Streptococcus agalactiae (group B streptococcus; GBS) is a significant cause of perinatal and neonatal infections worldwide. To detect GBS colonization in pregnant women, the CDC recommends isolation of the bacterium from vaginal and anorectal swab samples by growth in a selective enrichment medium, such as Lim broth (Todd-Hewitt broth supplemented with selective antibiotics), followed by subculture on sheep blood agar. However, this procedure may require 48 h to complete. We compared different sampling and culture techniques for the detection of GBS. A total of 300 swabs was taken from 100 pregnant women at 35-37 weeks of gestation. For each subject, one rectovaginal, one vaginal and one rectal ESwab were collected. Plating onto Columbia CNA agar (CNA), group B streptococcus differential agar (GBSDA) (Granada Medium) and chromID Strepto B agar (CA), with and without Lim broth enrichment, were compared. The isolates were confirmed as S. agalactiae using the CAMP test on blood agar and by molecular identification with tDNA-PCR or by 16S rRNA gene sequence determination. The overall GBS colonization rate was 22%. GBS positivity for rectovaginal sampling (100%) was significantly higher than detection on the basis of vaginal sampling (50%), but not significantly higher than for rectal sampling (82%). Direct plating of the rectovaginal swab on CNA, GBSDA and CA resulted in detection of 59, 91 and 95% of the carriers, respectively, whereas subculturing of Lim broth yielded 77, 95 and 100% positivity, respectively. Lim broth enrichment enabled the detection of only one additional GBS positive subject. There was no significant difference between GBSDA and CA, whereas both were more sensitive than CNA. Direct culture onto GBSDA or CA (91 and 95%) detected more carriers than Lim broth enrichment and subculture onto CNA (77%). One false negative isolate was observed on GBSDA, and three false positives on CA. In conclusion, rectovaginal sampling increased the number GBS positive women detected, compared to vaginal and/or rectal sampling. Direct plating on CA and/or GBSDA provided rapid detection of GBS that was at least as sensitive and specific as the CDC recommended method of Lim broth subcultured onto non chromogenic agar.

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Comparison of different sampling techniques and of different culture methods for detection of group B streptococcus carriage in pregnant women.

Group B streptococcus (GBS) is a major cause of serious infections in neonates. The 2002 revised guidelines of the Centers for Disease Control and Prevention (CDC) for the prevention of perinatal GBS disease recommend that all pregnant women be screened for GBS carriage at between 35 and 37 weeks of gestation and that intrapartum antibiotic prophylaxis be given to carriers. We studied the performances of four different GBS detection assays in the context of antenatal screening. Between May and August 2004, the 605 vaginorectal swab specimens received at our bacteriology laboratory for GBS antenatal detection were tested by the four assays. The standard culture method was done according to the CDC recommendations. The three experimental assays performed with the growth from the selective enrichment (LIM) broth (Todd-Hewitt broth with 15 μg/ml nalidixic acid and 10 μg/ml colistin) after overnight incubation were a GBS antigen detection assay (PathoDx) and two PCR assays (for cfb and scpB). The most accurate assay was the scpB PCR (sensitivity, 99.6%; specificity, 100%), followed by the cfb PCR (sensitivity, 75.3%; specificity, 100%), GBS antigen detection (sensitivity, 57.3%; specificity, 99.5%), and standard culture (sensitivity, 42.3%; specificity, 100%). The GBS antigen detection assay was found to be more sensitive than the standard culture method, and moreover, the assay has a low cost and is easy to perform in all obstetrical centers which have access to the most basic of diagnostic microbiology services. We believe that antigen detection on incubated LIM broth should replace the standard culture method for screening for GBS carriage at 35 to 37 weeks of gestation. The impact of the greater sensitivities of PCR assays on the diminution of neonatal GBS infections remains to be demonstrated.

Sensitivities of Antigen Detection and PCR Assays Greatly Increased Compared to That of the Standard Culture Method for Screening for Group B Streptococcus Carriage in Pregnant Women

Direct inoculation onto Granada medium (GM) in plates and tubes was compared to inoculation into a selective Todd-Hewitt broth (with 8 μg of gentamicin per ml and 15 μg of nalidixic acid per ml) for detection of group B streptococci (GBS) in pregnant women with 800 vaginal and 450 vaginoanorectal samples. Comparatively, GM was found to be as sensitive as the selective broth for the detection of GBS in vaginal specimens and more sensitive than selective broth for the detection of GBS in vaginoanorectal samples (96 versus 82%). The use of GM improved the time to reporting of a GBS-positive result by at least 24 h and reduced the direct cost of screening. We have also found that the inconvenience of anaerobic incubation of GM plates can be avoided when a cover slide is placed upon the inoculum, because aerobic incubation in GM plates with cover slides causes GBS to develop the same pigmentation that it develops with incubation under anaerobic conditions. These data support the routine use of GM plates or tubes as a more accurate, easier, and cheaper method of identification of GBS-colonized women compared to the enrichment broth technique.

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Use of Granada Medium To Detect Group B Streptococcal Colonization in Pregnant Women

Objective: To investigate asymptomatic vagino-rectal carriage of group B streptococcus (GBS) in pregnant women. Methods: Women in the final trimester of pregnancy were recruited. A single vagino-rectal swab was taken, with consent, for culture of GBS. Two microbiological methods for isolation of GBS from vagino-ractal swabs were compared. The distribution of capsular serotypes of the GBS identified was determined. Epidemiological data for a subset (n = 167) of the pregnant women participating were examined. Results: 21.3% were colonised vagino-rectally with GBS. Risk factors for neonatal GBS disease (maternal fever, prolonged rupture of membranes, and preterm delivery) were present in 34 of 167 women (20.4%), and the presence of these factors correlated poorly with GBS carriage. Capsular serotypes III (26.4%), IA (25.8%), V (18.9%), and IB (15.7%) were prevalent in the GBS isolates. Selective broth culture of vagino-rectal swabs was superior to selective plate culture, but the combination of both methods was associated with increased detection of GBS (7.5%). An algorithm for the identification of GBS from vagino-rectal swabs was developed. Conclusions: GBS carriage is prevalent in pregnant women in Oxfordshire, UK. The poor correlation between risk factors and GBS carriage requires further investigation in larger groups, given that the identification of these surrogate markers is recommended to guide administration of intrapartum antibiotic prophylaxis by the Royal College of Obstetricians of the UK. A selective broth culture detected more GBS carriers than a selective plate culture.

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Carriage of group B streptococcus in pregnant women from Oxford, UK

Granada medium was evaluated for the detection of group B streptococci (GBS) in vaginal and rectal swabs compared with selective Columbia blood agar and selective Lim broth. From May 1996 to March 1998, 702 pregnant women (35 to 37 weeks of gestation) participated in this three-phase study; 103 (14.7%) of these women carried GBS. In the first phase of the experiment (n = 273 women), vaginorectal specimens were collected on the same swab; the sensitivities of Granada tube, selective Columbia blood agar, and Lim broth were 31.4, 94.3, and 74.3%, respectively. In the second and third phases (n = 429 women), vaginal and rectal specimens were collected separately; the sensitivities of Granada plate, selective Columbia blood agar, and Lim broth (subcultured at 4 h on selective Columbia agar in the second phase and at 18 to 24 h in Granada plate in the third phase) were 91.1, 83.9, and 75%, respectively, in the second phase and 88.5, 90.4, and 63.5%, respectively, in the third phase. There were no statistically significant differences in GBS recovery between the Granada agar plate and selective Columbia blood agar, but the Granada plate provided a clear advantage; the characteristic red-orange colonies produced overnight by GBS can be identified by the naked eye and is so specific that further identification is unnecessary. The use of the Granada tube and Lim broth did not result in increased isolation of GBS. In conclusion, the Granada agar plate is highly sensitive for detecting GBS in vaginal and rectal swabs from pregnant women and can provide results in 18 to 24 h.

Evaluation of the Granada Agar Plate for Detection of Vaginal and Rectal Group B Streptococci in Pregnant Women

In 1996, the Centers for Disease Control and Prevention recommended the use of a selective broth culture for the improved detection of genital tract or anorectal carriage of group B streptococci (GBS) in pregnant women. In order to verify this recommendation in our laboratory, we compared the sensitivity of Todd-Hewitt medium with gentamicin and nalidixic acid (SBM) with our current method of direct plating on blood agar medium containing neomycin and nalidixic acid (NNA). Five hundred consecutive cervicovaginal and anorectal specimens submitted for GBS culture were included in the study. Swabs were plated onto NNA and the swabs were immersed in SBM, followed by overnight incubation at 35°C. On the following day, the NNA plates were examined for colonies typical of GBS and the organisms were identified by the CAMP test or by latex agglutination. SBM cultures were subcultured onto blood agar and CNA agar plates, and the plates were reincubated for 24 h. Negative specimens from either medium were incubated for an additional 24 h and were examined again before finalization of the results. GBS were recovered from 78 specimens by both methods; from SBM only for 17 specimens (sensitivity, 86%) and from NNA only for 16 specimens (sensitivity, 85%). A moderate to heavy growth of Enterococcus faecalis was observed on plates containing NNA-positive, SBM-negative specimens. Competitive growth studies suggested that E. faecalis suppressed the growth potential of GBS in SBM. Our study suggests that direct plating on NNA, as a single method, is equivalent in sensitivity to SBM for the recovery of GBS, and the results are often available 24 h sooner. However, it appears that both direct plating and selective broth amplification techniques are required for the maximum level of identification of colonization with GBS in pregnant women.

W. Michael Dunne

Papers

Comparison of NNA Agar Culture and Selective Broth Culture for Detection of Group B Streptococcal Colonization in Women