Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 28, Num. 4, 2010, pp. 320-325

Indian Journal of Medical Microbiology, Vol. 28, No. 4, October-December, 2010, pp. 320-325

Original Article

Comparison of scpB gene and cfb gene polymerase chain reaction assays with culture on Islam medium to detect Group B Streptococcus in pregnancy

Sarah Shabayek¹, Salah Abdalla¹, Abouzeid M.H Abouzeid²

¹ Department of Microbiology and Immunology, Faculty of Pharmacy, Suez Canal University, Ismailia, Egypt
² Departments of Gynaecology and Obstetrics, Ismailia General Hospital, Ismailia, Egypt
Correspondence Address: Sarah Shabayek, Department of Microbiology and Immunology, Faculty of Pharmacy, Suez Canal University, Ismailia, Egypt, sarahshabayek@yahoo.com

Date of Submission: 02-Dec-2009
Date of Acceptance: 01-Jul-2010

Code Number: mb10098

PMID: 20966562
DOI: 10.4103/0255-0857.71821

Abstract

Purpose: The purpose of the current study was to evaluate two low-costing PCR assays for rapid detection of Group B Streptococcus (GBS) in comparison to a pigment-based culture method.

Materials and Methods: One-hundred and fifty vaginal swabs were collected from pregnant women at 35-40 weeks of gestation. Vaginal swabs were inoculated in selective enrichment broth medium, and examined using Islam medium, cfb PCR and scpB PCR assays. The demographic data were analysed to identify independent predictors of GBS colonization (age and gravidity), with GBS status as the dependent variable.

Results: There was a significant association of age and gravidity with GBS colonization. GBS was detected in 25.3% of isolates by Islam medium, in 30.6% by using the cfb PCR assay and in 30% by using the scpB PCR assay.

Conclusion: older pregnant women (≥30 years) and multigravida (>3 pregnancies) are at higher risk of GBS colonization. Both scpB-gene and cfb-gene-based PCR methods are highly sensitive techniques (100% sensitivity) compared to culture method. However, the specificities of the scpB and cfb PCR assays were 93.75 and 92.85%, respectively.

Keywords: Group B Streptococcus, Vaginal swab, Islam medium, scpB-gene-based PCR, cfb-gene-based PCR

Introduction

Group B Streptococcus (GBS) is a leading cause of sepsis, meningitis, pneumonia and death among newborn infants in developed countries. ^[1] GBS diseases have two clinical presentations: early-onset infections, which occur within the first seven days of life and account for 80% of neonatal GBS infections and late-onset infections, which occur between the first week of life and three months of age. Approximately 10-35% of pregnant women are colonized with GBS as a part of normal flora of the vagina and/or rectum and children born to these women may develop disease due to exposure to bacteria before birth or during the neonatal period. ^[1] Women who are carriers are also at risk for severe infections. ^[1]

Neonatal infections can be prevented in most cases by providing intrapartum antibiotic prophylaxis (IAP) to the colonized mother. ^[2] Hence, determination of colonization by GBS at time of labour is essential. The revised Centres for Disease Control and Prevention (CDC) guidelines issued in 2002, recommended a culture-based screening for all pregnant women at 35-37 weeks of gestation for the prevention of early-onset GBS disease and that IAP is offered to the carriers. ^[3] Implementation of these guidelines has been associated with a falling incidence of neonatal GBS disease in developed countries. ^[4]

Currently, prenatal screening culture, including broth culture in selective medium, is the gold standard method for detection of anogenital GBS colonization. ^[3] However, the culture methods require several days to yield results. Rapid tests have been developed, such as antigen-based tests, hybridization-based methods and PCR assays, in order to detect GBS. ^[5] PCR-based assays offer promising tools for sensitive, specific, and rapid detection of GBS directly from clinical specimens at the time of delivery, avoiding the need for lengthy culture of GBS. PCR assays for the identification of GBS have been developed to detect a variety of genetic targets, including genes encoding C protein, the 16S rRNA, and the 16S-23S spacer region. ^[5] By the early 2000s, GBS-specific PCR assays had been developed. Such assays targeted the cfb gene, which encodes the CAMP factor, ^[6] and the scpB gene, which encodes the C5a peptidase. ^[7]

No epidemiological data has been published on the colonization rate with GBS of pregnant women in Egypt except for a recent preliminary study by Elbaradie et al.[8] There is no awareness of prenatal screening policy. Moreover, clinical problems related to maternal and neonatal GBS are not reported. Previously, we determined the colonization rate of GBS in Ismailia, Egypt, and the antibiotic susceptibility profile of the clinical isolates. ^[9] The purpose of the current study was to evaluate two low-costing PCR assays for rapid detection of GBS in comparison to a pigment-based culture method.

Materials and Methods

Culture and bacteria identification

One-hundred and fifty pregnant women at 35-40 weeks of gestation were enrolled in this study. Identical case report forms were used to record demographic details for each patient, including age, weeks of gestation, number of previous pregnancies, times of marriage, and current health state. One vaginal swab was collected from each patient with an informed consent, placed in Amies transport medium (Lab M, Limited Topley House, Wash Lane, Bury, Lancashire, United Kingdom) and kept at 4˚C. Afterwards, swabs were inoculated in selective enrichment broth medium (Todd Hewitt Broth; Oxoid LTD, Basingstoke, Hampshire, England), supplemented with nalidixic acid 15 μg/ml and colistin 10 μg/ml. After 18 to 24 h incubation at 37˚C in 5% CO ₂ , broths were divided into two portions. One portion was sub-cultured onto Islam medium (Oxoid LTD, Basingstoke, Hampshire, England). The other portion was stored at -80˚C for further PCR detection assays. Islam media plates were examined for orange-pigmented colonies after 24 to 48 h of anaerobic incubation. Positive selective broth cultures for GBS were further confirmed by the traditional culture method through sub-culturing onto 5% Columbia blood agar (Oxoid LTD, Basingstoke, Hampshire, England) supplemented with nalidixic acid 15 μg/ml and colistin 10 μg/ml (CNA medium), incubated for 18-24 h at 37˚C in 5% CO ₂ . Colonies on CNA plates were identified by Gram stain, haemolysis, catalase, CAMP test, hippurate hydrolysis and bile esculin hydrolysis tests. ^[3],[10]

PCR detection on incubated selective broth cultures

One and half millimetres of each primary selective enrichment broth culture preserved at -80˚C was allowed to thaw at room temperature. Cells were then pelleted by centrifugation at 6000 rpm for 5 minutes. The supernatant was discarded and the pellet was resuspended in 0.5 ml 2Χ TENS buffer as described by Kuske et al.[11] Samples were vortexed briefly and incubated at 70ºC for 20 minutes. During this time, samples were suspended and mixed by vortexing for 5 seconds every 10 minutes. Then DNA was prepared from lysed bacterial cells by phenol/chloroform extraction procedure. The DNA pellet was resuspended in 100 μl TE buffer and stored in -20ºC until use.

GBS nucleic acid detection was based on two different PCR assays; one targeting the cfb gene, which encodes the CAMP factor and the other one targeting the scpB gene, which encodes C5a peptidase. PCR reaction for each assay was performed in a 25 μl volume containing 1 μl DNA template, 5Χ PCR buffer (Promega, San Luis Obispo, CA, USA), 2.5 mM MgCl ₂ (Promega, San Luis Obispo, CA, USA), 200 μM PCR nucleotide Mix 10 mM each (Promega, San Luis Obispo, CA, USA), 0.4 μM each primer, 1 U Go Tag DNA polymerase (Promega, San Luis Obispo, CA, USA). ^[6] The volume for each PCR reaction was completed to 25 μl with nuclease free water (Promega, San Luis Obispo, CA, USA). For the cfb PCR, primers Sag59 and Sag190 defined by Ke et al.[6]were used. For the scpB PCR, the primers described by Dmitriev et al.[7] were used. Amplifications included initial denaturation at 94 ^o C for 3 minutes followed by 40 cycles of 1 second at 95 ^o C, and 30 seconds at 55 ^o C, then final elongation at 72 ^o C for 2 minutes. ^[6]DNA amplification was carried-out in a Gradient Thermacycler (MyCycler, BIO-RAD, USA). The sizes of the PCR products were determined by comparing them with the migration of 100 bp DNA ladder (Axygen Biosciences, CA, USA).

Statistical analysis

The only available demographic data of 137 out of 150 pregnant women were analysed using Chi-square and Fischer-exact two-tailed tests to compare GBS-positive and GBS-negative groups according to age and gravidity. Linear regression was applied to identify independent predictors of GBS colonization (age and gravidity), with GBS status as the dependent variable. P < 0.05 was considered statistically significant.

Sensitivity, specificity, positive and negative predictive values were calculated according to the standard formulas. ^[12]

Results

Demographics

Participants' ages ranged from 17 to 44 years with a mean age of 28 years. The mean gravidity was 3. Demographic data are summarized in [Table - 1]. GBS colonization rate profiles according to the age and gravidity are shown in [Table - 2] and [Table - 3], respectively. There was a significant association of age (P = 0.03, [Table - 4]) and gravidity (P = 0.03, [Table - 5]) with GBS colonization. Colonization rates were significantly higher in pregnant women ≥30 years (38.6%) compared to those <30 years (20.4%). Concerning gravidity, colonization rates were higher after the third pregnancy (37.7%) compared to previous pregnancies (19.56%). This holds also true when applying linear regression to identify age (R2 = 80%, at 0.01 level) and gravidity (R2 = 85%, at 0.01 level) as independent predictors of GBS status (dependent variable). Each additional year of age of pregnant woman increases the probability of being a GBS carrier by 0.87 and after each pregnancy, probability of being a GBS carrier increases by 5.44. Linear regression results are summarized in [Table - 6].

Culture and PCR

A total of 38 specimens (25.3%) were found to be positive for GBS by Islam medium. Forty six specimens (30.6%) were positive for GBS using the cfb PCR assay and forty five specimens (30%) were positive for GBS using the scpB PCR assay. Positive specimens for the cfb PCR assay showed characteristic bands of approximately 153 bp in size [Figure - 1], while samples positive for GBS using the scpB PCR assay showed characteristic bands of approximately 255 bp in size [Figure - 2]. Culture and the two PCR assays were positive for 38 specimens and negative for 104 specimens. One specimen was found to be negative by both culture and the scpB PCR, but positive for GBS by the cfb PCR. Seven culture negative specimens were found to be positive for GBS by the two PCR assays. PCR assays were repeated twice to confirm the positive results of the culture negative specimens. GBS detection profile from primary selective broth cultures for each assay is summarized in [Table - 7].

In comparison to culture method, the scpB PCR assay revealed 100% sensitivity and 93.75% specificity. The positive predictive value was 84.4% and the negative predictive value was 100%. The cfb PCR assay showed 100% sensitivity and 92.85% specificity. The positive predictive value was 82.6% and the negative predictive value was 100%.

Discussion

The colonization rate was found to be 25.3% in our region. Such finding was in accordance with many studies from USA, Europe, and the Middle East. ^{[13],[14],[15],[16]} Maternal age, parity, ethnicity, marital status, education, smoking, and frequent intercourse with multiple partners may influence the prevalence of colonization. The present study showed that generally older age and greater parity were associated with GBS colonization, where older pregnant women and multi-gravidae are at a higher risk for GBS colonization. These results were in agreement with those reported by El-Kersh et al. ^[16] in respect to maternal age but different in respect to multiparity. However, Collins et al. ^[17] found no association of GBS with the previously identified risk factors such as age and parity. Due to this inconsistency, Benitz et al. ^[18] concluded thatthese risk factors may only raise clinical suspicion for GBS colonization but do not identify the high risk women for whom selective screening might be appropriate.

A rapid, sensitive, and accurate method would provide the best means of GBS detection allowing the highest percentage of women to be treated, and reduce the rate of GBS transmission to the newborn. This method would be highly beneficial in the case of pre-term labour, pre-term premature rupture of membranes, and in patients with limited or no prenatal care.

However, the culture method has a slow turnaround time requiring 48-72 hours before results can be issued. Besides requiring experienced technicians, suppression of GBS growth by enterococci present in the vagina and rectal flora could lead to false negative results. ^[19] A rapid screening test for GBS-like PCR would accurately identify pregnant women who are carrying the bacteria at delivery time.

In the present study, two PCR assays have been investigated for GBS detection directly from incubated selective enrichment broth cultures: the cfb PCR assay and the scpB PCR assay. In comparison to culture, both PCR assays revealed a sensitivity of 100%. The scpB PCR assay showed a specificity of 93.75% and the cfb PCR assay had a specificity of 92.85%. Sensitivity of the cfb PCR method was higher than that reported by Bergeron et al. ^[20] and published by Atkins et al.[21] as the reported sensitivities were 97.7 and 86.8%, respectively. However, the demonstrated specificity for the cfb PCR method was lower as the reported specificities were 100 and 95.2%, respectively.

Concerning the PCR program, Ke et al.[6] was the first to develop a conventional cfb-gene-based PCR for GBS utilizing a one-hour program, and Dmitriev et al. ^[7] was the first to develop a conventional scpB-gene-based PCR utilizing a three-hour program. In the present study, it was interesting to say that the scpB-gene-based PCR could be done using the one-hour program and the same master mix concentrations described by Ke et al.[6] for the cfb-gene-based PCR.

Eight women exhibited positive GBS by PCR but not by culture. These discrepancies could be attributed to the presence of nonviable bacteria in the specimen or low bacterial numbers that could not be detected by culture. Moreover, GBS detection by culture could also be inhibited by antibiotics and feminine hygiene products. ^[22] Inadequate specimen collection and transport from obstetrical clinics to the laboratory may also have some contribution especially in case of light colonization. ^[23] All these factors could explain the higher detection rates obtained by the two PCR assays with a main concern on the ability to detect both viable and non-viable bacteria even in those with minute bacterial loads non-detectable by culture. Thus the major implication of false-positive PCR results could be the unnecessary administration of antimicrobial agents for false-positive carriers.

The increased awareness of the impact of GBS early-onset disease has led to great efforts in the treatment and prevention of GBS infection. GBS infection is now a public-health concern. Recognition of the nature of maternal-to-infant transmission, the efficacy of intrapartum maternal chemoprophylaxis, and the optimized detection methods for identification of GBS carriers allow for an efficient way to manage infections associated with GBS. Implementation of consensus guidelines by the CDC, the ACOG, and the AAP has proven to be associated with a substantial decline in the incidence of GBS diseases in the newborns. ^[4]

However, preventive strategies have not been widely implemented. Antenatal screening for GBS and intrapartum antibiotic prophylaxis is not universally adopted by all countries and physicians. The clinical problems relevant to GBS infection among parturient mothers and their newborns have not declined in many parts of the world and still to be unknown in other parts. The GBS issue is brand new to Egypt. There are no data available neither on the prevalence nor on neonatal GBS infections and consequently no antenatal screening policies are followed. Reporting a vaginal colonization rate of 25.3% in our region draws the attention to a group of women whose neonates are at high risk especially when knowing that such rate might reach 38% if vaginorectal specimens were collected. ^[24] Such findings strengthen the need to implement the international guidelines to prevent neonatal GBS infections or even formulate national guidelines that are suitable and cost-effective in a developing country like Egypt. There should be large efforts to estimate the magnitude of the neonatal GBS infections in comparison to the neonatal morbidity and mortality in Egypt.

In conclusion, our study showed that older pregnant women (≥30 years) and multigravida (>3 pregnancies) are at higher risk of GBS colonization. Both scpB-gene and cfb-gene-based PCR methods are highly sensitive techniques (100% sensitivity) compared to culture method. However, the specificities of the scpB and cfb PCR assays were 93.75 and 92.85%, respectively.

References

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