Barry M. Gray

ELISA methodology for polysaccharide antigens: protein coupling of polysaccharides for adsorption to plastic tubes.

A method is described which permits the adaption of ELISA techniques for measurement of antibody against bacterial polysaccharides. First, the polysaccharides antigen is covalently bound to poly-L-lysine, using cyanuric chloride as the coupling agent. The poly-L-lysine then adsorbs to the walls of plastic tubes, thus immobilizing the polysaccharide coupled to the poly-L-lysine. The method is simple, rapid, and utilizes small amounts of polysaccharide antigen.

Prospective studies of group B streptococcal infections in infants.

The incidence of GBS colonization at birth was determined prospectively among 96% of 2,407 infants born over a 12-month period in an urban community hospital. GBS were recovered from one or more of the four sites cultured in 290 (12.5%) of these newborn infants, 91 of whom were heavily colonized (3 to 4 sites positive). Sepsis or meningitis occurred in 13 infants, an attack rate of 5.4/1,000 live births. Attack rates for early and late onset disease, respectively, were 3.7 and 1.7 per 1,000 live births. All serotypes were found to cause disease. The incidence of early sepsis was strikingly high (8%) in heavily colonized infants. Those colonized at 1 to 2 sites were at no greater risk than noncolonized infants. The maternal vaginal colonization rate at delivery was 19%, with acquisition from the mother documented as the primary source of the organism in early onset infections. Perinatal events, including maternal complications and signs of illness at or immediately after birth, suggested ascending infection with exposure in utero to be likely in six infants. Passive acquisition of GBS (intrapartum exposure) probably occurred in the three remaining early onset cases. This mechanism was also likely responsible for five nonbacteremic infections. The four infants with late onset sepsis or meningitis were not colonized at birth or when discharged from the nursery (day 3); a possible maternal source for infection was found in only one of these infants.

Clinical and epidemiologic studies of pneumococcal infection in children

Streptococcus pneumoniae was isolated from 1310 children in a 5-year period from 1979 through 1984. There were 44 cases of meningitis, 172 bacteremic infections, 787 cases of otitis media and 307 respiratory and miscellaneous isolates. The majority of infections could be accounted for by a small number of serotypes, with types 3, 6, 14, 19 and 23 predominating. Most infections (70%) occurred in infants younger than 2 years of age. However, nearly one-fourth of those suffering systemic illness had some underlying condition which may have contributed to their risk for infection, even beyond 2 years of age. Ten of the 12 deaths occurred in patients with altered host defenses. Characteristics of pneumococcal disease and the distribution of serotypes are discussed in relation to the work of other investigators over the past 50 years.

Immunity to Cross-Reactive Serotypes Induced by Pneumococcal Conjugate Vaccines in Infants

Infants were immunized with 1 of the 3 experimental pneumococcal conjugate vaccines that contain 6B and 19F but not 6A or 19A serotypes. Their sera were studied for the capacity to opsonize Streptococcus pneumoniae 6A, 6B, 19A, and 19F serotypes and the level of IgG antibody to the 4 serotypes. Significant increases were observed in the number of infants with detectable opsonophagocytic titers with 3 conjugate vaccines for 6B (vaccine) serotype but with only 2 vaccines for 6A (cross-reactive) serotype. Significant increases were observed with 2 conjugate vaccines for 19F serotype but with only 1 vaccine for 19A serotype. Thus, some conjugate vaccines may elicit cross-protection better than others. In addition, correlations between opsonophagocytic titers and IgG antibody levels by ELISA were high for 6B and 19F serotypes but low for 6A and 19A serotypes. Thus, ELISA may be an inadequate surrogate assay of vaccine response for cross-reactive serotypes.

Level of Maternal IgG Anti-Group B Streptococcus Type III Antibody Correlated with Protection of Neonates against Early-Onset Disease Caused by This Pathogen

The present study estimates the level of maternal immunoglobulin (Ig) G anti-group B streptococcus (GBS) type III required to protect neonates against early-onset disease (EOD) caused by this pathogen. Levels of maternal serum IgG anti-GBS type III, measured by enzyme-linked immunosorbent assay, in 26 case patients (neonates with EOD caused by GBS type III) and 143 matched control subjects (neonates colonized by GBS type III who did not develop EOD) of > or = 34 weeks gestation were compared. The probability of EOD decreased with increasing levels of maternal IgG anti-GBS type III (P = .01). Neonates whose mothers had > or = 10 microg/mL IgG anti-GBS type III had a 91% lower risk for EOD, compared with those whose mothers had levels of < 2 microg/mL. A vaccine that induces IgG anti-GBS type III levels of > or = 10 microg/mL in mothers can be predicted to offer a significant degree of protection against EOD caused by this pathogen.

Level of Maternal Antibody Required to Protect Neonates against Early-Onset Disease Caused by Group B Streptococcus Type Ia: A Multicenter, Seroepidemiology Study

Because of the difficulty of conducting efficacy trials of vaccines against group B streptococcus (GBS), the licensure of these vaccines may have to rely on studies that measure vaccine-induced antibody levels that correlate with protection. This study estimates the level of maternal antibody required to protect neonates against early-onset disease (EOD) caused by GBS type Ia. Levels of maternal serum IgG GBS Ia antibodies, measured by ELISAs in 45 case patients (neonates with EOD caused by GBS Ia) and in 319 control subjects (neonates colonized by GBS Ia but without EOD) born at > or =34 weeks gestation were compared. The probability of developing EOD declined with increasing maternal levels of IgG GBS Ia antibody (P = .03). Neonates whose mothers had levels of IgG GBS Ia antibody > or =5 microg/mL had an 88% lower risk (95% confidence interval, 7%-98%) of developing type-specific EOD, compared with those whose mothers had levels < 0.5 microg/mL. A vaccine that induces IgG GBS Ia antibody levels > or =5 microg/mL in mothers can be predicted to confer a high degree of type-specific immunity to EOD to their infants.

Puerperal and perinatal infections with group B streptococci

Twenty-one patients were seen with puerperal sepsis owing to group B streptococci (GBS), resulting in an attack rate of 2/1,000 deliveries. Most were young primiparous black women from a population with a known high incidence of GBS carriage. The association among abdominal delivery, endometritis, and puerperal sepsis was striking. Cultures of the birth canal or lochia were commonly positive for the same serotype recovered from the blood. Forty-seven patients with nonbacteremic GBS infections were seen; 27 had endometritis or amnionitis. Twenty patients had GBS urinary tract infection: Eight infections occurred prenatally, seven at delivery, and five post partum. Seven neonates developed serious GBS infections; intrauterine exposure occurred in at least four cases. Fetal exposure to GBS also occurred in three of four cases in which parturients with GBS bacteremia were delivered of their infants by cesarean section. Because of the high incidence of puerperal and perinatal GBS infections in this population, antibiotic prophylaxis regimens may be beneficial.

Variation in the molecular weight of PspA (pneumococcal surface protein A) among Streptococcus pneumoniae.

Pneumococcal surface protein A (PspA) has been shown to be a virulence factor of pneumococci and to elicit protective anti-pneumococcal antibodies in mice. PspAs from different pneumococcal isolates have been shown to exhibit antigenic variability. In previous studies with three strains, two different apparent molecular weights of PspA were observed. In this report we have studied the variation in molecular weight of PspA from 43 pneumococcal strains reactive with anti-PspA monoclonal antibodies, Xi64 and/or Xi126. The relative molecular mass (Mr) of the major PspA band ranged from 67 k to 99 k in the different strains. Variations in Mr of PspA were observed even within strains of the same capsular type. The molecular size of PspA from strain Rx1 was not affected by treatment with a variety of chemical, enzymatic, and physical procedures, suggesting that the differences in Mr of PspA among different strains, was not due to uncontrolled variations in PspA preparation. The Mr of PspA of a given strain was found to be stable both in vivo and in vitro. As a result variations in the Mr of PspA from clinical isolates, should allow discrimination between strains within a given capsular type in epidemiologic studies.

Development of a reverse transcription-polymerase chain reaction assay for diagnosis of lymphocytic choriomeningitis virus infection and its use in a prospective surveillance study

Lymphocytic choriomeningitis virus (LCMV), which is one of several arenaviruses that are pathogenic for humans, causes encephalitis and meningitis in man. In this study, single‐stage and nested reverse transcription‐polymerase chain reaction (RT‐PCR) assays were developed that targeted the GPC and N genes of LCMV. Both assays detected <1 TCID50 unit of LCMV. These assays were used to measure the incidence of LCMV infection by testing cerebrospinal fluid (CSF) samples with ⩾10 leukocytes/μI collected over 1 year from patients undergoing lumbar puncture for diagnostic reasons at two Birmingham hospitals. Samples were tested for the presence of LCMV RNA by using the RT‐PCR assay and for LCMV‐specific IgM antibody by using an ELISA assay. None of the specimens collected from 813 patients was positive by either assay. Although no cases of acute infection were detected, 4% (11/272) of serum collected from a subset of patients was positive for LCMV‐specific IgG. A significantly greater rate of seropositivity was found among subjects over 60 years of age (9.4%; P < 0.025) than was found in younger subjects (2.4% at 30–59 years of age; 0% at <30 years of age). These data suggest that serious central nervous system disease due to LCMV infection is not common in this population. The high rate of seropositivity in those over 60 years of age suggest that infection was once more common. J Med Virol 51:107–114, 1997.

Quantitative levels of C-reactive protein in cerebrospinal fluid in patients with bacterial meningitis and other conditions.

We measured levels of C-reactive protein (CRP) in the cerebrospinal fluid in 145 children, using a solid-phase radioimmunoassay. The CRP levels in 49 patients with culture-proved bacterial meningitis ranged from 0 to 51,000 ng/ml (median 1460 ng/ml). In 33 patients with aseptic meningitis, values were much lower range 0 to 438 ng/ml; (median 17 ng/ml). In patients with CSF pleocytosis (greater than 10 WBC/microliter), CRP greater than 100 ng/ml was 95% accurate in identifying those with bacterial meningitis. However, a few patients with bacterial meningitis and little or no CSF pleocytosis had low levels of CRP at admission. Among the 63 patients with nonmeningitic conditions, those with bacterial infections frequently (10 of 13 had CRP greater than 100 ng/ml, whereas CRP elevations were infrequent (seven (18%) of 40) in patients with viral infections and other conditions. CRP diffuses into the CSF as readily as other proteins, but in bacterial meningitis the CSF/serum ratio of CRP was lower than that of albumin and IgG. The measurement of CRP in CSF is potentially a very useful diagnostic tool, but certain inherent limitations must be recognized, because some patients may fail to mount a prompt inflammatory response.