Abstract
Introduction
Group B streptococcus (GBS) infection during pregnancy is caused by Streptococcus agalactiae and it is recognized globally as a leading cause of chorioamnionitis, puerperal endometritis, andneonatal sepsis, meningitis and pneumonia [1, 2]. The prevalence of vaginal GBS colonization in industrialized countries has been reported as 5 to 35% [3]. In some developing countries, lower rates in Libya (5%), India (5.8%) and East Asia (8%) [4, 5] were described whereas rates >20% have been reported in Zimbabwe [6]. Approximately 50% of babies of carriers become colonized perinatally and 1 to 2% infected [7]. Case-fatality rates are estimated at 4 to 10%, with early-onset disease (i.e. <7 days of life) accounting for over 80% of GBS disease during infancy [7, 8].
The Centers for Disease Control (CDC) has recommended a culture based screening approach for all pregnant women between 35 and 37 weeks of gestation, while the American Academy of Pediatrics recommends treatment with intravenous antibiotics to all women with risk factors prior to screening, until their GBS status is established. However, identification of a high-risk population to be screened is a considerable challenge, as colonization is also observed in mothers without the classically known risk factors. Moreover, the occurrence of early neonatal GBS disease in this group of neonates is 25 to 30% [9–11]. Intrapartum prophylaxis has been reported to decrease the incidence of early onset disease from 1.7 to 0.6 per 1000 live births [9, 12] but the widespread use of antibiotics carries the potential for antibiotic resistance emergence [13, 14].
GBS colonization and infection has emerged as an important neonatal problem in Southern and Eastern Africa, raising the possibility of an association with HIV infection [15]; a higher risk of invasive GBS infection in HIV positive patients. In a study from Brazil, a higher rate of GBS colonization was observed in HIV infected pregnant women (19.8%) when compared to HIV negative (14.1%) [16], but the contribution of HIV infection to GBS colonization and disease remains uncertain. This study was therefore undertaken to estimate the rate of anogenital GBS colonization in our institution and compare the prevalence of GBS colonization and HIV status.
Materials and methods
Study location
This study was conducted from July to December 2011 in the Obstetrics and Gynecology department of the Obafemi Awolowo University TeachingHospitals Complex (OAUTHC) in South-western Nigeria. The department has two obstetric units: Ife Hospital Unit (IHU) and Wesley Guild Hospital (WGH) Ilesha, which serve as tertiary referral centers for secondary and primary tiers of hospitals within the zone and other neighboring states.
Study design
Prospective cohort study that included all pregnant women cared for at our obstetric clinics, with gestational age between 35 to 37 weeks. Patients with a history of previous early neonatal sepsis (i.e. ≤7 days of delivery), and use of antibiotics within 2 weeks prior to recruitment were excluded. The study was approved by the Institutional Ethics Board and informed consent was obtained from the patients.
Methods
A predefined data collection form was used to obtain information on socio-demographic characteristics, and factors that could potentially be associated with GBS colonization. As part of our standard of care, GBS positive subjects were treated with appropriate antibiotics and all patients were screened for HIV 1 and 2. HIV positive participants had samples taken for CD4 count and other tests for pre-assessment of eligibility for antiretroviral treatment or Prevention of Mother-to-Child Transmission (PMTCT) services.
Anogenital swabs for GBS colonization were obtained from participants in dorsal position with both knees raised. The vaginal swab was obtained about 2 cm proximal to the introitus after parting the labia minora. A separate swab was used for the anal collection and all samples were put into appropriate transport media with proper labeling of the specimens. Samples were then taken to the microbiology laboratory for subsequent analysis.
All swabs were cultured within an hour of collection in the laboratory. Swabs were removed from transport medium and placed into an enrichment broth (Todd-Hewitt broth supplemented with 10 μg/ml colistin and 15 μg/ml nalidixic acid) which is selective for GBS. All media were prepared according to manufacturer’s specifications. Inoculated enrichment broth was incubated aerobically at 37C for 24 h, after which the broth was examined for evidence of bacterial growth (turbidity) andre-incubated for an additional 24 h if necessary. The broth was sub-cultured into non-selective 5% sheep blood agar (SBA) plates (Oxoid, England) using a standard platinum wire loop of 4 mm in diameter and incubated for 24 h at 37C; following which an examination of the plate for beta or non- hemolytic colonies suggestive of group B streptococci was done. Any SBA culture plate that had no growth after 24 h was re-incubated for an additional 24 h before reporting a negative result.
Streptococcal serological grouping was carried out on the enrichment broth cultures to test for the presence of group B specific antigen using latex agglutination antigen detection kit specific for GBS (Oxoid, England), according to the manufacturer’s instructions, to further validate the culture results obtained.
Pure distinct colonies on the sheep blood agar plate were identified, and colonies that appeared soft, smooth and whitish gray with or without a narrow zone of hemolysis were gram-stained. The colonies were also tested for the catalase enzyme to distinguish streptococci from staphylococci. Gram positive, non-motile, catalase-negative cocci, suggestive of Group B Streptococci were definitively identified using this process.
The antibiotic susceptibility testing of the pure GBS isolates was performed by the Bauer-Kirby method [17], as modified by the National Committee for Clinical Laboratory Standards (NCCLS) [18]. The different antibiotic discs used for the susceptibility testing were penicillin (PEN) 1I.U, ampicillin (AMP) 10 μg, cefotaxime (CTX) 30 μg, clindamycin (CA) 2 μg, erythromycin (ERY) 15 μg and vancomycin (VAN) 30 μg. Plates were incubated at 37C for 24 h, after which zones of inhibition were measured in millimeter diameter using a ruler. The results were interpreted as Resistant (R), Intermediate (I) or Susceptible (S) according to the CLSI (2008) interpretative chart [19]. The disc diffusion of each isolate was done in duplicate for comparison to ascertain the susceptibility pattern.
Primary outcome
To determine the rates of anogenital GBS colonization in our institution and compare GBS colonization rates between HIV positive and negative pregnant women.
Secondary outcomes
Risk factors for GBS colonization, antibiotic sensitivity pattern of the isolates, and possible association between CD4 count and the prevalence of GBS colonization among HIV positive pregnantwomen.
Sample size and statistical analysis
The sample size was determined from the formula for estimating sample size for the comparison of two independent populations as described by Armitage et al. [20]. We used a GBS carriage rate of 14% in HIV negative pregnant women [16] and estimated a 15% higher prevalence (29%) in the proportion of GBS colonization in HIV positive women [16]. Estimating an attrition rate of 10% and using a ratio of 2:1 for HIV negative/positive patients a sample size of 133 patients for HIV negative and 67 for HIV positive pregnant women would have a power of 80% to detect a significant difference between the groups. The Fischer’s exact test and Pearson’s Chi-square were used for the categorical variables and the student t-test for continuous variables. Logistic regression analysis was performed to determine risk factors for GBS colonization. The level of significance was set at 0.05. Analysis was done using SPSS version 15.0 (Chicago, Illinois).
Results
A total of 200 patients were enrolled; 67 HIV positive and 133 HIV negative. Two samples belonging to HIV negative pregnant women were found to be contaminated leaving a total of 198 analyzable samples. Patient demographics are described on Table 1. A significant difference was observed in the gravidity, which was higher among the HIV positive subjects (p = 0.01). Also, the body mass index (BMI) was significantly lower in the HIV positive pregnantwomen.
Population characteristics
Population characteristics
Legend: HIV = human immunodeficiency virus. Results are expressed as n (%) or mean±standard deviation.
Rates of anogenital GBS colonization and prevalence of GBS colonization and HIV status
Of the total participants, 36 (18.2%) were found to be GBS colonized. No difference in the prevalence of GBS colonization was noted between HIV positive and negative patients (p = 0.750): 19.4% (13) in HIV positive pregnant women (95% CI: 9.7–29.1) and 17.6% (23/131) in HIV negative patients (95% CI: 11.0–24.2).
Secondary outcomes
Risk factors for GBS colonization
GBS colonization was not associated with sexual practice, number of sexual partners, previous contraceptive use, hepatitis B status, and use of illicit drug or alcohol (Table 2). A base model was fitted for predictors of GBS colonization among the study groups at the time of enrollment using all variables considered a priori to be either potentially associated or confounders. The age of participants, parity, level of education, gravidity and previous use ofcontraception were not significant predictors of GBS colonization. However, mean BMI showed a significant association between GBS and positive HIV; an increased BMI being associated with increased odds of GBS colonization (OR = 1.25, 95% CI 1.04–1.51, p = 0.019; Table 3).
Risk factors for group B streptococcus colonization and HIV status
Risk factors for group B streptococcus colonization and HIV status
Legend: HBSAg = hepatitis B antigen. Results are expressed as n (% of the total # of patients HIV positive or HIV negative); *p < 0.05.
Predictors of group B streptococcus colonization
Legend: OR = Odds ratio, BMI = Body mass index, and CI = Confidence interval. areference = tertiary; breference = yes.
Over 80% of the GBS strains were found susceptible to penicillin and ampicillin. A sensitivity of less than 50% was noted for erythromycin, cefotaxime, clindamycin and vancomycin (Table 4).
Antibiotic sensitivity profile
Antibiotic sensitivity profile
Legend: GBS antibiotic sensitive analysis was performed on 198 samples.
No association was observed between GBS colonization and CD4 count. Forty nine (73%) of the HIV positive women had CD4 count≥250 cells/mm3; 41 (84%) of which were GBS positive. Of the 18 HIV positive with CD4 counts of <250 cells/mm3 seven (39%) were GBS positive (p = 0.312). The mean CD4 count of GBS negative was 410.7±227.9 cells/mm3 compared to 358.9±190.9 cells/mm3 of GBS positive women (p = 0.452).
Discussion
In this prospective cohort study among pregnant women cared for at the obstetric clinics in our institution in Nigeria, a high overall prevalence of anogenital GBS colonization was observed. HIV status did not affect GBS colonization rates and amongst HIV positive women a higher BMI was identified as a risk factor for GBS colonization.
Rates of anogenital GBS colonization
A previous study conducted in South-south Nigeria tested for vaginal GBS and reported a colonization rate of 9% [21]. Our finding is comparable to that obtained in South-east Nigeria (18.0%) [22] and Thailand (18.12%) [23] Stoll and Schuchat [24] in a study in selected developing countries reported variable colonization rates: Nigeria (20%), Togo (4%), Gambia (22%) and Mozambique (1%) [24]. Possible reasons for the significant differences in colonization rates include socioeconomic, ethnic and genetic factors, and variations in practices of samples collection and detection techniques.
Prevalence of GBS colonization and HIV status
In our study, the prevalence of GBS colonization observed in HIV positive (19.4%) and negative (17.6%) pregnant women was similar to other investigators [16, 25]. In Brazil, El Beitune et al. reported a GBS colonization rate of 19.8% in HIV positive women. Similar rates were observed in Malawi (19%) [25]. As in our study, no significant differences in GBS colonization rates were observed between HIV positive and negative patients. These results contrast with another study in a University hospital in Brazil in pregnant women attending PMTCT clinic. In that study, a much higher prevalence (31%) of GBS colonization was noted [26]. The difference in prevalence for GBS colonization in HIV positive women may be explained by difficult access to medical facilities or differences in sexual behavior. Thus, further studies are required to identify specific factors that may be responsible for the prevalence of GBS colonization in HIV positive pregnant women across countries.
Risk factors for GBS colonization
Multiple logistic regression analysis was used to identify independent predictors of GBS colonization. An independent association between mean BMI of HIV positive women and GBS colonization was observed. This finding was similar to the results of a study [27] that showed a high BMI as a risk for GBS colonization during pregnancy. Some studies have suggested that anogenital colonization by GBS is sexually transmitted [28, 29]. However, GBS colonization rate in this study was not affected by sexual behavior.
Antibiotic sensitivity pattern of the GBS isolates
The most efficient management option available at present in GBS colonized patients is intrapartum chemoprophylaxis with parenteral antibiotics[10, 30]. Antibiotic susceptibility patterns of GBS isolated from the subjects were studied. A total of 36 strains were tested for susceptibility to antibiotics that have been recommended for eradication of carriage and treatment of invasive GBS diseases [31]. Using disk diffusion technique, GBS isolates showed susceptibility to ampicillin (86.6%), penicillin (80.6%) and erythromycin (41.7%); while susceptibility to cefotaxime, vancomycin and clindamycin were found to be much lower. This differs from the report of Tazi et al. [32] that described a high susceptibility to these antibiotics and low-level of resistance to aminoglycosides. In fact, there has been a reported increase in the resistance rate of GBS to erythromycin and clindamycin from different parts of the world [33–35], with a study showing that black ethnicity and serotype V were the strongest predictors of colonization with an erythromycin- or clindamycin resistant GBS strain [35]. These variable results are likely due to multiple factors including antimicrobial usage, prophylaxis practice and serotype frequency from different regions [36]. A limitation of this study was the inability to determine the MIC of isolates and also assess antibiotic resistance at the molecular level.
Association between CD4 count and the prevalence of GBS colonization
GBS carriage was observed in more subjects with higher CD4 count (>250 cells/mm3) than those with lower CD4 count, although the colonization rates did not differ significantly. As most of the participants in this study were on antiretroviral drugs, this could have accounted for the relatively high CD4 count observed. According to a study done in Malawi [25], GBS carriage was significantly increased at higher CD4 counts but other investigations have not demonstrated any association between CD4 counts and anogenital colonization by GBS.
Conclusion
A relatively high prevalence of GBS colonization was observed in our institution which confirms the need for effective routine screening during pregnancy, so that intrapartum antimicrobial prophylaxis can be offered to carriers. In this cohort, colonization rates were independent of the HIV status but associated with a high BMI in HIV positive women. Larger population based studies of GBS colonization rate among HIV positive pregnant women, taking into account serotype distribution, and treatment protocols are recommended.
Disclosure
The authors declare no conflicts of interest.
Footnotes
Acknowledgments
The authors would like to acknowledge all the residents from the department of Obstetrics and Gynecology that assisted in the recruitment of subjects for the study.