Detection of Streptococcus agalactiae colonization in pregnant women by using combined swab cultures: cross-sectional prevalence study

ABSTRACT CONTEXT AND OBJECTIVE: Maternal Streptococcus agalactiae colonization and early-onset neonatal sepsis have aroused interest in the worldwide literature. Streptococcal neonatal disease is associated with significant morbidity and mortality in the perinatal period, especially among premature neonates. The aim of this study was to assess the prevalence of maternal streptococcal colonization by using combined swab cultures, compared with swab collection from a single site. DESIGN AND SETTING: Cross-sectional study at Faculdade de Medicina de Botucatu, Universidade Estadual Paulista. METHODS: Samples were obtained from 405 patients at gestational ages of 35 to 37 weeks. Swabs from the perianal (rectal) region, vaginal introitus and upper lateral vaginal vault were cultured in Todd-Hewitt selective broth. Colonies suggestive of Streptococcus agalactiae were subjected to the catalase and CAMP (Christie, Atkins, Munch-Petersen) tests. To evaluate the positivity of combined swab cultures, Tukey's test was used for comparison of proportions. RESULTS: The prevalence of streptococcal colonization was 25.4%. Among the patients with positive cultures, 28.1% had this at only one collection site, 24.2% simultaneously at two sites and 47.5% at all three sites. Associating the swabs from two collection sites significantly increased streptococcal isolation, compared with a single swab (P < 0.05), except for perianal (rectal) collection. Use of combined swabs from three collection sites showed statistically higher isolation rates. CONCLUSION: In combined swab cultures collected from three collection sites, the prevalence of maternal Streptococcus agalactiae colonization was higher than in swabs collected from a single site.

and upper lateral vaginal vault were cultured in Todd-Hewitt selective broth.Colonies suggestive of Streptococcus agalactiae were subjected to the catalase and CAMP (Christie, Atkins, Munch-Petersen) tests.To evaluate the positivity of combined swab cultures, Tukey's test was used for comparison of proportions.

RESULTS:
The prevalence of streptococcal colonization was 25.4%.Among the patients with positive cultures, 28.1% had this at only one collection site, 24.2% simultaneously at two sites and 47.5% at all three sites.Associating the swabs from two collection sites significantly increased streptococcal isolation, compared with a single swab (P < 0.05), except for perianal (rectal) collection.Use of combined swabs from three collection sites showed statistically higher isolation rates.

Patient isolation.
Culture media.

INTRODUCTION
During the last few decades, maternal Streptococcus agalactiae (S. agalactiae) colonization and early-onset neonatal sepsis have undergone intense worldwide study.Streptococcal neonatal disease is associated with significant morbidity and mortality during the perinatal period, 1 especially among premature neonates. 2ram-positive S. agalactiae can colonize the genitourinary and lower gastrointestinal tract of asymptomatic women.Colonization by this organism is associated with an increased risk of urinary tract infection 3 and pregnancy complications such as endometritis 3 and chorioamnionitis. 3,4oreover, neonates exposed to S. agalactiae can suffer severe consequences like pneumonia, sepsis and meningitis. 5ertical transmission of S. agalactiae occurs when the membranes rupture and the neonate comes into contact with a colonized maternal birth canal during labor and delivery. 6The Centers for Disease Control and Prevention (CDC) currently recommends that all pregnant women should be screened for S. agalactiae at a gestational age of between 35 and 37 weeks. 7For patients with positive vaginal or rectal cultures, intravenous penicillin prophylaxis is recommended during labor.Implementation of this strategy has reduced early-onset neonatal sepsis by 70%. 8ccording to the literature, between 6.5% 9 and 43.6% 10 of pregnant women are colonized with S. agalactiae in the vagina or rectum.Maternal colonization rates may differ according to the culture medium and collection site. 11Some studies have consistently shown that combined swabs from different collection sites give up to 30% better yield than single vaginal swabs do. 12

OBJECTIVE
The purpose of this study was to evaluate the prevalence of maternal S. agalactiae colonization in combined swab cultures, compared with swabs collected from a single site.

MATERIALS AND METHODS
This cross-sectional study was conducted at the Faculdade de Medicina de Botucatu (FMB), Universidade Estadual Paulista (Unesp) between February 2006 and January 2007.This university's hospital is located in a central area of the state of São Paulo and caters to patients of differing socioeconomic statuses.The samples were obtained from 405 women with a gestational age of between 35 and 37 weeks and singleton pregnancy who attended the FMB prenatal service.
The exclusion criteria were refusal to participate, symptoms of urinary infection over the preceding 60 days, antibiotic use over the preceding 60 days and sexual intercourse over the 72 hours preceding the examination.After the study had been approved by the ethics committee and written informed consent had been obtained from all patients, vaginal and rectal samples were collected to assess the patients' S. agalactiae status.
Samples were collected using separate sterile swabs, from the vaginal introitus (VI), upper lateral vaginal vault (LV), and rectal region (RR).The samples were immediately inoculated into nonnutritive Amies transport medium for transportation to the microbiology laboratory.The swabs were then removed from the Amies medium and inoculated in Todd-Hewitt broth supplemented with colistin (10 µg/ml) and nalidixic acid (15 µg/ml).The inoculated samples were incubated for 24 hours at 37 °C.The broth was then subcultured onto a Columbia agar base with 5% sheep blood, under the same conditions.Colonies suggestive of S. agalactiae (because they presented a narrow zone of beta-hemolysis) were subjected to the catalase and CAMP tests.Negative blood-agar plates were reincubated for an additional 18 to 24 hours and reexamined.
Statistical analyses were performed using the SigmaStat software, version 3.1.Comparisons were performed using the Tukey test with significance set at P < 0.05.

RESULTS
During the study period, 567 patients with a gestational age of 35 to 37 weeks were seen at the FMB prenatal service.A total of 162 patients were excluded: 106 because they had been taking antibiotics and 56 because they refused to participate in the study.Considering all the 405 women included in the study, the results from the cultures showed that the rate of maternal colonization with S. agalactiae was 25.4%.
With regard to the collection site, the prevalence of S. agalactiae detection was evaluated by means of single and combined swabs from VI, LV and RR.A total of 103 culture-positive samples were included in this analysis.Out of all of the women with S. agalactiae colonization, 14.5% presented positive cultures both from VI and from LV; 5.8% from VI and RR, and 3.9% from LV and RR.Positive cultures from all three collection sites were found in 47.5%, and from only one site in 28.1% (9.7% from VI; 3.9% from LV and 14.5% from RR).
Statistical analysis on the effectiveness of associating swabs from different sites revealed that using a combination of two swabs increased the yield of S. agalactiae in relation to using a single swab, except when the swab was obtained from the rectal region.As shown in Table 1, association of swabs from the three collection sites significantly increased the detection of maternal S. agalactiae colonization.

DISCUSSION
4][15] Given that the culture sensitivity may vary according to the anatomical collection site and microbiological methods, the procedures used in this study to detect S. agalactiae were in accordance with the CDC recommendations. 12ith regard to evaluating combined swabs from vaginal sites, vaginal samples (from the introitus and lateral vault) were able to isolate more S. agalactiae than were rectal samples.Th ese results are in agree-These results are in agreement with findings from other studies, which also found higher detection rates in vaginal swabs than in rectal swabs. 16,17onsidering the effect of each anatomical site on S. agalactiae detection, the rectal region showed higher isolation frequency.According to Platt et al., 18 there is a significant increase in the streptococcal isolation rate when the rectal region is also assessed than when just using vaginal cultures.One study conducted in northeastern Brazil 19 showed that 22% of the pregnant women colonized by S. agalactiae would have had negative culture results if the rectal region had not been evaluated.Because the gastrointestinal tract is a natural reservoir for S. agalactiae and it is the likely source of vaginal colonization, culturing of rectal swabs is of great importance in assessing the streptococcal status.
Our findings agree with the literature [19][20][21] that swabbing from two or three anatomical sites significantly increases the detection rate in relation to swabbing from one site.Furthermore, the use of three collection sites (vaginal introitus, lateral vaginal vault and rectal region) gave statistically superior S. agalactiae detection rates.

CONCLUSION
In combined swab cultures collected from three different sites, the prevalence of maternal Streptococcus agalactiae colonization was higher than the prevalence observed in swabs collected from a single site.

Table 1 .
Number of Streptococcus agalactiae isolates according to collection site