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ORIGINAL RESEARCH

Group B Streptococcal Colonization and Serotype-Specific Immunity in Pregnant Women at Delivery

CAMPBELL, JUDITH R. MD; HILLIER, SHARON L. PhD; KROHN, MARIJANE A. PhD; FERRIERI, PATRICIA MD; ZALEZNIK, DORI F. MD; BAKER, CAROL J. MD

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Group B streptococci are a leading cause of neonatal sepsis and meningitis. Women colonized with group B streptococcus might also develop pregnancy-associated infections including urinary tract infection, bacteremia, chorioamnionitis, and postpartum endometritis.1–3 Group B streptococci are encapsulated organisms, and protective immunity is related to antibodies directed at these serotype-specific capsular polysaccharide structures.4 The role of maternal antibodies in neonatal immunity to early- and late-onset infant type III, group B streptococcal disease was first described in 1976,5 and others have reported similar correlations for serotypes Ia, Ib, and II.4 Candidate group B streptococcal capsular polysaccharide–protein conjugate vaccines for types Ia, Ib, and III have been developed and have been tested for safety and immunogenicity in healthy adults.6,7 Conjugate vaccines for serotype II and the newly emerged type V also have been developed8,9 and recently have undergone phase 1 clinical trials.

Since neonatal group B streptococcal disease first was described in the 1970s, colonization rates among pregnant women have remained constant, ranging from 15% to 40%.4 In previous studies, group B streptococcal colonization was associated with a greater likelihood of higher levels of antibody to the colonizing serotype.10–12 However, most women had very low, presumably nonprotective concentrations of antibodies. These studies were conducted nearly two decades ago, and shifts in serotype prevalence or immunity might have occurred since then. The studies also used assays that measured all immunoglobulin (Ig) classes, including those not expected to cross the placenta (eg, IgM and IgA), and they did not consider serotype V and group B streptococcus, which has emerged as a predominant serotype since 1990.3,13 Thus, contemporary data pertaining to the prevalence of group B streptococcal colonization and serum capsular polysaccharide–specific IgG is important as a benchmark for further group B streptococcal vaccine development.

The purpose of this cross-sectional study was to describe the prevalence of group B streptococcal colonization and serotype-specific immunity in ethnically and geographically diverse populations of pregnant women. We describe the relationship between colonization status, race or ethnicity, age, and serum concentrations of capsular polysaccharide–specific IgG specific for type Ia, Ib, II, III, or V group B streptococci in women at the time of hospital admission for delivery.

Materials and Methods

Women were recruited between October 1992 and January 1995 from three hospitals in Houston (St. Luke's Episcopal, the Methodist Hospital, and Ben Taub General Hospital) and from one hospital in Seattle (University of Washington Medical Center). Before enrollment, the participating centers agreed on a uniform protocol that was approved by the institutional review board for human research at each study site.

Women were enrolled prospectively when they were admitted for active labor or rupture of membranes. In Houston, recruitment occurred when study personnel were available (Monday through Wednesday, 8 AM until 3 PM); 4.5% of women admitted in labor were enrolled. In Seattle, recruitment was possible 24 hours a day, 7 days a week; 78% of women admitted in labor were enrolled. Recruitment continued in each city until there were at least 25 women with group B streptococcal colonization in the target ethnic groups. In Houston, target groups included Hispanic, black, and white women. In Seattle, two additional groups were targeted: 1) Asians and 2) Native Americans and Alaska Natives. Demographic and obstetric information obtained from study participants included maternal age, self-described race or ethnicity, delivery hospital, payer status, parity, gravidity, and gestational age. Data were recorded on standardized forms.

Cultures and blood specimens were obtained during the vaginal examination and before antibiotic administration. Lower vaginal and rectal sites were sampled with rayon (in Houston) or Dacron (in Seattle) swabs. These swabs were placed immediately into Amies transport media without charcoal (Difco, West Molesey, Surrey, UK) and transported within 24 hours to the investigator's laboratory at each study site. Swabs then were placed into selective broth medium (Baltimore Biologics Laboratories, Cockeysville, MD), incubated overnight at 35C, inoculated onto 5% sheep blood agar (Remel BAP, Lenexa, KS [Houston]; Prepared Media Laboratories, Tualatin, OR [Seattle]), and streaked for isolation. After overnight incubation at 35C, the blood agar plates were inspected for β-hemolytic colonies, which were confirmed as being group B streptococcus by latex agglutination (Streptex; Murex, Temple Hill, Dartford, UK [Houston]; Pathodx; Diagnostics Products Corp., Los Angeles, CA [Seattle]). Group B streptococcal isolates were serotyped by previously described methods14 using rabbit antisera specific for types Ia, Ib, and II through VIII and by immunodiffusion in agarose. Whole blood was allowed to clot at room temperature. Sera then were separated by centrifugation and stored at −80C until testing.

In Houston, sera from all women with group B streptococcal colonization and available admission samples were tested; in Seattle, the percentage of women colonized with group B streptococci whose sera were tested varied by race or ethnicity: 10% of white women, 22% of blacks, 65% of Hispanics, 51% of Asians, and 65% of Native Americans or Alaska Natives. Samples (n = 294) were tested by group B streptococcal capsular polysaccharide–specific IgG enzyme-linked immunosorbent assay (ELISA) for their colonizing serotype. In this subset, the number of colonized subjects per group B streptococcal serotype varied from 25 (Ib) to 78 (Ia). A subset of noncolonized women (n = 193) whose demographics were similar to those of the study population also had sera tested by ELISA for concentrations of Ia, Ib, II, III, and V capsular polysaccharide–specific IgG. These noncolonized women were selected on the basis of serum availability and then were group-matched so that the racial distribution paralleled that of the women enrolled from Houston and Seattle. Parity and gravidity of the noncolonized subset were similar to those of the colonized subset; thus, these women served as the control group. Immunoglobulin G ELISAs were performed using group B streptococcal capsular polysaccharides conjugated to human serum albumin (provided by Lawrence C. Paoletti, PhD, Channing Laboratory, Boston, MA). These methods were detailed previously.15 The results were expressed in micrograms per milliliter and represented the mean of duplicate determinations for each serum. Internal standard sera were included in each assay, and reproducibility was within 5%.

Odds ratios (ORs) and 95% confidence intervals (CIs) were used to compare Asian, Hispanic, and black women with white women in terms of frequency of group B streptococcal colonization. The frequency of group B streptococcal serotypes by age, ethnic or racial, and study site groups were compared using χ2 statistics. Concentrations of serotype-specific IgG of colonized women and those of noncolonized women were compared using the Mann-Whitney U test. In the group of colonized women, concentrations were compared across age and racial or ethnic groups, using Kruskal-Wallis statistics. Statistical tests were two-sided, and P < .05 was considered significant.

Results

During the 28-month study, 3307 pregnant women were enrolled: 574 in Houston and 2733 in Seattle. Of these participants, 856 (26%) were colonized with group B streptococci at lower vaginal and/or rectal sites. Of the colonized women, most (69%) had group B streptococci isolated from both sites; 185 (22%) had rectal colonization only and 10% had vaginal colonization only. Colonization rates for women enrolled in Houston and Seattle were 28% and 26%, respectively. The demographics of the study population are summarized in Table 1.

Table 1
Table 1:
Demographics of the Study Population

The study population was racially and ethnically diverse; 45% of women were white, 19% were black, 18% were Hispanic, 12% were Asian, 3% were Native American or Alaska Natives, and 3% belonged to other racial or ethnic groups (or race or ethnicity was not ascertained). Black women (37%) were more likely (OR 1.7; 95% CI 1.4, 2.1) and Asian women (15%) were less likely (OR 0.5; 95% CI 0.4, 0.7) to be colonized with group B streptococci than were white women (25%). Colonization rates among Hispanic (24%) and Native American or Alaska Native women (26%) were similar to those among white women. The majority (79%) of study participants were receiving Medicaid or public assistance, as were the majority of participants in each racial or ethnic group. Group B streptococcal colonization rates among women without a third-party payer were similar to those among women with private insurance. Women less than 20 years of age constituted 17% of study participants. The group B streptococcal colonization rate among these young women was 26%, a rate identical to that among older women.

Among group B streptococcal isolates from the 856 colonized women, the most frequent serotypes were Ia (26.3%) and III (21.4%). Serotype V was third in frequency, occurring in 20.9% of colonized women. Serotypes Ib (7.5%), II (18.1%), and VI (0.2%) and nontypeable strains (1.3%) occurred in the remainder of women. The distribution of serotypes among study participants did not differ by race or ethnicity or study site. However, women less than 20 years of age were more likely (33.8%) to be colonized with a type Ia strain than were older women (24.7%) (P = .03, Fisher exact test).

A subset of 294 women colonized with group B streptococci who had admission sera available were selected for serologic testing. Noncolonized controls (n = 193) were selected to assess the effect of colonization on capsular polysaccharide–specific IgG concentrations. These two groups of women were racially and ethnically diverse, as was the total study population. The colonized subset (n = 294) was 19% white, 27% black, 39% Hispanic, 10% Asian, and 5% Native American or Alaska Native. The noncolonized subset (n = 193) was 33% white, 23% black, 32% Hispanic, 9% Asian, and 1% Native American or Alaska Native (in 4%, race was not ascertained). In the colonized cohort, 78 women (26%) were colonized with serotype Ia, 25 (9%) with serotype Ib, 67 (23%) with serotype II, 71 (24%) with serotype III, and 54 (18%) with serotype V. One woman was colonized with both serotype Ib and II and underwent serologic testing for both serotypes. The serotype distribution in the subset group approximated that in the group B streptococcus–colonized women in the total study population. Median serum concentrations of capsular polysaccharide–specific IgG for each serotype in colonized and noncolonized women are summarized in Figure 1. The noncolonized pregnant women (n = 193) had capsular polysaccharide–specific IgG levels measured for each of the five common serotypes. Women colonized with serotypes Ia, II, III, or V had significantly higher delivery serum concentrations of IgG specific for the capsular polysaccharide of their colonizing group B streptococcal strain than did noncolonized women (P < .001, Mann-Whitney U test). However, 140 (48%) of these colonized women had low capsular polysaccharide–specific IgG concentrations (less than 0.5 μg/mL) in their delivery sera. For each serotype tested, one-third or more of colonized women had low levels (less than 0.5 μg/mL) of capsular polysaccharide–specific IgG specific for the group B streptococcal strain that they carried (Figure 2). The proportion of colonized women with capsular polysaccharide–specific IgG levels less than 0.5 μg/mL varied from 35% for type Ia–to 61% for type V–colonized women.

Figure 1
Figure 1:
Median serum concentrations of capsular polysaccharide–specific immunoglobulin G (IgG) measured by enzyme-linked immunosorbent assay in a cohort of colonized (black bars) and noncolonized (white bars) pregnant women presenting for delivery. *P < .001, Mann-Whitney U test.
Figure 2
Figure 2:
Distribution of capsular polysaccharide–specific immunoglobulin G concentrations in delivery sera from women colonized with group B streptococci, indicated bywhite (less than 0.5 μg/mL), dotted (0.5–1.0 μg/mL), and black (more than 1.0 μg/mL) bars.

The distribution of concentrations of capsular polysaccharide–specific IgG categorized by increments in delivery sera from noncolonized women is depicted in Figure 3. Among the 193 noncolonized controls, more than half had low concentrations of capsular polysaccharide–specific IgG specific for each of the five serotypes tested. With regard to the three most frequent serotypes, 64, 76, and 89% of women, respectively, had less than 0.5 μg/mL of Ia, III, and V capsular polysaccharide–specific IgG in their sera. The proportion of noncolonized women with these low values did not vary significantly by serotype.

Figure 3
Figure 3:
Distribution of capsular polysaccharide–specific immunoglobulin G concentrations in delivery sera from noncolonized women, indicated bywhite (less than 0.5 μg/mL), dotted (0.5–1.0 μg/mL), and black (more than 1.0 μg/mL) bars.

Group B streptococcal capsular polysaccharide–specific IgG concentrations categorized as low (less than 0.5 μg/mL), moderate (0.5–1.0 μg/mL), or higher (more than 1.0 μg/mL) also were compared across age groups (Figure 4, Table 2). In sera from colonized women less than 20 years of age, the median serum concentration of Ia, Ib, II, III and V capsular polysaccharide–specific IgG was 0.185 μg/mL, a value that was significantly lower than the median among women 20–29 years old and those 30 years of age or older (P < .001, Kruskal-Wallis test). There was also an increase in median serum concentration of capsular polysaccharide–specific IgG by age; women 30 years of age or older were more likely to have concentrations in excess of 1 μg/mL (P = .001, Kruskal-Wallis test) than were younger women. Of the 52 colonized women less than 20 years of age, 35 (67%) had capsular polysaccharide–specific IgG levels less than 0.5 μg/mL, whereas only 35% of women 30 years of age or older had these low serum concentrations. In contrast, race or ethnicity influenced neither median serum values nor the proportion of women with low or higher concentrations (Table 2).

Figure 4
Figure 4:
Distribution of group B streptococcal capsular polysaccharide–specific immunoglobulin G (Ig) G concentrations in delivery sera of 294 colonized women categorized by age. Distribution is illustrated as the percentage of each age group with less than 0.5 (white bars), 0.5–1.0 (dotted bars), or more than 1.0 (black bars) μg/mL of specific IgG.
Table 2
Table 2:
Capsular Polysaccharide–Specific Immunoglobulin G Concentrations in Delivery Sera From 294 Pregnant Women Colonized With Group B Streptococcus

Discussion

This study identified several features of group B streptococcal colonization and serotype-specific immunity in pregnant women. Colonization was detected in approximately one of four pregnant women presenting for delivery, a frequency almost identical to that reported more than 20 years ago.4,10,11,16 However, the distribution of serotypes among colonizing strains differed somewhat from that described in the 1970s and 1980s, in that type Ia was more frequent, type II was less frequent, and type V was newly recognized.13 Although colonization was associated with higher serum concentrations of IgG specific for the capsular polysaccharide of the colonizing serotype, group B streptococcal capsular polysaccharide–specific IgG concentrations were low in most pregnant women (ie, nonimmune status). Black women were significantly more likely than white women to be colonized. However, colonized black women had serum capsular polysaccharide–specific IgG concentrations similar to those of women of other racial or ethnic groups. Although age did not influence overall colonization rates, women less than 20 years of age were colonized significantly more often with type Ia group B streptococcus and were more likely to have low serum concentrations (less than 0.5 μg/mL) of IgG specific to any of the five capsular polysaccharides than were older women.

Compared with previous studies, in our study serotype Ia was the most frequent colonizing group B streptococcal serotype in pregnant women, and this type was especially prevalent among women less than 20 years of age. Serotype III remained common (21%), and, as recently reported by Harrison et al,17 this serotype still accounts for a substantial proportion of maternal and the majority of cases of early- and late-onset infant group B streptococcal disease. Our study confirms the emergence of serotype V as a frequent colonizing strain18; it was the third most frequent serotype in pregnant women at delivery. This finding is consistent with recent reports describing serotype distribution among group B streptococcal isolates from pregnant women and neonates with group B streptococcal disease.3

The presence of sufficient concentrations of maternal serum antibodies to the capsular polysaccharide of type III, group B streptococcus has been correlated with reduced risk of infant disease caused by this serotype.5 This finding provides one explanation for the discrepancy between the rate of vertical transmission from colonized women to their neonates (50%) and the incidence of invasive early-onset infection among these neonates (1%).4 As described previously, colonization with type III is associated with higher concentrations of III capsular polysaccharide–specific antibodies in maternal sera. A similar correlation for serotypes Ia, II, and V, but not Ib, was demonstrated in the present study. This observation suggests that vaginal or rectal colonization with group B streptococci induces a systemic immune response in some women. However, the mechanism by which this occurs has not been defined. The interval from colonization to immune response cannot be inferred from this cross-sectional study. Although colonized women had higher serum concentrations of capsular polysaccharide–specific IgG compared with noncolonized women, half of this obstetric population had very low concentrations (less than 0.5 μg/mL) in their sera. Although the protective level for each serotype is not known, these low concentrations of capsular polysaccharide–specific IgG would not be expected to protect either the mother from chorioamnionitis or bacteremia, for example, or the exposed infant from early- or late-onset invasive group B streptococcal disease.5

Multicenter population-based studies suggest that maternal black race and young age enhance the risk of invasive group B streptococcal disease in neonates.19,20 Our data confirm this hypothesis and possibly elucidate factors that might contribute to this risk. Black women were more likely to be colonized than were women in other racial/ethnic groups, and yet they had low serum capsular polysaccharide–specific IgG concentrations at a frequency similar to that seen in other racial or ethnic groups with lower rates of group B streptococcal colonization. We speculate that this might explain in part the increased risk of disease in neonates born to black women.19,20 By contrast, the rate of colonization among women less than 20 years of age did not differ from that among older women, but these young women were more likely to have the lowest levels of capsular polysaccharide–specific IgG. The low seroprevalence of group B streptococcal immunity in this population of young women might contribute to the previously reported increased risk of disease caused by this organism in their infants.19,20

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© 2000 The American College of Obstetricians and Gynecologists