Menu
Menu
Menu
Menu
Introduction: Group B Streptococcus (GBS) is a leading cause of neonatal sepsis and meningitis. We aimed to evaluate the burden of invasive early-onset (0-6 days of life, EOD) and late-onset (7-89 days, LOD) GBS disease and subsequent neurological sequelae in infants from a setting with a high prevalence (29.5%) of HIV among pregnant women. Methods: A case-control study was undertaken at three secondary-tertiary care public hospitals in Johannesburg. Invasive cases in infants <3 months age were identified by surveillance of laboratories from November 2012 to February 2014. Neurodevelopmental screening was done in surviving cases and controls at 3 and 6 months of age. Results: We identified 122 cases of invasive GBS disease over a 12 month period. Although the incidence (per 1,000 live births) of EOD was similar between HIV-exposed and HIV-unexposed infants (1.13 vs. 1.46; p = 0.487), there was a 4.67-fold (95%CI: 2.24-9.74) greater risk for LOD in HIV-exposed infants (2.27 vs. 0.49; p 350 cells/mm3 and WHO stage 1 and 2 received antiretroviral prophylaxis with zidovudine (AZT); whilst those with CD4+ lymphocyte count ≤350 cells/mm3 or WHO stage 3 or 4 were initiated on triple antiretroviral therapy (ART). From April 2013, all pregnant women irrespective of CD4+ lymphocyte count were initiated on ART [16, 17]. Invasive GBS disease (cases) were defined as an infant <90 days of age in whom GBS was cultured from blood, CSF or other normally sterile sites; or when GBS was identified in CSF by latex agglutination. Cases were identified by ZD through daily surveillance of the pediatric wards and microbiology services at the three hospitals. Early-onset disease (EOD) was defined when GBS was isolated in infants younger than seven days of life, and infants between 7–89 days of age with GBS disease were regarded as having late-onset disease (LOD). Control subjects were matched for: (i) gestational age to term, or within 2 weeks for cases born 7 days of life) of chronological age for LOD cases. Controls for EOD were selected from admission and labor wards at CHBAH, whereas controls for LOD were identified through the birth registries and contacted telephonically for possible study-enrolment. For cases born at ≥34 weeks gestational age, at least 5 controls (mean: 7; range: 5–14) were matched for EOD and 3 controls (mean: 5; range: 3–7) for LOD. For cases born at <34 weeks gestational age, at least one control (mean: 2; range: 1–5) was matched for EOD and at least one control (mean: 2, range: 1–4) for LOD. All controls were clinically well at enrolment, and followed up to confirm they did not develop invasive GBS disease. Cases and controls were followed up at 3 and 6 months of the infant’s chronological age. These visits were carried out by either one of three trained research assistants or by ZD. At these visits, the infant’s underwent neurological and development examinations and were screened using the Denver Developmental Screening Test II (Denver-II). The Denver-II makes a valuable screening tool (83% sensitivity) with a high degree of test-retest and inter-examiner reliability [18, 19]. The Denver-II tests 4 domains; gross-motor, fine-motor, language and personal-social. Each test item is represented horizontally as a percentile age range (25–90%) for which it is normally estimated that the item can be achieved. A “fail” or “refusal” by the infant in an item to the left of the age line is classified as a “delay”, whilst a “fail” or “refusal” by the infant in an item through the 75–90% age percentile is classified as a “caution”. The final result was then scored as “normal” (no delays or 1 caution) or “suspect/abnormal” (≥2 cautions or ≥1 delay) in each of the four domains. We defined neurological sequelae as an abnormal Denver-II developmental screening test for any of the four domains or hypertonia and/or hyper-reflexia detected on examination. Infants with developmental delay were referred to occupational, physical and/or speech therapists. Visual and hearing assessments were not routinely tested on participants. GBS was isolated from blood samples using the Bact/Alert microbial system (Organon Teknika, Durham, NC). Positive specimens were subsequently plated on blood or chocolate agar incubated both aerobically and at 35 degrees under 5–10% CO2, and observed for colony growth for 72 hours. Gram-staining was performed on CSF samples, which were also plated onto blood or chocolate agar plates, inoculated into an enrichment broth (Brain Heart Infusion, Diagnostics Media Production) and observed for colony growth for 72 hours. Specimens were also analyzed by a GBS antigen agglutination test if the CSF cell counts were suggestive of bacterial meningitis. Positive GBS isolates were serotyped and stored. Although screening for maternal GBS colonization is not a routine investigation in Johannesburg, maternal colonization status was determined for participants enrolled in the study by separately swabbing the lower vagina and rectum using Rayon tipped swabs and charcoal-free Amies transport medium (Medical Wire Equipment Co. Ltd. Cat: MW170). In addition, a mid-stream urine specimen was also cultured. Mothers of cases and controls were swabbed at the time of enrolment, while controls matched to EOD were swabbed immediately after delivery. Swabs were plated onto CHROMAgar StrepB plates (Media Mage Cat: {"type":"entrez-nucleotide","attrs":{"text":"M10155","term_id":"1059793855"}}M10155) which were incubated at 37°C for 18–24 hours in aerobic conditions and examined for growth of mauve GBS-like colony morphologies. Identified colonies were subjected to further confirmatory tests, such as the catalase test, growth on bile esculin agar, inability to hydrolyze esculin, Christie Atkinson Munch-Petersen (CAMP) test and B antigen latex agglutination test [20]. Serotyping for GBS types Ia, Ib, II to IX was performed using latex agglutination (Statens Serum Institute, SSI, Sweden) [21]. Non-typeable and discordant isolates were further characterized by a single-plex PCR method for serotypes Ia, Ib, II, III, IV and V using primer sequences described by Poyart et al. [22]. The incidence (per 1,000 live births) of invasive GBS disease over a twelve month period was calculated as the number of cases (EOD or LOD) in black-African infants that specifically resided in regions D and G of the Johannesburg metropolitan area. We only included black African infants with GBS disease residing in these specified regions because the care-givers of these infants predominantly access health care at either CHBAH or RMMCH. We did not undertake incidence calculation for non-black African infants or black-African infants not residing in regions D and G because these infants were likely to utilize other health care facilities not under surveillance in the study. There were 31504 live births over 12 months in regions D and G; 8827 (28%) infants were born to HIV-infected women [23]. For proportions, Chi-square or Fischer’s exact test were used to compare demographic and clinical characteristics between cases of EOD and LOD. Medians were reported for non-parametric variables and compared using the Wilcoxon rank-sum (Mann-Whitney) test. Serotype distributions were reported as proportions of the total number of cases serotyped and stratified by EOD and LOD. Univariate analysis was used to identify risk factors for invasive GBS disease, predictors of infant mortality and to compare neurological sequelae. For the multivariate analysis, adjusted odds ratios (aOR) using conditional logistic regression was used to adjust for variables with p-values <0.15 detected by univariate analysis. For the identification of risk factors predisposing to invasive GBS disease, we also included gestational age, maternal age and HIV status. For neurological sequelae, we adjusted for factors that may impact on neurodevelopment; including, gender, gestational age, birth weight <2500 grams, perinatal asphyxia, mechanical ventilation, infant HIV-exposure status and previous non-GBS-related hospitalizations. Data was analyzed using STATA version 13.1 (College Station, Texas, USA). Two-tailed p-values <0.05 were considered statistically significant. The study was approved by the University of Witwatersrand Human Research Ethics Committee (HREC number: M120963). Written informed consent was obtained from mothers of infants at enrolment for participation in the study.
