Granada Medium Research Articles

Vaginal Carriage of Group B Streptococcus (GBS) in Pregnant Women, Antibiotic Sensitivity and Associated Risk Factors in Dakar, Senegal.

The eradication of neonatal Group B Streptococcus (GBS) infections, considered as a major public health priority, necessarily requires a mastery of the data on vaginal carriage in pregnant women. The aims of this study were to determine the prevalence of vaginal carriage of GBS in pregnant women, antibiotic susceptibility, and associated risk factors. This was a cross-sectional, descriptive study conducted over a period of 9 months (July 2020 to March 2021) in pregnant women between 34 and 38 weeks of gestation (WG) followed at the Nabil Choucair health center in Dakar. Identification and antibiotic susceptibility of GBS isolates were performed on the Vitek 2 from vaginal swabs cultured on Granada medium. Demographic and obstetric interview data were collected and analyzed on SPSS (version 25). The level of significance for all statistical tests was set at P < .05. The search of GBS vaginal carriage had involved 279 women aged 16 to 46 years, with a median pregnancy age of 34 (34-37) weeks' gestation. GBS was found in 43 women, for a vaginal carriage rate of 15.4%. In 27.9% (12/43) of volunteers screened, this carriage was monomicrobial, while in 72.1% (31/43) of women, GBS was associated with other pathogens such as Candida spp. (60.5%), Trichomonas vaginalis (2.3%), Gardnerella vaginalis (34.9%) and/or Mobiluncus spp. (11.6%). The level of resistance was 27.9% (12/43) for penicillin G, 53.5% (23/43) for erythromycin, 25.6% (11/43) for clindamycin and 100% for tetracycline. However, the strains had retained fully susceptible to vancomycin and teicoplanin. The main risk factor associated with maternal GBS carriage were ectocervical inflammation associated with contact bleeding (OR = 3.55; P = .005). The high rate of maternal vaginal GBS carriage and the levels of resistance to the various antibiotics tested confirm the importance of continuous GBS surveillance in our resource-limited countries.

Retrospective Study for the Clinical Evaluation of a Real-Time PCR Assay with Lyophilized and Ready-to-Use Reagents for Streptococcus agalactiae Detection in Prenatal Screening Specimens.

Streptococcus agalactiae is a leading cause of sepsis and meningitis in newborns and young infants. Screening programs and intrapartum antibiotic prophylaxis have reduced early neonatal onset of disease. The aim of this study was to evaluate a molecular assay with lyophilized and ready-to-use reagents: VIASURE® Streptococcus B Real Time PCR detection kit (CerTest Biotec) (Viasure qPCR assay) compared to both the GBS culture and a molecular assay with separated and frozen reagents: Strep B Real-TM Quant (Sacace Biotecnologies®) (Sacace qPCR assay). A total of 413 vaginal–rectal swabs from women between the 35th and 37th weeks of pregnancy were processed. GBS culture was firstly achieved through Granada medium and Columbia CNA agar at 35 °C in aerobic conditions. Then, nucleic acid extraction was performed for subsequent molecular analysis using both commercial assays. Discordant results were resolved via bidirectional Sanger sequencing. Viasure qPCR assay clinical sensitivity was 0.97 (0.92–0.99) and specificity 1 (0.98–1). This retrospective study demonstrated the good clinical parameters and the strong overall agreement (99.3%) between the Viasure qPCR assay and both reference assays. Finally, the added value observed of the assay under study was the stabilized and ready-to-use format, reducing the number of time-consuming steps, permitting the storage at room temperature, facilitating transport, being environmentally respectful, and reducing additional costs.

Evaluation of the New Brilliance GBS Chromogenic Medium for Screening of Streptococcus agalactiae Vaginal Colonization in Pregnant Women

Three commercial chromogenic agar media were evaluated for Streptococcus agalactiae screening in 200 vaginal swabs from pregnant women. The sensitivity and specificity were 94.3% and 100% for Granada medium (bioMérieux), 100% and 90.3% for Brilliance GBS medium (Thermo Fisher Scientific), and 100% and 98.8% for ChromID STRB medium (bioMérieux), respectively.

Immunochromatographic Detection of the Group B Streptococcus Antigen from Enrichment Cultures

Group B Streptococcus (GBS; Streptococcus agalactiae) is a leading cause of serious neonatal infections. The Centers for Disease Control and Prevention recommends GBS screening for all pregnant women during the 35th to 37th weeks of gestation. Although GBS screening has been performed mainly by the culture-based method, it takes several days to obtain a reliable result. In this study, we developed a rapid immunochromatographic test (ICT) for the detection of GBS-specific surface immunogenic protein in 15 min using an overnight enrichment culture. The ICT was prepared using two anti-Sip monoclonal antibodies. This ICT was able to detect recombinant Sip levels of 0.5 ng/ml, or about 10(6) CFU/ml of GBS cells, in tests with 9 GBS strains of different serotypes. The cross-reactivity test using 26 species of microorganism showed no detectable false-positive result. Reactivity of the ICT with 229 GBS strains showed one false-negative result that was attributable to the production of truncated Sip. Among 260 enrichment cultures of vaginal swabs, 17 produced red to orange pigments in Granada medium, and they were all GBS and Sip positive. Among 219 pigment-negative cultures, 12 were GBS positive and 10 were Sip positive. Two Sip-negative cultures contained GBS cells below the limit of detection by the ICT. Among 207 GBS-negative cultures, only one was Sip positive, which was attributable to GBS cell debris. Thus, the sensitivity and specificity of the ICT appeared to be 93.1% and 99.6%, respectively. The newly developed ICT is readily applicable to clinical use in the detection of GBS.

Diagnostic yield of instant liquid biphasic Granada medium for group B streptococcus detection in pregnant women

Diagnostic yield of instant liquid biphasic Granada medium for group B streptococcus detection in pregnant women

Possible Solution to the Problem of Nonhemolytic Group B Streptococcus on Granada Medium

Group B Streptococcus (GBS) is an important cause of neonatal sepsis. Screening for maternal GBS colonization is generally advised at 35 to 37 weeks of gestation. For laboratory detection of GBS, one of the methods recommended by the CDC ([2][1]) is culturing of a vulvorectal swab on Granada medium

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

BackgroundStreptococcus 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.MethodsA 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.ResultsThe 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.ConclusionsIn 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.

High stability of a new Granada medium agar that allows rapid and accurate detection of colonization with group B streptococci in pregnant women

High stability of a new Granada medium agar that allows rapid and accurate detection of colonization with group B streptococci in pregnant women

Genotyping of Streptococcus agalactiae(group B streptococci) isolated from vaginal and rectal swabs of women at 35-37 weeks of pregnancy

BackgroundGroup B streptococci (GBS), or Streptococcus agalactiae, are the leading bacterial cause of meningitis and bacterial sepsis in newborns. Here we compared different culture media for GBS detection and we compared the occurrence of different genotypes and serotypes of GBS isolates from the vagina and rectum.MethodsStreptococcus agalactiae was cultured separately from both rectum and vagina, for a total of 150 pregnant women, i) directly onto Columbia CNA agar, or indirectly onto ii) Granada agar resp. iii) Columbia CNA agar, after overnight incubation in Lim broth.ResultsThirty six women (24%) were colonized by GBS. Of these, 19 harbored GBS in both rectum and vagina, 9 only in the vagina and 8 exclusively in the rectum. The combination of Lim broth and subculture on Granada agar was the only culture method that detected all GBS positive women. Using RAPD-analysis, a total of 66 genotypes could be established among the 118 isolates from 32 women for which fingerprinting was carried out. Up to 4 different genotypes in total (rectal + vaginal) were found for 4 women, one woman carried 3 different genotypes vaginally and 14 women carried two 2 different genotypes vaginally. Only two subjects were found to carry strains with the same genotype, although the serotype of both of these strains was different.Eighteen of the 19 subjects with GBS at both sites had at least one vaginal and one rectal isolate with the same genotype.We report the presence of two to four different genotypes in 22 (61%) of the 36 GBS positive women and the presence of identical genotypes in both sites for all women but one.ConclusionThe combination of Lim broth and subculture on Granada medium provide high sensitivity for GBS detection from vaginal and rectal swabs from pregnant women. We established a higher genotypic diversity per individual than other studies, with up to four different genotypes among a maximum of 6 isolates per individual picked. Still, 18 of the 19 women with GBS from both rectum and vagina had at least one isolate from each sampling site with the same genotype.

腟スワブを検体としたB 群レンサ球菌の保菌調査における 選択増菌培地の有用性

As the most common cause of neonatal sepsis, Lancefield Group B Streptococcus (GBS) must be diagnosed as early as possible in pregnant women is prevent neonatal infection. A selective enrichment broth medium has been widely recommended to optimally recover GBS from genital and anorectal samples. To establish a culture suitable for screening vaginal swab specimens, we compared subcultures of three selective enrichment media to direct culture on agar medium. Vaginal swab samples were inoculated directly onto 5% sheep blood agar and into New Granada medium (Eiken), Lim broth (Becton, Dickinson, and Company), and Todd Hewitt broth with gentamicin and nalidixic acid (Becton, Dickinson, and Company, Todd). Of the 288 specimens tested, GBS was recovered from 43 samples (14.9%) on direct agar media, with 82 (28.5%), positive on New Granada medium subculture, 67 (23.3%) on Lim broth subculture, and 61 (21.2%) on Todd, subculture. These results demonstrates that selective enrichment broth media provides more superior sensitivity than direct agar media for detection of GBS colonization in vaginal specimens, underscoring the usefulness of selective enrichment broth media in GBS screening for vaginal swabs in pregnant woman.

Colonization Rate, Serotypes, and Distributions of Macrolide-Lincosamide-StreptograminBResistant Types of Group B Streptococci in Pregnant Women

Background The prevalence of neonatal group B streptococcal infection depends mainly on the colonization rate of pregnant women by group B streptococci (GBS). Although the colonization rate of Korean women by GBS is considered lower than in other countries, recent data on the maternal colonization rate of GBS are sparse. Methods From August 2008 to June 2009, swab specimens from the anorectus, vagina, and urethral orifice of a sample of 234 pregnant Korean women were placed in new Granada medium (NGM-H), tube medium (NGM-T), commercial NGM (NGM-B), and selective Todd-Hewitt broth (S-THB) for 18∼24 hours in 5% CO2 at 35oC. Agar dilutional antimicrobial susceptibility tests, serotyping, and PCR were performed for GBS isolates. Results The colonization rate of GBS in pregnant women was 11.5% (27/234). Of the specimen cultures, 9.8% of anorectal cultures were positive, 8.1% of urethral orifice cultures were positive, and 7.3% of vagina cultures were positive. The detection rate of GBS in the different culture media was S-THB (96.3%), NGM-B (92.6%), NGM-H (88.9%), and NGM-T (85.2%). The distribution of GBS serotypes was as follows: III (29.6%), V and VI (22.2%), Ib and II (11.1%), and Ia (3.7%). 33.3% of GBS isolates were resistant to erythromycin and 44.4% to clindamycin. Among the nine erythromycin-resistant isolates, eight were serotype V and VI, which are erm(B) positive serotypes. Conclusion The colonization of pregnant women by GBS, and the incidence of resistance of the GBS isolates to erythromycin and clindamycin were higher than those previously reported. Serotypes V and VI, GBS serotypes that carry the erm(B), are novel serotypes that have not previously been identified in pregnant Korean women.

Evaluation of liquid biphasic Granada medium and instant liquid biphasic Granada medium for group B streptococcus detection

Group B streptococci (GBS) are transmitted from the mother to the newborn. Prevention of neonatal infection is achieved by intrapartum prophylaxis given to mothers colonized with GBS at 35 to 37 weeks of pregnancy. Liquid biphasic Granada medium (LB) and instant liquid biphasic Granada medium (ILB) were evaluated for GBS detection. Vaginal swabs obtained from 300 women were inoculated onto LB or ILB, or onto Todd-Hewitt broth and analyzed with the ATB system (comparison method). Prevalence of GBS was 20% (61/300). LB and Todd-Hewitt with ATB detected GBS in 20% of women, and ILB in 19% of women. No growth was observed at four hours in any of the media studied. At 10 h and 14 h, identification of GBS was possible in 43/300 (14%) and 53/300 (18%) of ILB cultures, respectively, and in 32/300 (11%) and 46/300 (15%) of LB cultures. All the media used are suitable for GBS detection. The majority of GBS were identified in ILB and LB cultures at 10 h and 14 h.

Evaluation of a new chromogenic agar medium for isolation and identification of Group B streptococci

To evaluate a new chromogenic agar as a screening medium for the isolation of Group B streptococci from high vaginal swabs from pregnant women. The medium was evaluated with 195 high vaginal swabs referred for antenatal screening and compared with blood agar and Granada medium. The new chromogenic medium showed 100% sensitivity for the detection of Group B streptococci, and also showed a positive predictive value of 100%. Granada medium also showed excellent sensitivity and specificity and both media were superior to blood agar. The new chromogenic medium showed excellent performance for the detection of Group B streptococci from clinical samples. This is the first chromogenic medium described for the detection of Group B streptococci. The medium offers an effective and convenient alternative to conventional media, currently used in clinical laboratories.

Evaluation of Group B Streptococcus Differential Agar for detection and isolation of Streptococcus agalactiae

In total, 320 vaginal or rectal swabs were cultured on Granada medium (GM) or Group B Streptococcus Differential Agar (GBSDA), and were also inoculated into LIM broth (Todd-Hewitt broth supplemented with selective antibiotics), for detection of group B Streptococcus (GBS). Overall, GBS isolates were detected on 53 of the 320 swabs; 47 of these isolates grew on both GM and GBSDA, five only on GBSDA, and one only following subculture from LIM broth. GBSDA appears to be a valid alternative to GM for the growth of GBS isolates from pregnant women.

Liquid Granada Medium for Detection of Group B Streptococci

Heelan et al. (5) recently presented data on the sensitivity of a liquid Granada medium (9) (GBS broth) for detection of Streptococcus agalactiae. We have tested this medium, and it contains, as Granada medium, starch, proteose peptone 3, methotrexate, a Good’s buffer, serum, glucose, and selective agents. So, we agree with the authors that detection of orange-red colonies in GBS broth is 100% specific for the presence of beta-hemolytic group B streptococci (GBS). Nevertheless, some statements in this paper need clarification. The authors claim that sometimes Granada agar lacks sensitivity for GBS detection, but no comparison between GBS broth and Granada agar is reported. In the two references cited to support this claim (3, 7), it is acknowledged that the batches of Granada agar used were probably spoiled (10). Moreover, the flurry of references (1, 2, 4, 6, 8, 9, 14, 15) that support a very good sensitivity of Granada agar to detect GBS are not mentioned. Regarding sensitivity of GBS broth, the figures reported are somewhat confusing. One-hundred fifty-eight GBS strains were recovered from 580 swabs, but only in 108 cases were red colonies detected, so GBS broth sensitivity is 68.3% instead of the 87.8% or 90.3% reported. GBS pigment was not observed in 35 tubes that later (after subculture) proved to be positive (25% of all positives), and this figure makes the usefulness of this medium for direct GBS detection doubtful. When (study 1) GBS broth was used as a detection medium, 9 of 75 GBS strains (12%) were completely lost (positive in subculture from LIM broth but negative in subculture from GBS broth). When (study 2) GBS broth was used as a detection and transport medium, 6 of 83 GBS (7%) were also completely lost. This failure to detect (even after subculture) some GBS-positive vaginorectal samples can be an important drawback of GBS broth. Another fact is the dramatic failure of LIM broth to recover GBS (21 of 83 positive samples were lost in study 2) found in Heelan's work. This finding needs further evaluation, as enrichment in LIM broth is an accepted procedure for GBS detection in the Centers for Disease Control and Prevention guidelines (13). Poor performance in detecting GBS could be explained by the fact that when liquid Granada media are kept refrigerated the starch precipitates (retrogradation) (16), producing an opaque white mass, where GBS colonies are sometimes barely noticeable. However, if not kept refrigerated the medium deteriorates in a few days (12). We have observed that when media containing folate antagonists (as methotretaxate) are used as transport media and swabs are kept in these media for several hours before starting incubation, the number of GBS CFU nosedives. This renders the use of such media as transport media unsafe. For these reasons, in our view, the use of liquid selective Granada media as transport media for detection of GBS colonization in pregnant women cannot be recommended.

Use of Unmodified Starches and Partial Removal of Serum To Improve Granada Medium Stability

The use of 1% unmodified rice starch and 1% horse serum instead of 2% soluble starch and 5% serum in Granada medium is described. These components result in a medium of increased stability, preventing spoilage after a few days of storage at room temperature.

Comparison of two culture media and three sampling techniques for sensitive and rapid screening of vaginal colonization by group B streptococcus in pregnant women.

The Centers for Disease Control and Prevention (CDC) recommend universal screening of all pregnant women between 35 and 37 weeks of gestation for group B streptococci (GBS) by use of a selective broth medium. Recent reports suggest that Granada medium can be used for rapid and direct visual identification of GBS colonies. However, studies comparing the Granada medium method to the selective broth method are few, and while some report comparable sensitivities, others have found significant differences in detection rates between the two methods. This prospective study compared a method using Granada agar to a Todd-Hewitt broth method with subculture to blood agar in order to determine which GBS detection method is more sensitive and less labor-intensive and has a more rapid turnaround time. Detection rates for three sampling techniques (rectovaginal, vaginal only, and cervical only) were also compared. Consecutive specimens for GBS screening received over a 6-month period from 1,635 pregnant women were included. Overall, GBS was detected in 390 (23.8%) women. The Granada medium gave positive results for 348 of these women, and the selective broth gave positive results for 385, indicating sensitivities of 89.2% for the Granada medium and 98.7% for the selective broth. These findings show that the Granada medium method is less sensitive than the selective broth method and should not replace it as the only method for screening pregnant women for GBS. However, the Granada medium method reduced detection time to 1 day and also reduced the use of ancillary tests in approximately 90% of positive cases. Additionally, no significant differences were noted in the detection rates with rectovaginal, vaginal, and cervical specimens.

Estudio comparativo de tres medios de cultivo para detectar la colonización por estreptococo del grupo B en la mujer embarazada

To compare the efficacy of three culture media to detect vaginal and rectal colonization by group B streptococci (GBS) in pregnant women. We processed 1334 samples from 861 pregnant women: in 388 patients only vaginal swab was cultured (Group A) and in 473 vaginal and rectal swabs were cultured (Group B). Samples were inoculated on blood agar with colistin-nalidixic acid (BA-CNA), on plates with Granada medium, and on Todd-Hewitt broth (THB) with amikacin followed by subculture in BA-CNA. GBS was isolated in 181 samples (13.6%): 114 vaginal swabs (13.2%) and 67 rectal swabs (14.2%). Among the positive samples, GBS was isolated on BA-CNA in 60.5% of vaginal swabs and in 59.7% of rectal swabs, on Granada medium in 80.7% of vaginal swabs and in 91% of rectal swabs, and on THB in 97.4% of vaginal swabs and in 97% of rectal swabs.We detected 130 GBS carriers, 54 (13.9%) in Group A and 76 (16.1%) in Group B. The percent of carriers detected in groups A and B, respectively, was 59.3% and 75% with BA-CNA, 77.8% and 93.4% with Granada medium, and 96.3% and 97.4% with THB. THB was the most reliable medium for the detection of GBS. Use of Granada medium allows fast detection of about 87% of carriers. The combination of THB and an initial plate of Granada medium or BA-CNA allows detection of more than 99% of GBS carriers.

Screening Protocols for Group B Streptococcus: Are TransportMedia Appropriate?

Objective: To evaluate group B streptococcus (GBS) detection in an in vitro setting, using a low and controlled inoculum from swabs directly inoculated into a selective medium, as compared to delayed inoculation following a period in a commercial Amies transport medium with charcoal (Venturi Transystem™ Copan, Italy). Study design: Clinical isolates of GBS (n = 103), were inoculated into the Amies transport medium with charcoal in a concentration of 100 colony-forming units (cfu)/ml (10 cfu/swab). Swabs were then transferred to an enrichment broth (NPC) at time intervals of 0, 2, 4, 6 and 24 hours. Broths were then incubated for 18–24 hours at 35°C in air, before being transferred to New Granada Medium Modified (NGM) for GBS detection and incubated for a further 18–24 hours at 35°C in air. If the characteristic orange pigmented colonies were observed after this period, the specimen was recorded as + (1–10 colonies) or ++ (more than 10 colonies). Results: Overall 92.2% (95/103) of isolates were detected in all tubes and at all times. An additional two isolates were non-hemolytic, non-pigment forming GBS. Of note, 3.9% (4/103) were negative until 2 hours delayed inoculation and 1.9% (2/103) gave inconsistent results, likely due to the low inoculum used. Conclusion: Delayed inoculation into selective enrichment broth following a period in transport medium, even with a low inoculum, gave a similar and acceptable GBS detection rate to direct inoculation. Hence, Amies transport medium with charcoal is an appropriate transport medium to use, where it is not practical for clinical specimens to be directly inoculated into selective enrichment broth and as endorsed in the Centers for Diseases Control (CDC) Guidelines, 2002.

Modified Granada Agar Medium for the detection of group B streptococcus carriage in pregnant women

To improve the detection rate of group B streptococci (GBS) in pregnant women, aiming at the prevention of early-onset septicemia in the newborn. The yield from culturing two sites, vaginal and anorectal, on a Modified Granada Medium (MGM) was compared with our standard approach of culturing a vaginal swab on blood agar (BA). Samples were processed from 430 consecutive pregnant women. GBS was isolated from the vagina in 11.6% with BA, and in 13.7% with MGM. In 17.0% of anorectal samples, GBS was identified with MGM. The combination of both sites and media had a yield of 20.0%. MGM identified all but six (2%) of 310 GBS strains after aerobic incubation, with use of a cover slide, and missed only three strains (1%) after anaerobic incubation. Separate culture of vaginal and anorectal samples using the same MGM agar plate resulted in an increase in detection rate for GBS of 76% as compared to BA alone. The technique is simple and results are available after overnight incubation. MGM was confirmed as a specific medium for the identification of GBS, with a sensitivity of 98-99%.