Investigation of different group A immunoassays following one dose of meningococcal group A conjugate vaccine or A/C polysaccharide vaccine in adults.

A double-blind, randomized, controlled phase I study to assess the safety, immunogenicity, and antibody persistence of a new group A conjugate vaccine (PsA-TT) in volunteers aged 18 to 35 years was previously performed. Subjects received one dose of either the PsA-TT conjugate vaccine, meningococcal A/C polysaccharide vaccine (PsA/C), or tetanus toxoid vaccine. The conjugate vaccine was shown to be safe and immunogenic as demonstrated by a standardized group A-specific immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) and by a serum bactericidal antibody (SBA) assay using rabbit complement (rSBA). This report details further analysis of the sera using four additional immunologic assays to investigate the relationship between the different immunoassays. The immunoassays used were an SBA assay that used human complement (hSBA), a group A-specific IgG multiplexed bead assay, and two opsonophagocytic antibody (OPA) assays which used two different methodologies. For each vaccine group, geometric mean concentrations or geometric mean titers were determined for all assays before and 4, 24, and 48 weeks after vaccination. Pearson's correlation coefficients were used to assess the relationship between the six assays using data from all available visits. An excellent correlation was observed between the group A-specific IgG concentrations obtained by ELISA and those obtained by the multiplexed bead assay. hSBA and rSBA titers correlated moderately, although proportions of subjects with putatively protective titers and those demonstrating a > or = 4-fold rise were similar. The two OPA methods correlated weakly and achieved only a low correlation with the other immunoassays. The correlation between hSBA and group A-specific IgG was higher for the PsA-TT group than for the PsA/C group.

Evaluation of PorB variable region typing of Neisseria gonorrhoeae using PCR-ELISA in samples collected from men who have sex with men.

A polymerase chain reaction (PCR)-based enzyme-linked immunosorbent assay (ELISA) methodology was developed to characterize Neisseria gonorrhoeae porB gene variable regions (VR); the methodology was evaluated in comparison to porB VR typing by checkerboard hybridization. Clinical noncultured samples from 35 men who have sex with men (MSM), positive by nucleic amplification assays for N. gonorrhoeae, were typed using a panel of 40 oligonucleotide probes to porB VRs and compared to checkerboard hybridization. Complete concordance was observed between the two methods at PIB VRs 1, 3, and 7. At the more degenerate VRs 5 and 6, PCR ELISA resulted in obtaining more typeable VRs than checkerboard hybridization due to single nucleotide mismatches. By PCR ELISA, two predominant PIB porB types were identified in 58% of the samples and the remaining 16 samples had one of six other porB types. Both PCR ELISA and checkerboard hybridization methods of porB VR typing allowed characterization of N. gonorrhoeae from noncultured clinical samples including throat and rectal swabs and discriminated N. gonorrhoeae from N. meningitidis present in some of the samples. PCR ELISA is a rapid, relatively inexpensive and alternative molecular typing method for N. gonorrhoeae, suitable for use in conjunction with molecular diagnostic tests.

Genetic and immunologic characterization of a novel serotype 4, 15 strain of Neisseria meningitidis.

The porin proteins of Neisseria meningitidis are important components of outer membrane protein (OMP) vaccines. The class 3 porin gene, porB, of a novel serogroup B, serotype 4, 15 isolate from Chile (Ch501) was found to be VR1-4, VR2-15, VR3-15 and VR4-15 by porB variable region (VR) typing. Rabbit immunization studies using outer membrane vesicles revealed immunodominance of individual PorB (class 3) VR epitopes. The predominant anti-Ch501 PorB response was directed to the VR1 epitope. Anti-PorB VR1 mediated killing was suggested by the bactericidal activity of Ch501 anti-sera against a type 4 strain not expressing PorA or class 5 OMPs. Studies that examine the molecular epidemiology of individual porB VRs, and the immune responses to PorB epitopes, may contribute to the development of broadly protective group B meningococcal vaccines.

A typing system for neisseria gonorrhoeae based on biotinylated oligonucleotide probes to PIB gene variable regions.

The porin proteins PIA and PIB of Neisseria gonorrhoeae are serotyping antigens for the serovar classification system and leading candidates for gonococcal vaccine development. Although serotyping has been a useful tool, this method can be insensitive to critical sequence changes in the por gene, including those in surface-exposed variable regions (VRs). A sensitive and specific typing system for N. gonorrhoeae has been developed that uses biotin-labeled oligonucleotide probes with chemiluminescence detection to type PIB gene VRs. The PIB VR types of geographically and temporally diverse gonococcal strains and sexual contact isolates were determined. por VR typing discriminated between most unrelated isolates and provided information about individual VR type that was not apparent from serovar designations. PIB VR typing avoids limited monoclonal antibody availability, interlaboratory variation, and the requirement for culture-based surveillance associated with gonococcal serotyping, and provides useful information about the molecular epidemiology of individual por gene VRs.

Analysis of Neisseria meningitidis class 3 outer membrane protein gene variable regions and type identification using genetic techniques.

The class 3 porin proteins of Neisseria meningitidis stimulate bactericidal antibodies and express serotype-specific antigenic epitopes. Sequence analysis of porB genes for the class 3 proteins revealed regions of variability that map to surface-exposed loops. To evaluate the relationship between serotype and variable-region (VR) genotype, sequences from the 11 class 3-expressing serotype strains and 3 additional serotype 4 strains were analyzed by molecular techniques. Multiple-sequence alignment revealed a limited number of unique sequences at each of four VRs (VR1 to VR4), ranging from four unique sequences at VR1 to seven sequence patterns at VR2 and VR4. Serotype-specific VR sequences were found in each of the four VRs, suggesting that each VR has immunologic importance. Five serotypes had at least one VR sequence that was unique. Three serotypes which had sequences in common with other serotypes at each VR were distinguished by examining multiple VRs. Serotype 3 was identical to serotype 19 at each VR, and serotype 8 was identical to serotype 18 at each VR. Serotypes 4 and 21 were identical at VR1 and significantly different at VR3 and VR4. A subpopulation of serotype 4 strains with a unique VR2 sequence was identified. The serotypes which were grouped with closely related or identical sequences at one VR were grouped with different serotypes at other VRs consistent with the pattern of genetic mosaicism described for the porA (class 1 protein) gene. Hybridization assays demonstrated the ability to identify VR genotypes and distinguish serotypes using biotin-labelled oligonucleotide probes. This information may be useful in strain selection for vaccine development, in epidemiologic studies to determine the prevalence of the individual VR genotype (especially among nonserotypeable strains) and, combined with PCR, in the identification of culture-negative suspected meningococcal cases.

Stomatococcus mucilaginosus catheter-related infection in an adolescent with osteosarcoma.

Ours is apparently the first reported pediatric case of Stomatococcus mucilaginosus bacteremia. It is the second reported case of S mucilaginosus catheter-associated infection, but the first case successfully treated without removing the central venous catheter. This unusual organism should be added to the list of opportunistic pathogens that can be isolated from immunocompromised pediatric patients.