Comparison of slide coagglutination test and countercurrent immunoelectrophoresis for detection of group B streptococcal antigen in cerebrospinal fluid from infants with meningitis.

The usefulness of Phadebact streptococcus reagents for the detection of group B streptococcal antigen in cerebrospinal fluid was evaluated in 54 infants with meningitis and in 22 normal infants. Antigens was detected by slide coagglutination in 19 (82.6%) and by countercurrent immunoelectrophoresis in 20 (87.0%) of 23 cerebrospinal fluid specimens from infants with group B streptococcal meningitis at admission. After initiation of antimicrobial therapy, antigen could be detected in 11 of 19 (by slide coagglutination) and 7 of 18 (by countercurrent immunoelectrophoresis) cerebrospinal fluids. False-positive reactions were noted by slide coagglutination in one infant with S. bovis meningitis and one with group B streptococcal bacteremia without meningitis; none occurred with countercurrent immunoelectrophoresis. The commercial availiability, simplicity, sensitivity (82.6%), and specificity (96.4%) of the Phadebact slide coaggluatination test for detecting group B streptococcal antigen in cerebrospinal fluid suggest that it may be useful for the early and rapid diagnosis of group B streptococcal meningitis.     

Increased frequency of high serum IgM among mothers of infants with neonatal group-B streptococcal septicemia

Total serum IgM levels were studied in 84 mothers of infants with group-B streptococcal (GBS) septicemia/meningitis and compared to IgM concentrations in 91 parturients who were urogenital carriers of GBS but nevertheless gave birth to healthy infants. In all, 22 (27%) in the study group showed IgM levels above the arbitrarily selected limit of 2.40 g/l, in contrast to 12 (13%) of 91 controls (p = 0.02). Among the study group members whose infants were infected with GBS type III, 8 of 34 (24%) were high in serum IgM, compared to only 2 of 34 (6%) of the corresponding controls (p = 0.04). The total serum IgG levels did not differ between the two groups.     

Association of bacterial carbohydrate-specific cold agglutinin antibody production with immunization by group C, group B type III, and Streptococcus pneumoniae type XIV streptococcal vaccines.

Rabbits immunized with group B type III, group C, and Streptococcus pneumoniae type XIV streptococcal vaccines developed autoantibodies reactive with autologous and isologous erythrocytes and human O-positive erythrocytes at reduced temperatures. The cold agglutinin antibodies were present in both the immunoglobulin M (IgM) and IgG fractions of group C streptococcal antiserum and in the IgM fraction of group B type III and S. pneumoniae type XIV antisera. BALB/c, CF1, and local strains of mice immunized with group B type III and S. pneumoniae type XIV streptococcal vaccines also produced a cold agglutinin antibody reactive with rabbit and human erythrocytes. The cold agglutinin antibodies were reactive with saccharide compounds representative of the determinants present on the individual bacterial carbohydrate structures, individual vaccine preparations, and isolated polysaccharides. The group C antibodies in rabbits were reactive with sugar ligands in the following order: N-acetylgalactosamine greater than melibiose greater than lactose greater than galactose greater than glucose. Group B type III and S. pneumoniae type XIV cold agglutinin antibodies in rabbit antisera, however, displayed reactivities different from group C antibodies and from each other. Group B type III antibodies reacted with galactose greater than lactose greater than N-acetylgalactosamine greater than glucose greater than rhamnose; S. pneumoniae type XIV antibodies reacted with lactose greater than melibiose greater than galactose greater than glucose greater than N-acetylgalactosamine. The same relative ligand specificity was observed for the cold agglutinin antibodies in S. pneumoniae type XIV mouse antisera. The cold agglutinin antibodies in group B type III and S. pneumoniae type XIV antiserum reacted with erythrocytes at higher temperatures (up to 31 degrees C) than did group C antibodies (up to 14 degrees C). In addition, S. pneumoniae type XIV antibodies did not discriminate between I- or i-bearing human erythrocytes to a significant extent. The results obtained provide substantial evidence that autoreactive cold agglutinin antibodies produced by immunization with these vaccines represent subpopulations of bacterial carbohydrate-specific antibodies that cross-react with mammalian carbohydrate structures.     

Human B Lymphocyte In Vitro Response to the Group B Streptococcal Type III Capsular Carbohydrate

Anti-group B streptococcal type III polysaccharide-specific human B cells from the peripheral circulation can be activated and delected in an in vitro culture system. It is possible to detect both IgM- and IgG-producing cells from both seropositive and seronegative donors. The specificity of the response was demonstrated by inhibition with excess liquid phase antigen and the use of related but antigenically distinct control antigens. The response was absent without the addition of T cells, optimal at 10% and 25% T cells respectively for IgM- and IgG-secreting cells, and undetectable using 50% T cells. The optimal antigen concentration for in vitro B-cell activation is 2.5 × 10⁻⁴ μ /ml. Cells from 5 of 6 seropositive donors and 3 of 7 seronegative donors produced specific IgM antibody after culture with antigen. We conclude that the control of the human antibody response to the group B streptococcal type III polysaccharide is influenced by T cells. The response seen in the culture system may be of value in assessing future vaccine candidates designed to prevent neonatal infections.     

Influence of preimmunization antibody levels on the specificity of the immune response to related polysaccharide antigens

We studied the influence of preimmunization antibody level on the immune response of adults to one of two structurally related yet immunologically distinct type-specific polysaccharides from Type III Group B streptococcus and Type 14 pneumonococcus. Four weeks after immunization with multivalent pneumococcal vaccine, 20 subjects with low levels of antibody to Type III Group B streptococcus antigen had no significant increase in antibody to this antigen (P greater than 0.05), but all volunteers with moderate to high preimmunization antibody levels who were immunized with Pneumovax had significant increases (P less than 0.01). However, the streptococcal antibody response to pneumococcal Type 14 antigen was weaker and briefer than that in 10 adults given Type III Group B streptococcus vaccine(P less than 0.05). Preimmunization antibody levels influenced the immune response to a structurally similar polysaccharide antigen, but specific Type III polysaccharide antigen appeared necessary to induce a primary antibody response in "nonimmune" adults. We conclude that immunization of mothers with pneumococcal vaccine is not likely to prevent neonatal Type III Group B streptococcal infection, despite immunologic similarities between the two organisms.     

Commercial latex agglutination test for rapid diagnosis of group B streptococcal infection in infants.

Although latex agglutination assays for detection of a variety of bacterial antigens in body fluids from patients with systemic infection have been shown to be useful as rapid diagnostic techniques, lack of commercial availability has restricted their application. The Streptex latex test kit for the detection of group B streptococcal (GBS) antigen in admission body fluid specimens was evaluated for sensitivity and specificity in 54 infants with meningitis and in 10 infants with normal cerebrospinal fluid (CSF) parameters. GBS antigen was detected in 22 of 28 (78.6%) CSF specimens by latex agglutination and in 23 of 28 (82.1%) by countercurrent immunoelectrophoresis. Antigen was present in 21 of 28 (latex agglutination) and 19 of 26 (countercurrent immunoelectrophoresis) CSF specimens after the initiation of antimicrobial therapy. Heat-labile factors accounted for nonspecific agglutination reactions with latex suspensions other than group B in 3 of 28 CSF samples from patients with GBS meningitis. These nonspecific reactions were readily eliminated by heating specimens for 10 min at 100 degrees C. Fifteen patients with GBS meningitis had admission serum and urine samples collected in addition to CSF. Antigen was detected by latex agglutination and countercurrent immunoelelectrophoresis in 14 of 15 (93.3%) and 13 of 15 (86.7%) concentrated urine specimens, respectively, and in 12 of 15 (80%) CSF specimens and 4 of 15 (27%) sera by each method. These findings indicate that the Streptex latex test is a rapid, sensitive, and readily available method for detection of GBS antigen in admission body fluid specimens from infants with meningitis.     

Detection of mycobacterial antigen and antibodies in the cerebrospinal fluid of patients with tuberculous meningitis

An immunodiagnostic test for the detection of a soluble nonprotein mycobacterial antigen by reverse passive haemagglutination with IgM murine monoclonal antibody was developed. The test was used to analyse the cerebrospinal fluid of 89 patients with tuberculous meningitis (TBM) from India and 127 control subjects from India and the UK. The antigen was demonstrable in 88% of culture-positive and 73% of culture-negative TBM patients. However, it was also detected in 21% of Indian patients with pyogenic meningitis, and in 8% of Indian and 1% of UK control subjects. Antibodies binding to a soluble mycobacterial extract were detected at low titre in 68% of all subjects with TBM and in 37% of Indian cases of pyogenic meningitis. Because patients with TBM had raised levels of the antigen and of antibodies to the antigen, the possible role of immune complexes in the pathogenesis of the disease is briefly discussed.     

Role of Adherence in the Pathogenesis of Neonatal Group B Streptococcal Infection

The ability of group B streptococci to attach to buccal epithelial cells from adult volunteers, healthy neonates, and infants with invasive group B streptococcal infection was assessed by using (3)H-labeled bacteria incubated at a bacteria-to-cells ratio of 1,000:1. Type III group B streptococcal clinical isolates adhered significantly better to the epithelial cells of healthy neonates than to those of adults (mean bacteria per cell of 31 versus 7, respectively; P < 0.005). In contrast, no statistically significant differences in adherence of type Ia or type II strains to cells of neonates and adults were noted. The adherence of strains isolated from 15 infants with invasive group B streptococcal infection was significantly greater to the cells of infected infants than to those of age-matched controls (mean bacteria per cell of 39 versus 18, respectively; P < 0.005). In contrast, no significant difference was noted in the adherence of a usually adherent type Ia strain and a nonadherent type III strain to the cells of infected infants compared with control infants. These results indicate that the serotype of group B streptococci with the greatest virulence for neonates (type III) adheres better to neonatal than to adult epithelial cells. Infants who develop invasive infection may have an increased number of epithelial cell surface receptor sites for attachment of group B streptococci, the bacteria may elaborate products which unmask receptor sites, or both.     

Evaluation of group B streptococcal opsonins by radiolabeled bacterial uptake.

The incidence of neonatal group B streptococcal sepsis and meningitis has increased in the last decade. Antibiotic therapy has proved inadequate in controlling this problem, and in developing immunological methods of disease prevention and control it is important to define the critical bacterial and host factors involved in the immune response. We have used a radiolabeled bacterial uptake method to assess the presence of functional opsonins for a strain of type III group B streptococci. Bacteria, labeled with tritiated leucine, were opsonized in fresh, heated or Mg-EGTA treated serum. The opsonized streptococci were added to monolayers of adult human phagocytes 90% adherent to glass coverslips. At 10-min intervals for 60 min the coverslips were washed free of uningested bacteria and placed in a scintillation counter to determine the degree of phagocytosis. Maximum opsonic activity was obtained with specific antibody containing serum. No uptake of unopsonized bacteria was observed. Markedly reduced activity with heated and Mg-EGTA treated serum indicated a major role for the classical complement pathway in host defense against group B streptococci. Serum lacking specific antibody demonstrated almost no uptake indicating a minor role for the alternative pathway in opsonizing these organisms. Reduced levels of phagocytosis were also measured in every case where sera from patients with known type III infections were used for opsonization. These results agree with previous data from our laboratory using a chemiluminescence assay of opsonic activity.     

Immune responses to the R4 protein antigen of group B streptococci and its relationship to other streptococcal R4 proteins.

The R antigen, a trypsin-resistant protein observed in group A, C, F, G, and L streptococci, has also been found in group B streptococci (GBS). Although four species of the R antigen have been described for GBS, the R4 protein is the most prevalent in GBS isolates recovered from humans. This study examined the prevalence of antibodies against the R4 antigen by Western blot (immunoblot) (WB) in sera from 40 mothers colonized with GBS serotype II and III and from 26 noncolonized mothers; 92.5% of the colonized mothers had anti-R4 antibodies, compared with 54% of the noncolonized mothers (P < 0.001). Findings of antibodies in neonatal cord sera (n = 14) were concordant with maternal results by WB analysis for 71% of mother-infant pairs colonized with serotype II and for 57% of pairs colonized with serotype III. Of mothers known to be colonized with type II/R4 or III/R4, 100% (n = 12) had antibody against R4 by WB. This study also evaluated the prevalence of antibody to the GBS R4 antigen in 48 sera from individuals with high and low group A streptococcal anti-DNase B titers. Of those individuals with an anti-DNase B titer of > 640, 64% had a positive WB for anti-R4 antibody, compared with 30% of individuals with low anti-DNase B titers (P < 0.05). The R4 antigen of GBS had immunologic identity to the R4 antigen of group A streptococci. Overall, the findings suggested that antibodies to the streptococcal R4 antigen were commonly present in GBS-colonized mothers and that transplacental passage of these antibodies occurred. The presence of antibody to R4 in non-GBS-colonized individuals may be due to immunologic responses to past exposure to the R antigen present in GBS or other streptococcal groups.     

Detection of group B streptococcal antigen in early-onset and late-onset group B streptococcal disease with the Wellcogen Strep B latex agglutination test.

The Wellcogen Strep B latex agglutination test (Wellcome Diagnostics, Dartford, England) was evaluated as a method of detecting group B streptococcal antigen in urine, cerebrospinal fluid, and serum from neonates with early-onset (less than or equal to 7 days of age) and late-onset group B streptococcal disease. Urine was the best source of antigen, which was detected in 100% of six neonates with early-onset group B streptococcal disease who had urine available in the first 12 h of illness and in 88% of 17 group B streptococcus-infected neonates with urine available in the first 48 h of illness. Antigen was not detected in any samples from patients without group B streptococcal disease except in the urine of one patient with Proteus mirabilis meningitis. The Wellcogen Strep B latex test of the lot tested compares favorably with a noncommercially available latex agglutination test.     

Chronic liver disease: the detection and characterization of circulating immune complexes.

Circulating immune complexes containing IgG, IgM and hepatitis B surface antigen (HBsAg) in sera from groups of patients with various liver diseases were detected by both the C1q and conglutinin solid phase assays. Elevated levels of antigen non-specific immune complexes were observed in sera from all groups and complexes containing IgG were present to a greater extent than were IgM-containing complexes. Higher levels of complexes were generally obtained using the conglutinin assay than the C1q assay and the two assays were shown to preferentially bind complexes of different size ranges and antigen-antibody ratios. Only sera from HBsAg-positive patients had complexes containing HBsAg, and although serum HBsAg titres and levels of HBsAg-containing complexes were correlated, the correlation coefficient was low. The mean levels of immune complexes and the frequency of positive sera varied between different disease categories, but there was little correlation between levels of the three types of complexes detected by the two tests. Assay of immune complexes in sequential serum samples from an individual patient revealed considerable variation in the levels of the three complex types, demonstrating that the measurement of complexes in single serum samples is of limited value in assessing the potential significance of circulating immune complexes in hepatitis B.     

Association of elevated levels of cellular lipoteichoic acids of group B streptococci with human neonatal disease.

Cell-associated lipoteichoic acids (LTAs) from late-exponential-phase cultures (serotypes Ia, Ib, Ic, II, and III) of group B streptococci isolated from infected and asymptomatically colonized infants were quantitated and characterized by growing the organisms in a chemically defined medium containing [3H]glycerol and [14C]acetate. Cell pellets were extracted with 45% aqueous phenol and chloroform-methanol and subjected to DEAE-Sephacel anion-exchange chromatography. Elution profiles resolved three major peaks, I, II, and III, with glycerol and phosphate present in a 1:1 molar ratio in each peak, and results obtained by Ouchterlony immunodiffusion analysis confirmed the presence of poly(glycerol phosphate). Saponification indicated that [14C]acetate was incorporated into fatty acids of peaks I and II only, suggesting that these were cell-associated LTAs. Peak II was of small molecular weight (less than 10,000) and probably represented another species of LTA. Peaks I and II were further demonstrated to be LTA by their ability to sensitize human type O erythrocytes. Peak III lacked fatty acids and was shown to probably be deacylated LTA. Quantitation of cell-associated teichoic acid material produced by the group B streptococcal strains indicated that the clinical isolates from infants with early- or late-onset disease possessed significantly higher levels than did the asymptomatic (clinical isolates from infants without symptoms of disease) group B streptococcal strains.     

Analysis of group B streptococcal types associated with disease in human infants and adults.

It is important to resolve existing differences of opinion regarding group B streptococcal type distribution in human disease because of the relevance of type prevalence to future programs of prevention. This report compares data obtained from typing 392 group B streptococci isolated from systemic infections in both infants and adults in the United States from 1972 through 1975. The data showed a substantial predominance of type III among strains isolated from cases of infant meningitis and from "late-onset" septicemia but did not confirm a prior report that type Ia causes most cases of "early-onset" infant septicemia. Type II was the predominant serotype among 11 cerebrospinal fluid isolates from adults. The fact that over one-fourth of the isolates were types other than Ia or III means that future epidemiological studies, including definition of immunological factors, must include all five group B types.     

Prognostic value of HBsAg/IgM complexes in hepatitis B patients: nature of the proteins involved

Hepatitis B surface antigen (HBsAg)/IgM complexes were measured using a solid phase radioimmunoassay in sera of patients with acute or chronic hepatitis B. These complexes were found in 6 out of 18 patients four weeks after the onset of the disease and only one of them developed chronic hepatitis. HBsAg/IgM complexes correlated with HBsAg, hepatitis B e antigen (HBeAg), and pre-S2 concentration. The precipitation of HBsAg/IgM reactivity by polyethylene-glycol (PEG) and the binding of this activity to the surface of certain uncoated enzyme linked immunosorbent assay (ELISA) plates indicates that HBsAg/IgM positivity may reflect the presence of circulating complexes in serum. HBsAg and pre-S2 were found as components of the complexes but anti-HBs, anti-pre-S2, and polymerized human serum albumin (pHSA) were not. An immune binding between HBsAg and IgM is still questionable. Whatever the nature of the HBsAg/IgM complexes, their detection does not seem to be an earlier indicator of prognosis than HBeAg and/or pre-S2.     

Relative functional activity of purified human immunoglobulin G against a type III group B streptococcal strain.

Human immunoglobulin G (IgG) separated from whole serum by a quaternary aminoethyl-Sephadex A-50 ion exchanger was evaluated for its activity against a type III group B streptococcal strain in the newborn rat model. Separated IgG yielded approximately 70 to 80% of whole serum IgG and did not contain detectable IgM or IgA. This IgG preparation also contained similar ratios of specific type III group B streptococcal antibody to total IgG in comparison with whole serum. In vivo, 50% protection from death was achieved by 3.9 ng of type III-specific antibody per 10 g of rat body weight. This value was considerably lower than 50% protective doses obtained in our previous studies with different human IgG preparations. Further studies are needed to understand the mechanisms responsible for these differences in the functional activity of IgG antibody.     

Functional activity of antibodies to the group B polysaccharide of group B streptococci elicited by a polysaccharide-protein conjugate vaccine.

Group B streptococci (GBS) are a major cause of sepsis and meningitis in infants. While antibodies directed to the type-specific GBS capsule have been shown to be protective, it is less clear whether antibodies to the group B polysaccharide, a noncapsular, cell wall-associated antigen, may play a role in immunity. To investigate the functional activity of group B polysaccharide-specific antibodies, we tested sera from rabbits vaccinated with group B polysaccharide coupled to tetanus toxoid (B-TT). Anti-B-TT was weakly opsonic in vitro for a highly encapsulated type III strain, while antiserum elicited by vaccination with type III capsular polysaccharide linked to tetanus toxoid (III-TT) was a very effective opsonin. In contrast to anti-III-TT, anti-B-TT given before or after bacterial challenge was only marginally effective in protecting newborn mice against lethal infection with type III GBS. The number of C3 molecules bound to type III GBS was augmented by anti-III-TT but not by high antibody concentrations of anti-B-TT. These results suggest that the difference in opsonic activity between anti-B-TT and anti-III-TT may be due to a difference in their ability to deposit C3. In addition, the maximum number of antibody molecules bound to the bacterial surface was greater for anti-III-TT than for anti-B-TT. That anti-B-TT binds to fewer sites than anti-III-TT may explain the differences in complement activation and in opsonic and protective efficacy of antibodies to group B polysaccharide compared with antibodies to the type-specific capsular polysaccharide.     

Detection of group B streptococcal antigen in body fluids by a latex-coupled monoclonal antibody assay.

A commercially available latex agglutination reagent, Directigen (Hynson, Westcott & Dunning, Div. Becton Dickinson & Co., Baltimore, Md.), in which a murine monoclonal antibody to group B streptococcal (GBS) antigen is the active component, was evaluated by using body fluid specimens from 94 sick infants. Antigen was detected in one or more admission specimens from 18 of 19 (94.7%) infants with symptomatic GBS infection. In 15 patients with GBS meningitis, cerebrospinal fluid, serum, and 10-fold-concentrated urine specimens were positive in 87, 50, and 100%, respectively. GBS antigen was detected in 50% of unconcentrated urines, but in no sera from infants with nonmeningitic GBS sepsis. Among specimens from 27 infants with invasive infection due to organisms other than GBS and from 48 culture-negative sick infants, false-positive latex agglutination reactions occurred in only 4 (9.5%) urine specimens and in no cerebrospinal fluid or serum specimens. The 94.7% sensitivity and specificity of the Directigen GBS test indicate that it is a useful reagent for the rapid diagnosis of invasive GBS infection in young infants.     

Reduced incidence of slowly progressive Heymann nephritis in rats immunized with a modified vaccination technique

A slowly progressive Heymann nephritis (SPHN) was induced in three groups of rats by weekly injections of a chemically modified renal tubular antigen in an aqueous medium. A control group of rats received the chemically unmodified version of the antigen in an aqueous solution. One group of SPHN rats were pre- and post-treated with weekly injections of IC made up of rKF3 and rarKF3 IgM antibody at antigen excess (MIC) (immune complexes [ICs] containing sonicated ultracentrifuged [u/c] rat kidney fraction 3 [rKF3] antigen and IgM antibodies specific against the antigen, at slight antigen excess). One group of SPHN rats were post-treated with MIC 3 weeks after the induction of the disease and one group of SPHN animals received no treatment. The control group of rats received pre- and post-treatment with sonicated u/c rKF3. The incidence of immune-complex glomerulonephritis (ICGN) in the untreated SPHN rats was 87%, in the pre- and post-treated animals 13%, and in the post-treated-only rats 20%. Rats receiving sonicated ultracentrifuged rKF3 antigen did not develop ICGN. The present experiment demonstrates that the development of SPHN can be not only prevented but also effectively terminated by our newly developed modified vaccination technique.     

The interaction of rheumatoid factor with hepatitis B surface antigen-antibody complexes.

A solid phase radioimmunoassay was developed in which the hepatitis B surface antigen was adsorbed to the surface of plastic beads. When the antigen-coated beads were incubated with human IgG antibody against hepatitis B surface antigen, immune complexes were formed on the solid phase surface. IgM rheumatoid factor was found to bind to the hepatitis B surface antigen-antibody complexes but not to the antigen or the IgG antibody alone. Since both hepatitis B surface antigen-antibody complexes and rheumatoid factor are commonly present in type B viral hepatitis, it is suggested that rheumatoid factor may play a role in the pathogenesis of this viral disease in man.