the current panel of MAbs underestimated, by at least 50% the PorB variability, presumably because reagents for several PorB variable regions (VRs) are lacking, or because a number of VR variants are not recognized by serotype-defining MAbs. We analyzed the use of oligonucleotide probes to characterize serotype 10 and serotype 19 of N. meningitidis. The porB gene sequence for the prototype strain of serotype 10 was determined, aligned with 7 other porB sequences from different serotypes, and analysis of individual VRs were performed. The results of DNA probes 21U (VR1-A) and 615U (VR3-B) used against 72 N. meningitidis strains confirm that VR1 type A and VR3 type B encode epitopes for serotype-defined MAbs 19 and 10, respectively. The use of probes for characterizing serotypes possible can type 100% of the PorB VR diversity. It is a simple and rapid method specially useful for analysis of large number of samples.

Genetic structure of Neisseria meningitidis serogroup C epidemic strains in south Brazil.

In the present study we report the results of an analysis, based on serotyping, multilocus enzyme electrophoresis (MEE), and ribotyping of N. meningitidis serogroup C strains isolated from patients with meningococcal disease (MD) in Rio Grande do Sul (RS) and Santa Catarina (SC) States, Brazil, as the Center of Epidemiology Control of Ministry of Health detected an increasing of MD cases due to this serogroup in the last two years (1992-1993). We have demonstrated that the MD due to N.meningitidis serogroup C strains in RS and SC States occurring in the last 4 years were caused mainly by one clone of strains (ET 40), with isolates indistinguishable by serogroup, serotype, subtype and even by ribotyping. One small number of cases that were not due to an ET 40 strains, represent closely related clones that probably are new lineages generated from the ET 40 clone referred as ET 11A complex. We have also analyzed N.meningitidis serogroup C strains isolated in the greater São Paulo in 1976 as representative of the first post epidemic year in that region. The ribotyping method, as well as MEE, could provide useful information about the clonal characteristics of those isolates and also of strains isolated in south Brazil. The strains from 1976 have more similarity with the actual endemic than epidemic strains, by the ribotyping, sulfonamide sensitivity, and MEE results. In conclusion, serotyping with monoclonal antibodies (C:2b:P1.3), MEE (ET 11 and ET 11A complex), and ribotyping by using ClaI restriction enzyme (Rb2), were useful to characterize these epidemic strains of N.meningitidis related to the increased incidence of MD in different States of south Brazil. It is mostly probable that these N.meningitidis serogroup C strains have poor or no genetic correlation with 1971-1975 epidemic serogroup C strains. The genetic similarity of members of the ET 11 and ET 11A complex were confirmed by the ribotyping method by using three restriction endonucleases.

[Immune response to anti-meningococcal vaccines]. / Imunidade conferida por vacinas anti-meningocócicas.

In view of a recent epidemic of meningococcal disease caused by serogroup B N. meningitidis in the Greater S. Paulo area (Brazil), a review of the epidemics that occurred in Brazil during the period from 1900 to 1990 is presented. The current status of vaccines against N. meningitidis A.C.Y. and W135 is analysed. The recent advances in the development and effectiveness of B. meningococci vaccines are discussed.

Comparative activity of six beta-lactam antibiotics against strains of Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae.

The minimum inhibitory concentrations (MIC) of penicillin G, ampicillin, mezlocillin, azlocillin, cephalothin and cefoxitin were determined for 47 strains of Haemophilus influenzae, 68 strains of Neisseria meningitidis and 45 strains of Streptococcus pneumoniae. These strains were isolated during the past three years from patients with acute bacterial meningitis. Three strains of H. influenzae were ampicillin-resistant while no pneumococcus or meningococcus strain was penicillin-resistant. Mezlocillin was the most potent antibiotic against the Haemophilus and pneumococcus strains, followed closely by azlocillin. Mezlocillin inhibited 77.7% of the meningococci strains tested at a concentration of 0.03 mg/l. Penicillin G was the most effective of the drugs against these strains. It inhibited 100% at a concentration of 0.5 mg/l. The cephalosporins were the least active of the six beta-lactam antibiotics tested.

Characterization of epidemic Neisseria meningitidis serogroup C strains in several Brazilian states.

Epidemic strains of the Neisseria meningitidis C:2b:P1.3 electrophoretic type 11 complex were responsible for an outbreak in Curitiba, Parana State, Brazil, from 1990 to 1991. Strains of this complex were also isolated in other Brazilian states and were responsible for a meningococcal disease epidemic in São Paulo State in 1990. Serotyping both with monoclonal antibodies and by multilocus enzyme electrophoresis was useful for typing these epidemic strains related to the increased incidence of meningococcal disease. The genetic similarity of members of the electrophoretic type 11 complex was confirmed by the ribotyping method by using EcoRI or ClaI endonuclease restriction enzymes.

Distribution of serotypes of Streptococcus pneumoniae isolated from invasive infections over a 16-year period in the greater São Paulo area, Brazil.

Capsular types of pneumococci from normally sterile body sites of 1,622 patients in Brazil were analyzed. Of 1,477 isolates from cerebrospinal fluid, 76.1% were of types represented in the currently available pneumococcal vaccine. The importance of age, time, and place in determining the optimal formulation of pneumococcal vaccine is considered.

Correlation between serological and sequencing analyses of the PorB outer membrane protein in the Neisseria meningitidis serotyping system.

The current serological typing scheme for Neisseria meningitidis is not comprehensive; a proportion of isolates are not serotypeable. DNA sequence analysis and predicted amino acid sequences were used to characterize the structures of variable-region (VR) epitopes on N. meningitidis PorB proteins (PorB VR typing). Twenty-six porB gene sequences were obtained from GenBank and aligned with 41 new sequences. Primary amino acid structures predicted from those genes were grouped into 30 VR families of related variants that displayed at least 60% similarity. We correlated VR families with monoclonal antibody (MAb) reactivities, establishing a relationship between VR families and epitope locations for 15 serotype-defining MAbs. The current panel of serotype-defining MAbs underestimates by at least 50% the PorB VR variability because reagents for several major VR families are lacking or because a number of VR variants within some families are not recognized by serotype-defining MAbs. These difficulties, also reported for serosubtyping based on the PorA protein, are shown as inconsistent results between serological and sequence analyses, leading to inaccurate strain identification and incomplete epidemiological data. The information from this study enabled the expansion of the panel of MAbs currently available for serotyping, by including MAbs of previously undetermined specificities. Use of the expanded serotype panel enabled us to improve the sensitivity of serotyping by resolving a number of formerly nonserotypeable strains. In most cases, this information can be used to predict the VR family placement of unknown PorB proteins without sequencing the entire porB gene. PorB VR typing complements serotyping, and a combination of both techniques may be used for full characterization of meningococcal strains. The present work represents the most complete and integrated data set of PorB VR sequences and MAb reactivities of serogroup B and C meningococci produced to date.

Proposed standardization of Neisseria meningitidis PorA variable-region typing nomenclature.

Neisseria meningitidis isolates are conventionally classified by serosubtyping, which characterizes the reactivities of the PorA outer membrane protein variable-region (VR) epitopes with monoclonal antibodies (MAbs). A newer method (PorA VR typing) uses predicted amino acid sequences derived from DNA sequence analysis. The resulting classification schemes are not standardized, offering conflicting and sometimes irreconcilable data from the two methods. In this paper, we propose a standardization of the PorA VR typing nomenclature that incorporates serologic information from traditional PorA serosubtyping with molecular data from predicted VR sequences. We performed a comprehensive literature and database search, generating a collection of strains and DNA sequences that reflects the diversity within PorA that exists to date. We have arranged this information in a comprehensive logical model that includes both serosubtype and PorA VR type assignments. Our data demonstrate that the current panel of serosubtype-defining MAbs underestimates PorA VR variability by at least 50%. Our proposal for VR typing is informative because amino acid sequence and serologic information, when serosubtype-defining MAbs are available, can be deduced simultaneously from the PorA VR designation. This scheme will be useful in future classification and applied epidemiologic studies of N. meningitidis, being a systematic way of selecting PorA vaccine candidates and analyzing vaccine coverage and failure.

Immune response of Brazilian children to a Neisseria meningitidis serogroup B outer membrane protein vaccine: comparison with efficacy.

Since 1986, serogroup B Neisseria meningitidis has caused approximately 80% of the meningococcal disease in Brazil. In 1988, an epidemic caused by N. meningitidis B:4:P1.15 was recognized in the greater São Paulo area of Brazil. The São Paulo state government decided to vaccinate children from 3 to 83 months of age with a vaccine consisting of serotype 4 outer membrane protein and group C meningococcal polysaccharide that was produced in Cuba. About 2.7 million children were vaccinated during two immunization campaigns conducted in 1989 and 1990. Because of this, a case-control study was designed to determine vaccine efficacy against group B meningococcal disease. The purpose of our study was to compare the antibody response with the protection from disease estimated from the case-control study. We measured the immune responses of vaccinees by enzyme-linked immunosorbent assay (ELISA), immunoblot, and bactericidal assay. The development of bactericidal antibodies was age dependent and in good agreement with the results of the case-control study. Only 40% of vaccinees showed fourfold or greater increases in bactericidal antibody titers after vaccination. A poor correlation between antibody levels detected by ELISA and those by bactericidal assay was found. Immunoblot analysis showed that about 50% of the serum samples with bactericidal titers higher than 1:4 were reactive with class 1 outer membrane protein. We conclude that the bactericidal assay is a good, laboratory-based, functional assay for the study of vaccine immunogenicity and that an effective solution to group B meningococcal disease remains to be demonstrated.

Ongoing group B Neisseria meningitidis epidemic in São Paulo, Brazil, due to increased prevalence of a single clone of the ET-5 complex.

Beginning in 1988, the incidence of meningococcal disease in the area of greater São Paulo began to surpass the upper confidence limit of an 8-year average incidence (from 1979 to 1986), thus characterizing a new epidemic in the region of greater São Paulo. This epidemic, which extended to 1990, was different from previous epidemics in that it was caused by serogroup B. The increased incidence of meningococcal disease was paralleled by an increased prevalence of a single group B clone, B:4:P1.15, of the ET-5 complex. ET-5 strains have been present in the greater São Paulo area since 1979; however, they have been associated with a high percentage of the group B disease only from 1987 to the present. On the basis of the increased incidence of group B disease in São Paulo, a mass vaccination program with a serotype 4:P1.15 meningococcal protein vaccine was undertaken. The impact of this vaccination program is under analysis.

Emergence of a new clone of serogroup C Neisseria meningitidis in São Paulo, Brazil.

Serogroup C isolates of Neisseria meningitidis recovered from 121 patients with meningitis or septicemia in Greater São Paulo, Brazil, between 1976 and 1990 were analyzed with respect to serotype and multilocus enzyme genotype. The distribution of serotypes has changed since 1989 when serotype 2b started to replace serotype 2a. There were 48 distinct multilocus genotypes (electrophoretic types [ETs]) and 13 distinct complexes. Among the 41 serotype C:2b:- strains analyzed, 38 (93%) were found in complex 11. The percentage of complex 11 increased from 8% in 1988 to 50 and 66% in 1989 and 1990, respectively. Although we have been in an epidemic situation due to serogroup B:4:P1.15 ET-5 complex since 1988, the appearance and increase of a new unrelated strain, C:2b:- of ET-11 complex, in 1989 and 1990 provide enough data to conclude that the presence of two different complexes, ET-5 and -11, of N. meningitidis were responsible for the high levels of meningococcal disease in Greater São Paulo during this period.

Serosubtypes and PorA types of Neisseria meningitidis serogroup B isolated in Brazil during 1997--1998: overview and implications for vaccine development.

Meningococcal disease caused by N. meningitidis serogroup B (MenB) has been endemic in Brazil since 1997. In this study, we determined the prevalence of serosubtypes of MenB isolated in 10 Brazilian states and the Federal District during 1997 and 1998 and investigated the extent of PorA VR sequence variation among the most prevalent serosubtypes to evaluate the possible use of an outer membrane vesicle (OMV)-, PorA-based vaccine to prevent meningococcal disease in Brazil. During this period, a total of 8,932 cases of meningococcal disease were reported. Only 42% (n = 3,751) of the reported cases were laboratory confirmed, and about 60% (n = 2,255) of those were identified as MenB. Among 1,297 MenB strains selected for this study, the most prevalent serosubtypes were P1.19,15 (66%), P1.7,1 (11%), and P1.7,16 (4%). PorA VR typing showed that 91% of the P1.19,15 strains analyzed had VR1 and VR2 sequences identical to those of the prototype strain. No sequence variation was detected among the 40 strains representing all isolated MenB P1.7,16 strains in the three southern states, where this serosubtype accounts for 75% of the serosubtypes identified. Similarly, all P1.7,1 strains were identified by PorA typing as P1.7-1,1. Although further improvements in the reporting of cases and collection of strains in Brazil are needed, our data suggest that a trivalent OMV-based vaccine prepared with PorA types P1.19,15, P1.7-1,1, and P1.7,16 may be appropriate to control serogroup B meningococcal disease in most of the Brazilian states.