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Group B streptococcal colonization and transmission dynamics in pregnant women and their newborns in Nigeria: implications for prevention strategies
Accepted Manuscript Group B streptococcal colonization and transmission dynamics in pregnant women and their newborns in Nigeria: implications for prevention strategies Nubwa Medugu, F.W.A.C.P., Kenneth C. Iregbu, F.M.C.Path, Robert E. Parker, Ph.D., Jessica Plemmons, Pallavi Singh, Ph.D., Lamidi I. Audu, F.M.C.Paed, Efena Efetie, F.W.A.C.P., H. Dele Davies, M.D., Shannon D. Manning, Ph.D. PII:
S1198-743X(17)30126-X
DOI:
10.1016/j.cmi.2017.02.029
Reference:
CMI 877
To appear in:
Clinical Microbiology and Infection
Received Date: 20 October 2016 Revised Date:
24 February 2017
Accepted Date: 25 February 2017
Please cite this article as: Medugu N, Iregbu KC, Parker RE, Plemmons J, Singh P, Audu LI, Efetie E, Davies HD, Manning SD, Group B streptococcal colonization and transmission dynamics in pregnant women and their newborns in Nigeria: implications for prevention strategies, Clinical Microbiology and Infection (2017), doi: 10.1016/j.cmi.2017.02.029. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Original Article
Group B streptococcal colonization and transmission dynamics in pregnant women and
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their newborns in Nigeria: implications for prevention strategies
Nubwa Medugu, F.W.A.C.P.1, Kenneth C. Iregbu, F.M.C.Path1, Robert E. Parker, Ph.D.2,
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Jessica Plemmons2, Pallavi Singh Ph.D. 2, Lamidi I. Audu, F.M.C.Paed3,
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Efena Efetie, F.W.A.C.P. 4, H. Dele Davies, M.D.5, and Shannon D. Manning, Ph.D.2*
Departments of 1Medical Microbiology and Parasitology, 3Paediatrics, and 4Obstetrics and Gynaecology, National Hospital Abuja, Abuja, Nigeria; 2Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, U.S.A.; Department of Pediatrics, University of Nebraska Medical Center, Omaha, NE, U.S.A.
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Running title: GBS colonization and disease in Nigeria
*Corresponding author
Shannon D. Manning, Department of Microbiology and Molecular Genetics, Michigan State University, 1129 Farm Lane, East Lansing, MI 48824 Phone: (517) 884-2033; Email: [email protected]
Word count: Abstract (226 words); text (3,385 words)
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ACCEPTED MANUSCRIPT ABSTRACT
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Objectives: Because few studies have been conducted on group B Streptococcus (GBS) in
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Nigeria, we sought to estimate GBS colonization and transmission frequencies for 500
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women and their newborns and identify risk factors for both outcomes.
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Methods: GBS strains were characterized for antibiotic susceptibilities, capsule (cps)
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genotype, pilus island profile, and multilocus sequence type (ST).
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Results: In all, 171 (34.2%) mothers and 95 (19.0%) of their newborns were colonized with
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GBS; the vertical transmission rate was 48.5%. One newborn developed early onset disease,
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yielding an incidence of 2.0 cases per 1,000 live births (95% confidence interval (CI): 0.50-
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7.30). Rectal maternal colonization (Odds ratio (OR): 26.6; 95% CI: 13.69-51.58) and
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prolonged rupture of membranes (OR: 4.2; 95% CI: 1.03-17.17) were associated with
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neonatal colonization, while prolonged membrane rupture (OR: 3.4; 95% CI: 1.04-11.39) and
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young maternal age (OR: 2.0; 95% CI: 1.22-3.39) were associated with maternal
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colonization. Women reporting ≥4 intrapartum vaginal exams (OR: 6.1; 95% CI: 3.41-10.93)
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and douching (OR: 3.7; 95% CI: 2.26-6.11) were also more likely to be colonized. Twelve
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STs were identified among 35 mother-baby pairs with evidence of transmission; strains of
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cpsV ST-19 (n=9; 25.7%) and cpsIII ST-182 (n=7; 20.0%) predominated.
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Conclusions: These data demonstrate high rates of colonization and transmission in a
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population that does not utilize antibiotics to prevent neonatal infections, a strategy that
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should be considered in the future.
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INTRODUCTION Group B Streptococcus (GBS) colonization of the genitourinary tract occurs in 15-35% of pregnant women [1], who can transmit the pathogen to their neonate during childbirth and
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contribute to early onset disease (EOD) [1], a leading cause of sepsis and meningitis
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worldwide. Because maternal colonization is the primary risk factor, intrapartum antibiotic
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prophylaxis (IAP) is recommended for colonized women and women with premature rupture
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of membranes, prolonged membrane rupture (≥18 hours), fever, and preterm birth in the U.S.
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[2]. Although IAP has resulted in a 80% reduction of GBS EOD in the U.S. [3], not all
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countries have established prophylaxis protocols or accurate disease estimates.
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Earlier studies in South Africa estimated the incidence of EOD to be 2.1 cases per 1,000
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live births [4], prompting the recommendation for IAP. Recent reports estimate the incidence
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to be 1.1-1.5 cases per 1,000 live births depending on HIV status, though neither IAP or GBS
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screening are routine [5]. In Nigeria, prior studies have reported low rates of EOD [6–9];
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however, the detection methods utilized previously have not been sufficient and therefore,
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EOD incidence has likely been underestimated. To better understand the epidemiology of
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GBS in Nigeria, we conducted a longitudinal study of 500 pregnant women and their
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neonates to calculate disease incidence, identify risk factors for colonization and
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transmission, and characterize the strains using molecular methods. Such studies are
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warranted to guide disease prevention practices in developing countries where underreporting
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is a concern, IAP use is not common, and neonatal morbidity and mortality rates are high.
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ACCEPTED MANUSCRIPT METHODS
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Study population and design. In this longitudinal study, 500 women delivering at National
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Hospital Abuja, Kubwa General Hospital, Garki General Hospital and Wuse General
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Hospital in Abuja, Nigeria were enrolled May-September, 2014. Based on a 40% prevalence
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of maternal GBS colonization, a sample of 500 provided sufficient power (>80%) to detect
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associations. Ethical approval was granted by the Federal Capital Territory Health Research
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Ethics Committee and review boards of each hospital.
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Women were eligible if in their third trimester and they provided informed consent.
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Assent from a parent/guardian was required for women <18 years of age. Exclusion criteria
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included: multifetal gestation, placenta previa or elective Caesarean sections. Newborns of
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eligible mothers were enrolled and followed for ~12 weeks. Epidemiological data were
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collected through interviews, questionnaires and hospital records.
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Rectal and vaginal samples were collected before vaginal examinations. Vaginal swabs
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were inserted into the lower third of the vagina and rotated 360° while touching the vaginal
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walls. Rectal swabs were inserted ~2 cm beyond the anal sphincter and rotated to touch the
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anal crypts. The external auditory meatus was swabbed in neonates within 2-24 hours of birth
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to prevent contamination from colonized mothers during childbirth.
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Swabs were inoculated in StrepB Carrot (Hardy Diagnostics) or LIM broth and
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transported to the National Hospital Abuja laboratory for culture within four hours. No
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women received IAP regardless of the GBS culture results. Blood cultures from newborns
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were transported within four hours for BACTEC 9050 testing (Becton Dickinson). When
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GBS status was unknown, babies were considered to have signs of sepsis if presenting with:
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fever, hypothermia, respiratory distress, irritability, lethargy, and appetite loss.
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ACCEPTED MANUSCRIPT GBS identification and susceptibility testing. Samples were subcultured on CHROMagar
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StrepB; suspect colonies were confirmed using the PathoDxtra Strep Grouping Kit (Thermo
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Fisher Scientific). Susceptibilities to penicillin G (10U), ampicillin (10µg), clindamycin
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(2µg), erythromycin (15µg), vancomycin (30µg), ofloxacin (5µg), and ceftriaxone (30µg)
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were determined by disk diffusion using published breakpoints [10]; Streptococcus
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pneumoniae ATCC 49619 was the control. Inducible clindamycin resistance was determined
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using the “D-test” as described [10] and multidrug resistance was defined as resistance to >1
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antimicrobial class.
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Serotyping and multilocus sequence typing (MLST). DNA was extracted and cps types
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were determined as described [11]. Libraries were prepared for 70 strains from mother-baby
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pairs and one EOD strain using the Nextera XT kit (Illumina). Bioanalyzer (Agilent
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Technologies) and library quantification kits (Kapa Biosystems) were used to validate the
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libraries, which were pooled for denaturing and sequencing using the MiSeq (Illumina)
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(2x250). Sequences were assembled de novo using Velvet,1.2.07 [12] after trimming in
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Trimmomatic [13] and quality checking with FastQC (www.bioinformatics.babraham.
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ac.uk/projects/fastqc/). Sequence type (ST) and pilus island (PI) sequences were extracted
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using BLAST; PubMLST was used for allele and ST assignments (pubmlst.org/sagalactiae/).
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Data analysis. Statistical analyses were performed in SAS 9.3 (SAS Institute) using
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dichotomous variables. Univariate analyses were performed using χ2 tests with odds ratios
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(OR) and 95% confidence intervals (CI), and potential confounders and interactions were
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identified. The four primary outcomes included: maternal GBS colonization (n=171 of 500
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mothers), neonatal GBS colonization (n=95 of 500 neonates), sepsis development (n=21 of
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500 babies), and transmission (n=80 of 165 mothers and babies with molecular data).
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Multivariate analyses were performed using stepwise logistic regression. Significant explanatory variables were evaluated based on a chi-squared distribution using -2 LOG L and
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the Akaike and Schwartz Information Criterion, which examined the fit of each variable
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while adjusting for all variables and observations. Variables identified at P≤0.20 were
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evaluated using backward elimination to remove insignificant factors and solidify the base
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model. Wald CIs for adjusted ORs were calculated and other variables, which were selected
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for inclusion based on biological plausibility and univariate associations (P≤ 0.20), were
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added to each model. Caution was used to ensure that addition of each variable did not
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change the effect sizes of base model variables. The variance inflation factor was computed
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to ensure lack of multicollinearity and the Hosmer and Lemeshow test determined the
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goodness-of-fit [14]. The Wilson score index without continuity correction [15] was used to
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calculate the 95% CI around the EOD incidence estimate.
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Participant characteristics and GBS colonization frequencies. Enrolled women were 17-
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46 years of age (mean: 29.8 ±5 years) and most (61.0%) had a tertiary level of education,
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were married (98.0%), and had >2 prior pregnancies (80.0%). In all, 231 GBS isolates were
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recovered from the vagina, rectum or both sites in 171 women, yielding a maternal
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colonization rate of 34.2%; none of the women received IAP. The rectum was most
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commonly colonized (n=83; 48.5%), but some women had only vaginal colonization (n=28;
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16.3%) or were colonized at both sites (n=60; 35.1%). All four hospitals had similar
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populations with regard to age, marital status, parity and socioeconomic indicators, though
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colonization frequencies ranged between 26.0%-39.3% (P=0.15).
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Among the 500 babies of enrolled mothers, the majority were ≥37 weeks gestation (n=429; 85.8%) and ≥2.5 kg (n=401; 80.2%). Almost half of the neonates were male (n=239;
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47.8%) and 95 (19.0%) were colonized with GBS. One preterm male neonate developed GBS
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EOD at birth that was confirmed by culture. Three babies were stillborn and one died
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immediately after birth; no bacterial cultures were available. Two neonates died in the first
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month leaving 494 babies to follow for 12 weeks. Twenty-one of the 500 babies (4.2%)
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developed sepsis or had clinical signs of sepsis; all but four babies were admitted to the
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hospital. Among these 21 sepsis cases, one tested positive for GBS and three each were
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caused by Staphylococcus aureus and Klebsiella pneumoniae; the remainder were culture-
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negative.
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Associations with maternal GBS colonization. Following the univariate analysis (Table
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1), stepwise logistic regression identified maternal age, prolonged membrane rupture,
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douching, and number of vaginal exams to be associated with maternal GBS colonization.
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Preterm rupture of membranes was included in the based model given the correlation with
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ACCEPTED MANUSCRIPT prolonged membrane rupture (P<0.0001) and preterm birth (P<0.04). Women reporting ≥4
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vaginal exams and douching were 6.1 (95% CI: 3.41-10.93) and 3.7 (95% CI: 2.26-6.10)
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times more likely to be colonized, respectively, while women ≤25 years were twice (95% CI:
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1.22-3.39) as likely. Similarly, women with prolonged membrane rupture were more
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frequently colonized (OR: 3.4; 95% CI: 1.04-11.39), though no associations were observed
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for gestational diabetes, abnormal discharge, and having a neonate with sepsis. Women
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reporting ≥4 vaginal exams were more likely to have a preterm birth (OR: 2.1; 95% CI: 1.16-
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3.96) and more frequently developed premature (OR: 1.9; 95% CI: 0.78-4.60) and prolonged
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membrane rupture (OR: 2.6; 95% CI: 0.88-7.58), while women ≤25 years more commonly
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had preterm rupture (OR: 2.3; 95% CI: 1.02-5.01). Douching was not independently
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associated with any other variables in the model.
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Associations with neonatal colonization. Several factors were associated with neonatal
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colonization in the univariate analysis (Table 2). Colonized mothers were ~92 times (95%
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CI: 32.97-259.07) more likely to have a colonized neonate. Among the 91 (95.8%) colonized
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neonates born to colonized mothers, most had mothers with only rectal colonization (n=48;
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50.5%) followed by vaginal/rectal colonization (n=34; 35.8%), and vaginal colonization only
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(n=9; 9.5%). Four colonized neonates were born to women without GBS, while 80 (46.8%)
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colonized women had GBS-negative neonates.
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Stepwise logistic regression identified maternal colonization, number of vaginal exams,
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and prolonged membrane rupture to be most strongly associated with neonatal GBS
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colonization after adjusting for maternal age and premature membrane rupture (Table 3).
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Women receiving ≥4 vaginal exams and with prolonged membrane rupture were 3.0 times
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(95% CI: 1.52-5.97) and 4.2 times (95% CI: 1.03-17.17) more likely to have a colonized
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neonate. Maternal rectal GBS colonization was also important (OR: 26.6; 95% CI: 13.69-
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51.58), though baby gender, preterm birth, maternal age and douching were not.
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Associations with sepsis onset before 12 weeks of age. Using variables identified in the
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univariate analysis (Table 2), stepwise logistic regression identified young age, abnormal
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vaginal discharge, preterm birth, and low Apgar scores to be associated with sepsis (Table 3).
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Apgar scores ≤ 7 after 5 minutes yielded the strongest association (OR: 8.2; 95% CI: 3.00-
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22.30) followed by history of abnormal vaginal discharge (OR: 6.4; 95% CI: 2.08-19.79) and
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preterm birth (OR: 4.9; 95% CI: 1.64-14.55). Women ≤25 years of age and women with
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prolonged membrane rupture were ~3 times more likely to have a baby develop sepsis,
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though only maternal age was significant (P = 0.03). Abnormal vaginal discharge was
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independently associated with prolonged membrane rupture (OR: 6.8; 2.47-18.77) and low
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Apgar scores (OR: 2.3; 95% CI: 1.13-4.88).
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Phenotypic characteristics of GBS strains. Molecular serotypes were determined for 227
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(98.3%) strains from the 171 colonized women. Strains with cpsV (n=74, 43.3%), cpsII
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(n=39, 22.8%), cpsIa (n=22, 12.9%) and cpsIII (n=18, 10.3%) predominated. Three women
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(1.8%) had cpsIV, seven (4.1%) had non-typeable strains, and one had a strain with a unique
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RFLP pattern (cpsV variant). Most (78.3%) of the 60 women colonized in both the rectum
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and vagina had identical cps types, though nine women had discrepant types. The cps
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distribution was similar for the 95 neonates with cpsV (n=37, 39.0%), cpsII (n=21, 22.1%),
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cpsIa (n=12, 12.6%) and cpsIII (n=11, 11.6%) predominating.
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Resistance to clindamycin, erythromycin and ofloxacin was observed in 46 (26.9%), 34
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(19.9%) and 15 (8.8%) of the colonized mothers and 24 (25.3%), 13 (13.7%) and 6 (6.3%) of
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colonized neonates, respectively. All strains were susceptible to penicillin, ceftriaxone and
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ACCEPTED MANUSCRIPT vancomycin and no inducible resistance to clindamycin was observed. Mothers were not
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more likely to have resistant GBS than neonates (P=0.29) nor were they more likely to have
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multidrug resistant strains (P=0.41). Twenty-eight (16.4%) mothers and 12 (12.6%) neonates
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had strains resistant to >1 antibiotic and maternal cpsV strains were 2.5 times more likely to
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be resistant relative to other types (95% CI: 1.34-4.74).
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Vertical transmission. All but five mothers had the same cps types when compared to their
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neonates, yielding a vertical transmission rate of 48.5% (n=80) among the 165 colonized
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mothers with cps data available. Although cpsIII strains were most commonly transmitted,
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the difference was not statistically significant when comparing it to all other cps types
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(P=0.23). When maternal colonization site was examined, the transmission rate for rectal
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colonization (52.2%) was greater than vaginal colonization alone (29.6%) (P=0.03).
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Stepwise logistic regression adjusting for factors identified in the neonatal colonization model found ≥4 vaginal exams (OR: 5.2; 95% CI: 2.39, 11.13) and prolonged membrane
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rupture (OR: 4.0; 95% CI: 1.43, 11.10) to be associated with transmission of the same GBS
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strain. The likelihood of transmission increased according to the time of membrane rupture.
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The transmission rate was 66.7% for women delivering ≥1 hour before term, which increased
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to 83.3% for women experiencing ≥18 hours of ruptured membranes regardless of the
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gestational age. The association with premature rupture (OR: 2.0; 95% CI: 0.59-7.08),
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however, was not significant in the model.
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Thirty-five of the 80 mother-baby pairs with matching cps types were examined by
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MLST and PI profiling. Twelve STs were identified that grouped into five previously defined
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clonal complexes (CCs) [16] and one singleton (Figure 1). ST-19 predominated (n=9, 25.7%)
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followed by ST-182 (n=7, 20.0%), and ST-1 (n=4, 11.4%). Three pairs were colonized with
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ST-17 (8.6%) as well as ST-23 (8.6%), while STs 2, 8, 24, 26, 196, and 762, a novel ST
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ACCEPTED MANUSCRIPT identified in this population, were each found in one pair. Among the 11 pairs with cpsIII
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strains, most (n=7) were ST-182 and three were ST-17; one pair only had a maternal strain
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(ST-182) available. The invasive EOD strain was classified as cpsIII ST-182, which clustered
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within CC-19. ST-19 strains containing cpsV comprised 18 of the 26 (69.0%) cpsV strains
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with MLST data available. The PI distribution was less variable among the pairs, with the
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majority of strains possessing both PI-I and PI-2a (n=27, 77.1%) followed by PI-2a only
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(n=5, 14.3%) and PI-2b only (n=3, 8.6%). Strains possessing only the PI-2b belonged
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exclusively to ST-17 strains as described [17]. All cpsV/ST-19 and cpsIII/ST-182 strains had
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both PI-1 and PI-2a.
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DISCUSSION A high prevalence of GBS colonization was detected in third trimester pregnant women receiving antenatal care (34.2%) and their neonates (19.0%) in Nigeria. The maternal
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colonization rate was ~2 times higher than prior Nigerian reports [18,19] and is higher than
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the 22.4% prevalence estimate from four African studies [20]. Although GBS prevalence
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varies geographically and temporally, rate differences could also be due to use of more
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sensitive detection methods [2]. The incidence of EOD was 2.0 cases per 1,000 live births
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(95% CI: 0.50-7.30), which is similar to U.S. rates (1-3 cases/1,000) before maternal IAP was
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introduced [1]. Among the seven million births in Nigeria each year [21], we estimate that
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14,000 children will develop EOD with ~3,220 deaths and ~1,820 children with long term
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disabilities based on annual South African EOD mortality (23%) and long term sequelae
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(13%) estimates [5]. Consequently, it is recommended that GBS screening protocols and IAP
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be considered as a prevention strategy for Nigerian women.
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colonization and factors such as prematurity and premature membrane rupture [1]. Indeed,
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rectally colonized women were significantly more likely to have colonized neonates, which
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contradicts a study reporting higher transmission rates in mothers with genital versus rectal
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colonization [22]. Another study found GBS colonization of the rectum to be a predictor of
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vaginal colonization and women with only rectal colonization may become vaginally
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colonized during childbirth, thereby enhancing transmission risk to the neonate [24].
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Sampling neonates 2-24 hours after birth was performed to ensure that colonization of the
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neonate was not due to maternal contamination; however, it is possible that neonatal
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colonization frequencies were overestimated since other sites that more accurately represent
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colonization were not examined. Differences in neonatal colonization rates could also be
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attributable to variation in maternal colonization densities, which was not addressed and
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represents a study limitation. Moreover, the finding that some colonized neonates were born
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to GBS-negative mothers suggests that some mothers had low colonization densities that
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precluded detection or neonates acquired GBS from external sources.
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Young maternal age has been linked to colonization previously, [25] which could be due to higher levels of sexual activity and less stable relationships. The finding that 67% of
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women not living with their partners were 16-25 years old provides support for the latter.
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Associations have also been observed between young age and EOD [1], which could be due
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to lower levels of circulating antibodies and is consistent with our observed association
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between young age and sepsis development. Low Apgar scores and preterm birth were also
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associated with sepsis and supports studies that have identified preterm birth as the primary
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risk factor for late onset sepsis caused by many pathogens [26,27]. History of abnormal
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vaginal discharge was also associated with sepsis and may represent a marker for
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chorioamnionitis, a risk factor for both preterm birth and sepsis [28], that has also been linked
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to GBS. Still, 26 of 52 women reporting discharge had GBS and five had preterm birth and
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one was <25 years. One limitation, however, is that the definition of sepsis may have differed
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across hospitals since 17 (81%) of the 21 cases were diagnosed at one site. Indeed, the
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hospital with the most cases more frequently identified culture-negative cases (Fisher’s exact
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test P = 0.025), which could contribute to misclassification bias.
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Neonatal colonization was associated with prolonged membrane rupture (>18 hrs). The
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higher transmission rates associated with prolonged versus preterm rupture of membranes
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support the hypothesis that transmission risk increases as the time of GBS exposure increases
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inside the maternal genital tract [2]. Because choriodecidual infection with GBS in a
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nonhuman primate model was found to contribute to premature rupture of membranes [30],
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which was associated with prolonged membrane rupture in our study, it is important to
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identify factors for both conditions as well as GBS colonization. Behavioural factors like
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colonization, could cause alterations in the vaginal microbiota or disruption of the vaginal
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mucosa to promote colonization. Since colonization frequencies differed among the douche
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preparations, it is possible that each type has varying effects on the vaginal milieu. Increased
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education of factors that impact colonization during pregnancy is important in Nigeria and
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elsewhere, particularly given that a greater number of intrapartum vaginal exams and
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prolonged membrane rupture were also risk factors for transmission and neonatal
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colonization, the first steps in disease.
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GBS strain diversity was low compared to other studies and ST-182 has not been previously reported in high frequency among cpsIII strains. Similarly, ST-19 strains
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possessing cpsV, a unique ST/cps combination, has been infrequently reported. A close
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evolutionary relationship was previously observed between the cpsV strain 2603V/R and ST-
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19 [31], suggesting the importance of capsule switching in the generation of new strain types
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that impact colonization frequencies. The low prevalence of ST-17, a lineage associated with
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neonatal disease [16], also suggests that ST-17 frequencies vary by location, thereby
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impacting disease incidence. Indeed, a 25-year GBS EOD study in the Netherlands identified
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a significantly lower frequency of ST-17 infections before IAP use [32], which may be due to
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variation in the ability of ST-17 strains to tolerate antibiotics and persist following IAP [25].
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Because GBS strain types were equally likely to be transmitted, continuous monitoring in
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Nigeria will enhance our knowledge of pathogen evolution after prevention practices are
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established. Surveillance efforts are also needed to ensure that antibiotic resistance rates do
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not increase in second-line agents and that penicillin-resistance does not emerge.
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Based on our findings, use of penicillin or vancomycin would be appropriate for IAP in
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women with GBS colonization, prolonged rupture of membranes and/or preterm birth, which
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was linked to late-onset sepsis. Other factors including young age and abnormal vaginal
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ACCEPTED MANUSCRIPT discharge should also be considered, though several challenges must be overcome prior to
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implementing a universal screening protocol. Challenges include shortages of antenatal care
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facilities, late presentation to the delivery ward, limited laboratory capacity, delayed
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dissemination of laboratory results, and limited availability and technicalities associated with
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intravenous drug administration. While a risk-based screening approach may be easier, ~348
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(69.6%) of GBS-colonized women would have been missed because they lacked premature
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rupture of membranes, preterm birth or fever. Antibiotics would have also been unnecessarily
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administered to 326 (65.2%) of GBS-negative women with these risk factors.
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In Nigeria, a universal GBS screening strategy with IAP would cost ₦300,000 to implement for 500 women using the same detection methods, which is less than the average
296
cost of managing a newborn with EOD (₦500,000) or long-term sequelae (₦1,000,000) in
297
surviving children. Alternative prevention strategies including a GBS vaccine should also be
298
considered as a vaccine targeting PI-2a would cover 91% of the strains identified in our
299
study; coverage would increase if serotypes II, III, and V were targeted. Such prevention
300
practices could reduce EOD incidence in Nigeria as well as the frequency of preterm delivery
301
and premature/prolonged rupture of membranes.
302
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Funding
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The study was primarily supported by the National Hospital Abuja in Nigeria [NM, KCI,
305
LIA, EE]. Additional support was provided by Michigan State University with salary support
306
from the Global Alliance to Prevent Prematurity and Stillbirth [N015615 to SDM]. The
307
authors declare no conflicts of interest.
308
Acknowledgements
309
We thank the many individuals who assisted with recruitment, data collection, and laboratory
310
work.
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References
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[19] Uhiara JE. Group B streptococcal carriage among parturients and their neonates in Zaria, Nigeria. Afr J Med Med Sci 1993;22:79–83. [20] Kwatra G, Cunnington MC, Merrall E, Adrian P V., Ip M, Klugman KP, et al. Prevalence of maternal colonisation with group B Streptococcus: a systematic review
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[27] Downey LC, Smith PB, Benjamin DK. Risk factors and prevention of late-onset sepsis in premature infants. Early Hum Dev 2010;86. [28] Galinsky R, Polglase GR, Hooper SB, Black MJ, Moss TJM. The consequences of chorioamnionitis: preterm birth and effects on development. J Pregnancy 18
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2013;2013:412831. [29] Yancey MK, Duff P, Clark P, Kurtzer T, Frentzen BH, Kubilis P. Peripartum infection associated with vaginal group B streptococcal colonization. Obstet Gynecol
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[30] Vanderhoeven JP, Bierle CJ, Kapur RP, McAdams RM, Beyer RP, Bammler TK, et al.
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[31] Davies HD, Jones N, Whittam TS, Elsayed S, Bisharat N, Baker CJ. Multilocus
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sequence typing of serotype III group B Streptococcus and correlation with pathogenic
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potential. J Infect Dis 2004;189:1097–102.
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[32] Bekker V, Bijlsma MW, van de Beek D, Kuijpers TW, Van der Ende A. Incidence of invasive group B streptococcal disease and pathogen genotype distribution in newborn
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babies in the Netherlands over 25 years: A nationwide surveillance study. Lancet
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Infect Dis 2014;14:1083–9.
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Table 1. Factors associated with maternal group B streptococcal colonization in the univariate
397
and multivariate analyses. No. of women
Characteristic
GBS positive No. (%)
Univariate OR (95% CI)
Multivariate⊥ ⊥ OR (95% CI)
95 349 56
41 116 14
(43.2) (33.2) (25.0)
2.3 (1.10-4.72) 1.5 (0.82-2.97) 1.0
Marital Status Married/living together Single/separated/divorced
494 6
168 3
(34·0) (50·0)
1.0 1.9 (0.39-9.72)*
Highest level of education Never attended Primary Secondary Tertiary
15 17 161 307
5 8 50 108
(33·3) (47·1) (31·1) (35·2)
Type of toilet Water cistern Aqua privy Pit toilet
429 63 8
146 20 5
(34·0) (31·8) (62·5)
1.0 0.9 (0.5-1.6) 3.2 (0.76-13.71)*
History of douching No Yes
162 338
28 143
(17.3) (42.3)
1.0 3.5 (2.21-5.56)
162 118 173 22 25
28 50 77 11 5
(17.3) (42.4) (45.0) (50.0) (20.0)
1.0 3.5 (2.04-6.08) 3.8 (2.31-6.37) 4.8 (1.89-12.13) 1.2 (0.41-3.46)*
Maternal medical history No. of prior pregnancies 1 2 ≥3
98 141 261
35 57 79
(35.7) (40.4) (30.0)
1.0 1.2 (0.72-2.08) 0.8 (0.48-1.28)
HIV Positive No Yes
479 21
162 9
(33.8) (42.9)
1.0 1.5 (0.6-3.56)
-
Prior preterm rupture of membranes No Yes
462 38
157 14
(34.0) (36.8)
1.0 1.1 (0.57-2.25)
-
Prior stillbirth or baby death No Yes
467 33
154 17
(33.0) (51.5)
1.0 2.2 (1.06-4.39)
1.3 (0.59-2.99)
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0.9 (0.31-2.76) 1.6 (0.61-4.37) 0.8 (0.55-1.25) 1.0
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Type of douche used None Antiseptic solution Bath soap and water Salt and water solution Native preparation
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Demographics, lifestyle, behaviors Age group (years) 17-25 26-35 ≥36
2.0 (1.22-3.39) -
-
-
-
3.7 (2.26-6.11)
-
-
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136 7
(33.4) (38.9)
1·0 1.3 (0.48-3.34)
-
Previous preterm delivery No Yes
414 86
138 33
(33·3) (38·4)
1.0 1.2 (0.74-2.06)
-
Current pregnancy history and complications No. of intrapartum vaginal exams 0-3 ≥4
428 72
120 51
(28.0) (70.8)
1.0 6.2 (3.60-10.11)
6.1 (3.41-10.93)
Antibiotic use in past 3 months No Yes
479 21
165 6
(34.5) (28.6)
1.0 0·7 (0.29-1.20)
-
448 52
145 26
(32.4) (50.0)
Intrapartum fever No Yes
468 32
470
160 11
(33.2) (61.1)
1.0 3.2 (1.20-8.31)
159 12
(34.0) (37.5)
1.0 1.2 (0.56, 2.45)
155
(33.0)
1.0
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Preterm rupture of membranes (>1 hr) No Yes
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1.0 2.1 (1.17-3.73)
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Abnormal vaginal discharge in the past 3 mos. No Yes Gestational Diabetes No Yes
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Prior baby with sepsis No Yes
1.8 (0.93, 3.46)
2.4 (0.83-7.10)
-
30
16
(53.3)
2.3 (1.11-4.88)
1.8 (0.78, 4.35)
483 17
159 12
(32.9) (70.6)
1.0 4.9 (1.69-14.12)
3.4 (1.04-11.39)
Prolonged labor (>18 hrs) No Yes
487 13
162 9
(33.3) (65·0)
1.0 4.9 (1.69-14.12)
2.2 (0.56-8.87)
Gestational age at birth < 37 weeks (preterm) ≥ 37 weeks (term)
71 429
34 137
(47.9) (31.9)
2.0 (1.18-3.25) 1.0
1.4 (0.81-2.61)
Birth weight ≤ 2.5 kg > 2.5 kg
99 401
41 130
(41.4) (32.4)
1.5 (0.94-2.31) 1.0
1.1 (0.68-1.88)
Apgar score (5 minutes) >7 ≤7
443 57
147 24
(33.2) (42.1)
1.0 1.5 (0.84-2.57)
1.1 (0.58-2.13)
Baby with sepsis within the 12 week follow up period No Yes
479 21
157 14
(32.8) (66.7)
1.0 4.2 (1.62-10.37)
2.4 (0.84-7.05)
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Prolonged rupture of membranes (> 18 hrs) No Yes
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399
Note: Not all denominators are equivalent as some data were missing.
400
*Fisher’s exact test was used for variables with ≤ 5 in at least one cell.
401
⊥ Stepwise logistic regression and backward elimination were used to identify explanatory
402
variables (age ≤ 25 years, ≥4 vaginal examinations, douching history, preterm rupture of
403
membranes and prolonged membrane rupture) to be included in the base model at P≤0.20
404
(Hosmer and Lemeshow test P=0.25). Age≤ 25 years was examined relative to both of the
405
other two age categories (≥26 years) combined. All additional variables were added separately
406
to this base model and ORs were adjusted for each of these five variables; adjusted ORs and
407
Wald confidence intervals are noted. Variables without a point estimate (-) were not examined
408
in the multivariate analysis because no association was identified with maternal GBS
409
colonization in the univariate analysis at P≤0.2.
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Table 2. Univariate analysis of factors associated with neonatal group B streptococcal
412
colonization and sepsis development up to 12 weeks following birth.
No. with characteristic
Characteristic
Neonatal GBS colonization GBS positive No. (%) OR (95% CI)
Sepsis signs or development Sepsis positive No. (%) OR (95% CI)
28 83 60
9 (32.1) 48 (57.8) 34 (56.7)
1.0 2.9 (1.17-7.16) 2.8 (1.07-7.09)
Age group (years) 17-25 26-35 ≥36
95 349 56
19 (20.0) 70 (20.1) 6 (10.7)
2.1 (0.78-5.58) 2.1 (0.86-5.07) 1.0
9 (9.5) 10 (2.9) 2 (3.6)
2.8 (0.59-13.58)* 0.7 (0.17-3.73)* 1.0
History of douching No Yes
162 338
13 (8.0) 82 (24.3)
1.0 3.7 (1.98-6.82)
4 (2.5) 17 (5.0)
1.0 2.1 (0.69-6.32)*
Type of douche used None Antiseptic solution Bath soap and water Salt and water solution Native preparation
162 118 173 22 25
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Maternal factors GBS colonization site Vagina Rectum Vagina and rectum
479 21
93 (34.5) 2 (28.6)
1.0 0·4 (0.10-1.91)*
19 (4.0) 2 (9.5)
1.0 2.5 (0.55-11.74)*
467 33
85 (18.2) 10 (30.2)
1·0 2.0 (0.90-4.26)
16 (3.4) 5 (15.2)
1.0 5.0 (1.72-14.74)*
407 18
73 (17.9) 3 (16.7)
1.0 0.9 (0.26-3.24)
16 (4.0) 3 (16.7)
1.0 4.9 (1.28-18.60)*
Abnormal vaginal discharge in the past 3 mos. No Yes
448 52
80 (17.) 15 (28.9)
1.0 1.9 (0.98-3.56)
13 (2.9) 8 (15.4)
1.0 6.1 (2.39-15.48)
Current pregnancy and neonatal factors Sex of neonate Female Male
261 239
36 (13.8) 59 (24.7)
1.0 2.0 (1.39-3.24)
10 (3.8) 11 (4.6)
1.0 1.2 (0.50-2.90)
Neonate colonized with GBS No Yes
405 95
-
12 (3.0) 9 (9.5)
1.0 3.4 (1.40-8.39)
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Prior baby with sepsis No Yes
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1.0 2.9 (1.42-6.02) 4.4 (2.28-8.49) 4.3 (1.44-12.86) 2.2 (0.65-7.32)*
Prior stillbirth or baby death No Yes
-
4 3 9 2 3
(2.5) (2.5) (5.2) (9.1) (12.0)
1.0 0.6 (0.15-2.79)* 0.8 (0.17-3.42)*
(8.0) (20.3) (27.8) (27.3) (16.0)
Antibiotic use in past 3 months No Yes
13 24 48 6 4
3 (10.7) 6 (7.2) 5 (8.3)
1.0 1.0 (0.23-4.69)* 2.2 (0.65-7.18)* 4.0 (0.68-22.96)* 5.4 (1.13-25.68)*
23
482 18
92 (19.1) 3 (16.7)
1.0 0.8 (0.24-2.99)*
18 (3.7) 3 (16.7)
1.0 5.2 (1.36, 19.42)*
No. of intrapartum vaginal exams 0-3 ≥4
428 72
56 (13.1) 39 (54.2)
1.0 7.9 (4.57-13.50)
15 (3.5) 6 (8.3)
1.0 2.5 (0.94-6.68)
Intrapartum fever No Yes
468 32
88 (18.8) 7 (21.9)
1.0 1.2 (0.51-2.88)
16 (3.4) 5 (15.6)
1.0 5.2 (1.8-15.35)*
Premature rupture of membranes (>1 hr) No Yes
470 30
83 (17.7) 12 (40.0)
1.0 3.1 (1.44-6.70)
18 (3.8) 3 (10.0)
1.0 2.8 (0.77-10.06)*
Prolonged rupture of membranes (>18 hrs) No Yes
483 17
85 (17.6) 10 (58.8)
17 (3.5) 4 (23.5)
1.0 8.4 (2.49-28.59)*
Prolonged labour (>18 hrs) No Yes
487 13
Gestational age at birth <37 weeks (preterm) ≥37 weeks (term)
71 429
Birth weight ≤ 2·5 kg > 2·5 kg
19 (3.9) 2 (15.4)
1.0 4.5 (0.93-21.63)*
20 (28.2) 75 (17.5)
1.9 (1.04-3.29) 1.0
10 (14.1) 11 (2.6)
6.2 (2.54-15.28) 1.0
99 401
22 (22.2) 73 (18.2)
1.3 (0.75-2.20) 1.0
10 (10.1) 11 (2.7)
4.0 (1.64-9.67) 1.0
443 57
80 (18.1) 15 (26.3)
1.0 1·6 (0.86-3.07)
10 (2.3) 11 (29.3)
1.0 10.4 (4.17-25.69)
86 (18.0) 9 (42.9)
1·0 3.4 (1.40-8.39)
479 21
-
-
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413
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1.0 5.3 (1.74-16.12)
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Baby with sepsis within the 12 week follow up period No Yes
1.0 6.7 (2.48-18.07)
88 (18.1) 7 (53·9)
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Apgar score (5 mins) >7 ≤7
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Gestational Diabetes No Yes
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No. = Number; OR= odds ratio; 95% CI = 95% confidence interval; P = P-value
415
Note: Not all denominators are equivalent as some data were missing.
416
*Fisher’s exact test was used for variables with ≤ 5 in at least one cell.
24
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Table 3. Multivariate analysis of factors associated with neonatal colonization and the
418
development of sepsis in the 12 weeks following birth. Neonatal GBS
Sepsis signs or
colonization
development
OR
95% CI
OR
95% CI
Maternal rectal GBS colonization*
26.6
13.69-51.58
1.4
0.50-3.96
Maternal age ≤ 25 years*⊥
0.9
0.41-1.77
3.1
1.10-8.82
History of douching
2.1
0.98-4.58
1.7
0.52-5.91
Prior stillbirth or baby death
0.8
0.28-2.28
3.6
0.90-14.25
Abnormal vaginal discharge⊥
1.2
0.51-3.02
6.4
2.08-19.79
≥4 intrapartum vaginal exams*
3.0
1.52-5.97
1.3
0.40-4.44
Intrapartum fever
0.8
0.24-2.61
3.4
0.92-12.80
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Characteristic
Prolonged rupture of membranes (>18 hrs)*⊥
4.2
1.03-17.17
3.0
0.68-13.03
Premature rupture of membranes (>1 hr)*⊥
1.7
0.60-5.04
1.3
0.30-5.48
Preterm birth (< 37 weeks)⊥
1.0
0.44-2.08
4.9
1.64-14.55
1.0
0.42-2.55
8.2
3.00-22.30
1.3
0.73-2.42
0.9
0.31-2.47
Prolonged labour (>18 hrs)
1.5
0.31-6.37
1.6
0.21-11.67
Low birth weight (≤ 2.5 kg)⊥
0.8
0.41-1.68
0.6
0.14-2.97
-
1.8
0.62-5.47
0.50-6.67
-
Apgar score ≤ 7 (5 mins)⊥
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Male gender
Neonatal GBS colonization
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Baby developed sepsis within 12 weeks
1.8
-
OR= odds ratio; 95% CI = 95% confidence interval; P = P-value
420
Note: Rectally colonized women included those with rectal only and rectal plus vaginal
421
colonization. For both outcomes (neonatal GBS colonization* and sepsis⊥), each model was
422
adjusted for the significant variables identified by stepwise selection to comprise the base
423
model; the Hosmer and Lemeshow P-value was >0.05 in each model examined. All
424
additional variables were added separately to each base model and were adjusted for each of
425
the variables included. The adjusted ORs and Wald confidence intervals are noted.
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ACCEPTED MANUSCRIPT Figure 1. Neighbor-joining phylogeny illustrating the type of group B Streptoccocus strains
428
shared between 35 mothers and their newborns. The number of mother-baby pairs with
429
specific sequence types (STs) is noted as well as the previously defined clonal complex (CC)
430
and distribution of cps types representing each lineage. S=singleton.
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ACCEPTED MANUSCRIPT
S1198-743X(17)30126-X
DOI:
10.1016/j.cmi.2017.02.029
Reference:
CMI 877
To appear in:
Clinical Microbiology and Infection
Received Date: 20 October 2016 Revised Date:
24 February 2017
Accepted Date: 25 February 2017
Please cite this article as: Medugu N, Iregbu KC, Parker RE, Plemmons J, Singh P, Audu LI, Efetie E, Davies HD, Manning SD, Group B streptococcal colonization and transmission dynamics in pregnant women and their newborns in Nigeria: implications for prevention strategies, Clinical Microbiology and Infection (2017), doi: 10.1016/j.cmi.2017.02.029. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Original Article
Group B streptococcal colonization and transmission dynamics in pregnant women and
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their newborns in Nigeria: implications for prevention strategies
Nubwa Medugu, F.W.A.C.P.1, Kenneth C. Iregbu, F.M.C.Path1, Robert E. Parker, Ph.D.2,
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Jessica Plemmons2, Pallavi Singh Ph.D. 2, Lamidi I. Audu, F.M.C.Paed3,
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Efena Efetie, F.W.A.C.P. 4, H. Dele Davies, M.D.5, and Shannon D. Manning, Ph.D.2*
Departments of 1Medical Microbiology and Parasitology, 3Paediatrics, and 4Obstetrics and Gynaecology, National Hospital Abuja, Abuja, Nigeria; 2Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, U.S.A.; Department of Pediatrics, University of Nebraska Medical Center, Omaha, NE, U.S.A.
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Running title: GBS colonization and disease in Nigeria
*Corresponding author
Shannon D. Manning, Department of Microbiology and Molecular Genetics, Michigan State University, 1129 Farm Lane, East Lansing, MI 48824 Phone: (517) 884-2033; Email: [email protected]
Word count: Abstract (226 words); text (3,385 words)
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ACCEPTED MANUSCRIPT ABSTRACT
2
Objectives: Because few studies have been conducted on group B Streptococcus (GBS) in
3
Nigeria, we sought to estimate GBS colonization and transmission frequencies for 500
4
women and their newborns and identify risk factors for both outcomes.
5
Methods: GBS strains were characterized for antibiotic susceptibilities, capsule (cps)
6
genotype, pilus island profile, and multilocus sequence type (ST).
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Results: In all, 171 (34.2%) mothers and 95 (19.0%) of their newborns were colonized with
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GBS; the vertical transmission rate was 48.5%. One newborn developed early onset disease,
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yielding an incidence of 2.0 cases per 1,000 live births (95% confidence interval (CI): 0.50-
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7.30). Rectal maternal colonization (Odds ratio (OR): 26.6; 95% CI: 13.69-51.58) and
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prolonged rupture of membranes (OR: 4.2; 95% CI: 1.03-17.17) were associated with
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neonatal colonization, while prolonged membrane rupture (OR: 3.4; 95% CI: 1.04-11.39) and
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young maternal age (OR: 2.0; 95% CI: 1.22-3.39) were associated with maternal
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colonization. Women reporting ≥4 intrapartum vaginal exams (OR: 6.1; 95% CI: 3.41-10.93)
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and douching (OR: 3.7; 95% CI: 2.26-6.11) were also more likely to be colonized. Twelve
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STs were identified among 35 mother-baby pairs with evidence of transmission; strains of
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cpsV ST-19 (n=9; 25.7%) and cpsIII ST-182 (n=7; 20.0%) predominated.
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Conclusions: These data demonstrate high rates of colonization and transmission in a
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population that does not utilize antibiotics to prevent neonatal infections, a strategy that
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should be considered in the future.
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INTRODUCTION Group B Streptococcus (GBS) colonization of the genitourinary tract occurs in 15-35% of pregnant women [1], who can transmit the pathogen to their neonate during childbirth and
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contribute to early onset disease (EOD) [1], a leading cause of sepsis and meningitis
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worldwide. Because maternal colonization is the primary risk factor, intrapartum antibiotic
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prophylaxis (IAP) is recommended for colonized women and women with premature rupture
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of membranes, prolonged membrane rupture (≥18 hours), fever, and preterm birth in the U.S.
28
[2]. Although IAP has resulted in a 80% reduction of GBS EOD in the U.S. [3], not all
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countries have established prophylaxis protocols or accurate disease estimates.
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live births [4], prompting the recommendation for IAP. Recent reports estimate the incidence
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to be 1.1-1.5 cases per 1,000 live births depending on HIV status, though neither IAP or GBS
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screening are routine [5]. In Nigeria, prior studies have reported low rates of EOD [6–9];
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however, the detection methods utilized previously have not been sufficient and therefore,
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EOD incidence has likely been underestimated. To better understand the epidemiology of
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GBS in Nigeria, we conducted a longitudinal study of 500 pregnant women and their
37
neonates to calculate disease incidence, identify risk factors for colonization and
38
transmission, and characterize the strains using molecular methods. Such studies are
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warranted to guide disease prevention practices in developing countries where underreporting
40
is a concern, IAP use is not common, and neonatal morbidity and mortality rates are high.
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Study population and design. In this longitudinal study, 500 women delivering at National
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Hospital Abuja, Kubwa General Hospital, Garki General Hospital and Wuse General
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Hospital in Abuja, Nigeria were enrolled May-September, 2014. Based on a 40% prevalence
45
of maternal GBS colonization, a sample of 500 provided sufficient power (>80%) to detect
46
associations. Ethical approval was granted by the Federal Capital Territory Health Research
47
Ethics Committee and review boards of each hospital.
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Women were eligible if in their third trimester and they provided informed consent.
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Assent from a parent/guardian was required for women <18 years of age. Exclusion criteria
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included: multifetal gestation, placenta previa or elective Caesarean sections. Newborns of
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eligible mothers were enrolled and followed for ~12 weeks. Epidemiological data were
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collected through interviews, questionnaires and hospital records.
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Rectal and vaginal samples were collected before vaginal examinations. Vaginal swabs
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were inserted into the lower third of the vagina and rotated 360° while touching the vaginal
55
walls. Rectal swabs were inserted ~2 cm beyond the anal sphincter and rotated to touch the
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anal crypts. The external auditory meatus was swabbed in neonates within 2-24 hours of birth
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to prevent contamination from colonized mothers during childbirth.
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Swabs were inoculated in StrepB Carrot (Hardy Diagnostics) or LIM broth and
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transported to the National Hospital Abuja laboratory for culture within four hours. No
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women received IAP regardless of the GBS culture results. Blood cultures from newborns
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were transported within four hours for BACTEC 9050 testing (Becton Dickinson). When
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GBS status was unknown, babies were considered to have signs of sepsis if presenting with:
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fever, hypothermia, respiratory distress, irritability, lethargy, and appetite loss.
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ACCEPTED MANUSCRIPT GBS identification and susceptibility testing. Samples were subcultured on CHROMagar
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StrepB; suspect colonies were confirmed using the PathoDxtra Strep Grouping Kit (Thermo
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Fisher Scientific). Susceptibilities to penicillin G (10U), ampicillin (10µg), clindamycin
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(2µg), erythromycin (15µg), vancomycin (30µg), ofloxacin (5µg), and ceftriaxone (30µg)
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were determined by disk diffusion using published breakpoints [10]; Streptococcus
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pneumoniae ATCC 49619 was the control. Inducible clindamycin resistance was determined
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using the “D-test” as described [10] and multidrug resistance was defined as resistance to >1
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antimicrobial class.
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Serotyping and multilocus sequence typing (MLST). DNA was extracted and cps types
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were determined as described [11]. Libraries were prepared for 70 strains from mother-baby
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pairs and one EOD strain using the Nextera XT kit (Illumina). Bioanalyzer (Agilent
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Technologies) and library quantification kits (Kapa Biosystems) were used to validate the
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libraries, which were pooled for denaturing and sequencing using the MiSeq (Illumina)
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(2x250). Sequences were assembled de novo using Velvet,1.2.07 [12] after trimming in
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Trimmomatic [13] and quality checking with FastQC (www.bioinformatics.babraham.
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ac.uk/projects/fastqc/). Sequence type (ST) and pilus island (PI) sequences were extracted
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using BLAST; PubMLST was used for allele and ST assignments (pubmlst.org/sagalactiae/).
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Data analysis. Statistical analyses were performed in SAS 9.3 (SAS Institute) using
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dichotomous variables. Univariate analyses were performed using χ2 tests with odds ratios
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(OR) and 95% confidence intervals (CI), and potential confounders and interactions were
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identified. The four primary outcomes included: maternal GBS colonization (n=171 of 500
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mothers), neonatal GBS colonization (n=95 of 500 neonates), sepsis development (n=21 of
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500 babies), and transmission (n=80 of 165 mothers and babies with molecular data).
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Multivariate analyses were performed using stepwise logistic regression. Significant explanatory variables were evaluated based on a chi-squared distribution using -2 LOG L and
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the Akaike and Schwartz Information Criterion, which examined the fit of each variable
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while adjusting for all variables and observations. Variables identified at P≤0.20 were
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evaluated using backward elimination to remove insignificant factors and solidify the base
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model. Wald CIs for adjusted ORs were calculated and other variables, which were selected
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for inclusion based on biological plausibility and univariate associations (P≤ 0.20), were
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added to each model. Caution was used to ensure that addition of each variable did not
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change the effect sizes of base model variables. The variance inflation factor was computed
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to ensure lack of multicollinearity and the Hosmer and Lemeshow test determined the
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goodness-of-fit [14]. The Wilson score index without continuity correction [15] was used to
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calculate the 95% CI around the EOD incidence estimate.
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Participant characteristics and GBS colonization frequencies. Enrolled women were 17-
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46 years of age (mean: 29.8 ±5 years) and most (61.0%) had a tertiary level of education,
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were married (98.0%), and had >2 prior pregnancies (80.0%). In all, 231 GBS isolates were
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recovered from the vagina, rectum or both sites in 171 women, yielding a maternal
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colonization rate of 34.2%; none of the women received IAP. The rectum was most
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commonly colonized (n=83; 48.5%), but some women had only vaginal colonization (n=28;
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16.3%) or were colonized at both sites (n=60; 35.1%). All four hospitals had similar
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populations with regard to age, marital status, parity and socioeconomic indicators, though
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colonization frequencies ranged between 26.0%-39.3% (P=0.15).
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Among the 500 babies of enrolled mothers, the majority were ≥37 weeks gestation (n=429; 85.8%) and ≥2.5 kg (n=401; 80.2%). Almost half of the neonates were male (n=239;
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47.8%) and 95 (19.0%) were colonized with GBS. One preterm male neonate developed GBS
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EOD at birth that was confirmed by culture. Three babies were stillborn and one died
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immediately after birth; no bacterial cultures were available. Two neonates died in the first
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month leaving 494 babies to follow for 12 weeks. Twenty-one of the 500 babies (4.2%)
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developed sepsis or had clinical signs of sepsis; all but four babies were admitted to the
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hospital. Among these 21 sepsis cases, one tested positive for GBS and three each were
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caused by Staphylococcus aureus and Klebsiella pneumoniae; the remainder were culture-
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negative.
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Associations with maternal GBS colonization. Following the univariate analysis (Table
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1), stepwise logistic regression identified maternal age, prolonged membrane rupture,
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douching, and number of vaginal exams to be associated with maternal GBS colonization.
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Preterm rupture of membranes was included in the based model given the correlation with
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ACCEPTED MANUSCRIPT prolonged membrane rupture (P<0.0001) and preterm birth (P<0.04). Women reporting ≥4
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vaginal exams and douching were 6.1 (95% CI: 3.41-10.93) and 3.7 (95% CI: 2.26-6.10)
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times more likely to be colonized, respectively, while women ≤25 years were twice (95% CI:
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1.22-3.39) as likely. Similarly, women with prolonged membrane rupture were more
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frequently colonized (OR: 3.4; 95% CI: 1.04-11.39), though no associations were observed
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for gestational diabetes, abnormal discharge, and having a neonate with sepsis. Women
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reporting ≥4 vaginal exams were more likely to have a preterm birth (OR: 2.1; 95% CI: 1.16-
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3.96) and more frequently developed premature (OR: 1.9; 95% CI: 0.78-4.60) and prolonged
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membrane rupture (OR: 2.6; 95% CI: 0.88-7.58), while women ≤25 years more commonly
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had preterm rupture (OR: 2.3; 95% CI: 1.02-5.01). Douching was not independently
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associated with any other variables in the model.
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Associations with neonatal colonization. Several factors were associated with neonatal
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colonization in the univariate analysis (Table 2). Colonized mothers were ~92 times (95%
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CI: 32.97-259.07) more likely to have a colonized neonate. Among the 91 (95.8%) colonized
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neonates born to colonized mothers, most had mothers with only rectal colonization (n=48;
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50.5%) followed by vaginal/rectal colonization (n=34; 35.8%), and vaginal colonization only
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(n=9; 9.5%). Four colonized neonates were born to women without GBS, while 80 (46.8%)
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colonized women had GBS-negative neonates.
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Stepwise logistic regression identified maternal colonization, number of vaginal exams,
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and prolonged membrane rupture to be most strongly associated with neonatal GBS
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colonization after adjusting for maternal age and premature membrane rupture (Table 3).
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Women receiving ≥4 vaginal exams and with prolonged membrane rupture were 3.0 times
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(95% CI: 1.52-5.97) and 4.2 times (95% CI: 1.03-17.17) more likely to have a colonized
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neonate. Maternal rectal GBS colonization was also important (OR: 26.6; 95% CI: 13.69-
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51.58), though baby gender, preterm birth, maternal age and douching were not.
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Associations with sepsis onset before 12 weeks of age. Using variables identified in the
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univariate analysis (Table 2), stepwise logistic regression identified young age, abnormal
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vaginal discharge, preterm birth, and low Apgar scores to be associated with sepsis (Table 3).
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Apgar scores ≤ 7 after 5 minutes yielded the strongest association (OR: 8.2; 95% CI: 3.00-
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22.30) followed by history of abnormal vaginal discharge (OR: 6.4; 95% CI: 2.08-19.79) and
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preterm birth (OR: 4.9; 95% CI: 1.64-14.55). Women ≤25 years of age and women with
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prolonged membrane rupture were ~3 times more likely to have a baby develop sepsis,
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though only maternal age was significant (P = 0.03). Abnormal vaginal discharge was
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independently associated with prolonged membrane rupture (OR: 6.8; 2.47-18.77) and low
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Apgar scores (OR: 2.3; 95% CI: 1.13-4.88).
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Phenotypic characteristics of GBS strains. Molecular serotypes were determined for 227
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(98.3%) strains from the 171 colonized women. Strains with cpsV (n=74, 43.3%), cpsII
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(n=39, 22.8%), cpsIa (n=22, 12.9%) and cpsIII (n=18, 10.3%) predominated. Three women
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(1.8%) had cpsIV, seven (4.1%) had non-typeable strains, and one had a strain with a unique
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RFLP pattern (cpsV variant). Most (78.3%) of the 60 women colonized in both the rectum
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and vagina had identical cps types, though nine women had discrepant types. The cps
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distribution was similar for the 95 neonates with cpsV (n=37, 39.0%), cpsII (n=21, 22.1%),
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cpsIa (n=12, 12.6%) and cpsIII (n=11, 11.6%) predominating.
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Resistance to clindamycin, erythromycin and ofloxacin was observed in 46 (26.9%), 34
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(19.9%) and 15 (8.8%) of the colonized mothers and 24 (25.3%), 13 (13.7%) and 6 (6.3%) of
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colonized neonates, respectively. All strains were susceptible to penicillin, ceftriaxone and
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more likely to have resistant GBS than neonates (P=0.29) nor were they more likely to have
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multidrug resistant strains (P=0.41). Twenty-eight (16.4%) mothers and 12 (12.6%) neonates
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had strains resistant to >1 antibiotic and maternal cpsV strains were 2.5 times more likely to
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be resistant relative to other types (95% CI: 1.34-4.74).
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Vertical transmission. All but five mothers had the same cps types when compared to their
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neonates, yielding a vertical transmission rate of 48.5% (n=80) among the 165 colonized
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mothers with cps data available. Although cpsIII strains were most commonly transmitted,
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the difference was not statistically significant when comparing it to all other cps types
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(P=0.23). When maternal colonization site was examined, the transmission rate for rectal
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colonization (52.2%) was greater than vaginal colonization alone (29.6%) (P=0.03).
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Stepwise logistic regression adjusting for factors identified in the neonatal colonization model found ≥4 vaginal exams (OR: 5.2; 95% CI: 2.39, 11.13) and prolonged membrane
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rupture (OR: 4.0; 95% CI: 1.43, 11.10) to be associated with transmission of the same GBS
192
strain. The likelihood of transmission increased according to the time of membrane rupture.
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The transmission rate was 66.7% for women delivering ≥1 hour before term, which increased
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to 83.3% for women experiencing ≥18 hours of ruptured membranes regardless of the
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gestational age. The association with premature rupture (OR: 2.0; 95% CI: 0.59-7.08),
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however, was not significant in the model.
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Thirty-five of the 80 mother-baby pairs with matching cps types were examined by
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MLST and PI profiling. Twelve STs were identified that grouped into five previously defined
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clonal complexes (CCs) [16] and one singleton (Figure 1). ST-19 predominated (n=9, 25.7%)
200
followed by ST-182 (n=7, 20.0%), and ST-1 (n=4, 11.4%). Three pairs were colonized with
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ST-17 (8.6%) as well as ST-23 (8.6%), while STs 2, 8, 24, 26, 196, and 762, a novel ST
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strains, most (n=7) were ST-182 and three were ST-17; one pair only had a maternal strain
204
(ST-182) available. The invasive EOD strain was classified as cpsIII ST-182, which clustered
205
within CC-19. ST-19 strains containing cpsV comprised 18 of the 26 (69.0%) cpsV strains
206
with MLST data available. The PI distribution was less variable among the pairs, with the
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majority of strains possessing both PI-I and PI-2a (n=27, 77.1%) followed by PI-2a only
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(n=5, 14.3%) and PI-2b only (n=3, 8.6%). Strains possessing only the PI-2b belonged
209
exclusively to ST-17 strains as described [17]. All cpsV/ST-19 and cpsIII/ST-182 strains had
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both PI-1 and PI-2a.
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DISCUSSION A high prevalence of GBS colonization was detected in third trimester pregnant women receiving antenatal care (34.2%) and their neonates (19.0%) in Nigeria. The maternal
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colonization rate was ~2 times higher than prior Nigerian reports [18,19] and is higher than
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the 22.4% prevalence estimate from four African studies [20]. Although GBS prevalence
216
varies geographically and temporally, rate differences could also be due to use of more
217
sensitive detection methods [2]. The incidence of EOD was 2.0 cases per 1,000 live births
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(95% CI: 0.50-7.30), which is similar to U.S. rates (1-3 cases/1,000) before maternal IAP was
219
introduced [1]. Among the seven million births in Nigeria each year [21], we estimate that
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14,000 children will develop EOD with ~3,220 deaths and ~1,820 children with long term
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disabilities based on annual South African EOD mortality (23%) and long term sequelae
222
(13%) estimates [5]. Consequently, it is recommended that GBS screening protocols and IAP
223
be considered as a prevention strategy for Nigerian women.
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colonization and factors such as prematurity and premature membrane rupture [1]. Indeed,
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rectally colonized women were significantly more likely to have colonized neonates, which
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contradicts a study reporting higher transmission rates in mothers with genital versus rectal
228
colonization [22]. Another study found GBS colonization of the rectum to be a predictor of
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vaginal colonization and women with only rectal colonization may become vaginally
230
colonized during childbirth, thereby enhancing transmission risk to the neonate [24].
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Sampling neonates 2-24 hours after birth was performed to ensure that colonization of the
232
neonate was not due to maternal contamination; however, it is possible that neonatal
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colonization frequencies were overestimated since other sites that more accurately represent
234
colonization were not examined. Differences in neonatal colonization rates could also be
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attributable to variation in maternal colonization densities, which was not addressed and
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represents a study limitation. Moreover, the finding that some colonized neonates were born
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to GBS-negative mothers suggests that some mothers had low colonization densities that
238
precluded detection or neonates acquired GBS from external sources.
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Young maternal age has been linked to colonization previously, [25] which could be due to higher levels of sexual activity and less stable relationships. The finding that 67% of
241
women not living with their partners were 16-25 years old provides support for the latter.
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Associations have also been observed between young age and EOD [1], which could be due
243
to lower levels of circulating antibodies and is consistent with our observed association
244
between young age and sepsis development. Low Apgar scores and preterm birth were also
245
associated with sepsis and supports studies that have identified preterm birth as the primary
246
risk factor for late onset sepsis caused by many pathogens [26,27]. History of abnormal
247
vaginal discharge was also associated with sepsis and may represent a marker for
248
chorioamnionitis, a risk factor for both preterm birth and sepsis [28], that has also been linked
249
to GBS. Still, 26 of 52 women reporting discharge had GBS and five had preterm birth and
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one was <25 years. One limitation, however, is that the definition of sepsis may have differed
251
across hospitals since 17 (81%) of the 21 cases were diagnosed at one site. Indeed, the
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hospital with the most cases more frequently identified culture-negative cases (Fisher’s exact
253
test P = 0.025), which could contribute to misclassification bias.
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Neonatal colonization was associated with prolonged membrane rupture (>18 hrs). The
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higher transmission rates associated with prolonged versus preterm rupture of membranes
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support the hypothesis that transmission risk increases as the time of GBS exposure increases
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inside the maternal genital tract [2]. Because choriodecidual infection with GBS in a
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nonhuman primate model was found to contribute to premature rupture of membranes [30],
259
which was associated with prolonged membrane rupture in our study, it is important to
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identify factors for both conditions as well as GBS colonization. Behavioural factors like
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colonization, could cause alterations in the vaginal microbiota or disruption of the vaginal
263
mucosa to promote colonization. Since colonization frequencies differed among the douche
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preparations, it is possible that each type has varying effects on the vaginal milieu. Increased
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education of factors that impact colonization during pregnancy is important in Nigeria and
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elsewhere, particularly given that a greater number of intrapartum vaginal exams and
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prolonged membrane rupture were also risk factors for transmission and neonatal
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colonization, the first steps in disease.
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GBS strain diversity was low compared to other studies and ST-182 has not been previously reported in high frequency among cpsIII strains. Similarly, ST-19 strains
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possessing cpsV, a unique ST/cps combination, has been infrequently reported. A close
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evolutionary relationship was previously observed between the cpsV strain 2603V/R and ST-
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19 [31], suggesting the importance of capsule switching in the generation of new strain types
274
that impact colonization frequencies. The low prevalence of ST-17, a lineage associated with
275
neonatal disease [16], also suggests that ST-17 frequencies vary by location, thereby
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impacting disease incidence. Indeed, a 25-year GBS EOD study in the Netherlands identified
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a significantly lower frequency of ST-17 infections before IAP use [32], which may be due to
278
variation in the ability of ST-17 strains to tolerate antibiotics and persist following IAP [25].
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Because GBS strain types were equally likely to be transmitted, continuous monitoring in
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Nigeria will enhance our knowledge of pathogen evolution after prevention practices are
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established. Surveillance efforts are also needed to ensure that antibiotic resistance rates do
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not increase in second-line agents and that penicillin-resistance does not emerge.
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Based on our findings, use of penicillin or vancomycin would be appropriate for IAP in
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women with GBS colonization, prolonged rupture of membranes and/or preterm birth, which
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was linked to late-onset sepsis. Other factors including young age and abnormal vaginal
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implementing a universal screening protocol. Challenges include shortages of antenatal care
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facilities, late presentation to the delivery ward, limited laboratory capacity, delayed
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dissemination of laboratory results, and limited availability and technicalities associated with
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intravenous drug administration. While a risk-based screening approach may be easier, ~348
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(69.6%) of GBS-colonized women would have been missed because they lacked premature
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rupture of membranes, preterm birth or fever. Antibiotics would have also been unnecessarily
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administered to 326 (65.2%) of GBS-negative women with these risk factors.
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In Nigeria, a universal GBS screening strategy with IAP would cost ₦300,000 to implement for 500 women using the same detection methods, which is less than the average
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cost of managing a newborn with EOD (₦500,000) or long-term sequelae (₦1,000,000) in
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surviving children. Alternative prevention strategies including a GBS vaccine should also be
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considered as a vaccine targeting PI-2a would cover 91% of the strains identified in our
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study; coverage would increase if serotypes II, III, and V were targeted. Such prevention
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practices could reduce EOD incidence in Nigeria as well as the frequency of preterm delivery
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and premature/prolonged rupture of membranes.
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Funding
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The study was primarily supported by the National Hospital Abuja in Nigeria [NM, KCI,
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LIA, EE]. Additional support was provided by Michigan State University with salary support
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from the Global Alliance to Prevent Prematurity and Stillbirth [N015615 to SDM]. The
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authors declare no conflicts of interest.
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Acknowledgements
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We thank the many individuals who assisted with recruitment, data collection, and laboratory
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work.
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Ogunlesi TA, Ogunfowora OB, Osinupebi O, Olanrewaju DM. Changing trends in
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newborn sepsis in Sagamu, Nigeria: bacterial aetiology, risk factors and antibiotic
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susceptibility. J Paediatr Child Health 2011;47:5–11.
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[10] Clinical and Laboratory Standards Institute (CLSI). Performance Standards for
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Antimicrobial Susceptibility Testing; Twenty-Fifth Informational Supplement. CLSI
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Document M100-S25. 2015. [11] Manning SD, Lacher DW, Davies HD, Foxman B, Whittam TS. DNA polymorphism
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and molecular subtyping of the capsular gene cluster of group B Streptococcus. J Clin
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Microbiol 2005;43:6113–6.
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[13] Bolger AM, Lohse M, Usadel B. Trimmomatic: A flexible trimmer for Illumina
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Bruijn graphs. Genome Res 2008;18:821–9.
sequence data. Bioinformatics 2014;30:2114–20.
[14] Hosmer DW, Lemeshow S. Assessing the Fit of the Model. 2nd ed. Hoboken, NJ: John Wiley & Sons, Inc.; 2000.
[15] Newcombe RG. Two-sided confidence intervals for the single proportion: Comparison
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[12] Zerbino DR, Birney E. Velvet: Algorithms for de novo short read assembly using de
of seven methods. Stat Med 1998;17:857–72. [16] Manning SD, Springman AC, Lehotzky E, Lewis MA, Whittam TS, Davies HD.
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Multilocus sequence types associated with neonatal group B streptococcal sepsis and
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meningitis in Canada. J Clin Microbiol 2009;47:1143–8.
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[17] Springman AC, Lacher DW, Waymire E a, Wengert SL, Singh P, Zadoks RN, et al. Pilus distribution among lineages of group B Streptococcus: an evolutionary and clinical perspective. BMC Microbiol 2014;14:159. [18] Dawodu AH, Damole IO, Onile BA. Epidemiology of group B streptococcal carriage
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among pregnant women and their neonates: an African experience. Trop Geogr Med
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1983;35:145–50.
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[19] Uhiara JE. Group B streptococcal carriage among parturients and their neonates in Zaria, Nigeria. Afr J Med Med Sci 1993;22:79–83. [20] Kwatra G, Cunnington MC, Merrall E, Adrian P V., Ip M, Klugman KP, et al. Prevalence of maternal colonisation with group B Streptococcus: a systematic review
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and meta-analysis. Lancet Infect Dis 2016;16:1076–84.
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[21] Nigeria Demographic and Health Survey 2013.
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https://dhsprogram.com/pubs/pdf/FR293/FR293.pdf (accessed May 11, 2016).
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[22] Hoogkamp-Korstanje JA, Gerards LJ, Cats BP. Maternal carriage and neonatal
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acquisition of group B streptococci. J Infect Dis 1982;145:800–3.
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[23] Meyn LA, Krohn MA, Hillier SL. Rectal colonization by group B Streptococcus as a predictor of vaginal colonization. Am J Obstet Gynecol 2009;201. [24] Badri MS, Zawaneh S, Cruz a C, Mantilla G, Baer H, Spellacy WN, et al. Rectal colonization with group B Streptococcus: relation to vaginal colonization of pregnant
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women. J Infect Dis 1977;135:308–12.
[25] Manning SD, Lewis MA, Springman a C, Lehotzky E, Whittam TS, Davies HD.
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Genotypic diversity and serotype distribution of group B Streptococcus isolated from
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women before and after delivery. Clin Infect Dis 2008;46:1829–37.
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[26] Seo K, McGregor JA, French JI. Preterm birth is associated with increased risk of maternal and neonatal infection. Obstet Gynecol 1992;79:75–80.
[27] Downey LC, Smith PB, Benjamin DK. Risk factors and prevention of late-onset sepsis in premature infants. Early Hum Dev 2010;86. [28] Galinsky R, Polglase GR, Hooper SB, Black MJ, Moss TJM. The consequences of chorioamnionitis: preterm birth and effects on development. J Pregnancy 18
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2013;2013:412831. [29] Yancey MK, Duff P, Clark P, Kurtzer T, Frentzen BH, Kubilis P. Peripartum infection associated with vaginal group B streptococcal colonization. Obstet Gynecol
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1994;84:816–9.
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[30] Vanderhoeven JP, Bierle CJ, Kapur RP, McAdams RM, Beyer RP, Bammler TK, et al.
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Group B streptococcal infection of the choriodecidua induces dysfunction of the
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cytokeratin network in amniotic epithelium: a pathway to membrane weakening. PLoS
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Pathog 2014;10:e1003920.
[31] Davies HD, Jones N, Whittam TS, Elsayed S, Bisharat N, Baker CJ. Multilocus
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sequence typing of serotype III group B Streptococcus and correlation with pathogenic
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potential. J Infect Dis 2004;189:1097–102.
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[32] Bekker V, Bijlsma MW, van de Beek D, Kuijpers TW, Van der Ende A. Incidence of invasive group B streptococcal disease and pathogen genotype distribution in newborn
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babies in the Netherlands over 25 years: A nationwide surveillance study. Lancet
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Infect Dis 2014;14:1083–9.
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Table 1. Factors associated with maternal group B streptococcal colonization in the univariate
397
and multivariate analyses. No. of women
Characteristic
GBS positive No. (%)
Univariate OR (95% CI)
Multivariate⊥ ⊥ OR (95% CI)
95 349 56
41 116 14
(43.2) (33.2) (25.0)
2.3 (1.10-4.72) 1.5 (0.82-2.97) 1.0
Marital Status Married/living together Single/separated/divorced
494 6
168 3
(34·0) (50·0)
1.0 1.9 (0.39-9.72)*
Highest level of education Never attended Primary Secondary Tertiary
15 17 161 307
5 8 50 108
(33·3) (47·1) (31·1) (35·2)
Type of toilet Water cistern Aqua privy Pit toilet
429 63 8
146 20 5
(34·0) (31·8) (62·5)
1.0 0.9 (0.5-1.6) 3.2 (0.76-13.71)*
History of douching No Yes
162 338
28 143
(17.3) (42.3)
1.0 3.5 (2.21-5.56)
162 118 173 22 25
28 50 77 11 5
(17.3) (42.4) (45.0) (50.0) (20.0)
1.0 3.5 (2.04-6.08) 3.8 (2.31-6.37) 4.8 (1.89-12.13) 1.2 (0.41-3.46)*
Maternal medical history No. of prior pregnancies 1 2 ≥3
98 141 261
35 57 79
(35.7) (40.4) (30.0)
1.0 1.2 (0.72-2.08) 0.8 (0.48-1.28)
HIV Positive No Yes
479 21
162 9
(33.8) (42.9)
1.0 1.5 (0.6-3.56)
-
Prior preterm rupture of membranes No Yes
462 38
157 14
(34.0) (36.8)
1.0 1.1 (0.57-2.25)
-
Prior stillbirth or baby death No Yes
467 33
154 17
(33.0) (51.5)
1.0 2.2 (1.06-4.39)
1.3 (0.59-2.99)
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0.9 (0.31-2.76) 1.6 (0.61-4.37) 0.8 (0.55-1.25) 1.0
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Type of douche used None Antiseptic solution Bath soap and water Salt and water solution Native preparation
RI PT
Demographics, lifestyle, behaviors Age group (years) 17-25 26-35 ≥36
2.0 (1.22-3.39) -
-
-
-
3.7 (2.26-6.11)
-
-
20
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136 7
(33.4) (38.9)
1·0 1.3 (0.48-3.34)
-
Previous preterm delivery No Yes
414 86
138 33
(33·3) (38·4)
1.0 1.2 (0.74-2.06)
-
Current pregnancy history and complications No. of intrapartum vaginal exams 0-3 ≥4
428 72
120 51
(28.0) (70.8)
1.0 6.2 (3.60-10.11)
6.1 (3.41-10.93)
Antibiotic use in past 3 months No Yes
479 21
165 6
(34.5) (28.6)
1.0 0·7 (0.29-1.20)
-
448 52
145 26
(32.4) (50.0)
Intrapartum fever No Yes
468 32
470
160 11
(33.2) (61.1)
1.0 3.2 (1.20-8.31)
159 12
(34.0) (37.5)
1.0 1.2 (0.56, 2.45)
155
(33.0)
1.0
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Preterm rupture of membranes (>1 hr) No Yes
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482 18
1.0 2.1 (1.17-3.73)
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Abnormal vaginal discharge in the past 3 mos. No Yes Gestational Diabetes No Yes
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Prior baby with sepsis No Yes
1.8 (0.93, 3.46)
2.4 (0.83-7.10)
-
30
16
(53.3)
2.3 (1.11-4.88)
1.8 (0.78, 4.35)
483 17
159 12
(32.9) (70.6)
1.0 4.9 (1.69-14.12)
3.4 (1.04-11.39)
Prolonged labor (>18 hrs) No Yes
487 13
162 9
(33.3) (65·0)
1.0 4.9 (1.69-14.12)
2.2 (0.56-8.87)
Gestational age at birth < 37 weeks (preterm) ≥ 37 weeks (term)
71 429
34 137
(47.9) (31.9)
2.0 (1.18-3.25) 1.0
1.4 (0.81-2.61)
Birth weight ≤ 2.5 kg > 2.5 kg
99 401
41 130
(41.4) (32.4)
1.5 (0.94-2.31) 1.0
1.1 (0.68-1.88)
Apgar score (5 minutes) >7 ≤7
443 57
147 24
(33.2) (42.1)
1.0 1.5 (0.84-2.57)
1.1 (0.58-2.13)
Baby with sepsis within the 12 week follow up period No Yes
479 21
157 14
(32.8) (66.7)
1.0 4.2 (1.62-10.37)
2.4 (0.84-7.05)
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Prolonged rupture of membranes (> 18 hrs) No Yes
21
ACCEPTED MANUSCRIPT No. = Number; OR= odds ratio; 95% CI = 95% confidence interval; P = P-value
399
Note: Not all denominators are equivalent as some data were missing.
400
*Fisher’s exact test was used for variables with ≤ 5 in at least one cell.
401
⊥ Stepwise logistic regression and backward elimination were used to identify explanatory
402
variables (age ≤ 25 years, ≥4 vaginal examinations, douching history, preterm rupture of
403
membranes and prolonged membrane rupture) to be included in the base model at P≤0.20
404
(Hosmer and Lemeshow test P=0.25). Age≤ 25 years was examined relative to both of the
405
other two age categories (≥26 years) combined. All additional variables were added separately
406
to this base model and ORs were adjusted for each of these five variables; adjusted ORs and
407
Wald confidence intervals are noted. Variables without a point estimate (-) were not examined
408
in the multivariate analysis because no association was identified with maternal GBS
409
colonization in the univariate analysis at P≤0.2.
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Table 2. Univariate analysis of factors associated with neonatal group B streptococcal
412
colonization and sepsis development up to 12 weeks following birth.
No. with characteristic
Characteristic
Neonatal GBS colonization GBS positive No. (%) OR (95% CI)
Sepsis signs or development Sepsis positive No. (%) OR (95% CI)
28 83 60
9 (32.1) 48 (57.8) 34 (56.7)
1.0 2.9 (1.17-7.16) 2.8 (1.07-7.09)
Age group (years) 17-25 26-35 ≥36
95 349 56
19 (20.0) 70 (20.1) 6 (10.7)
2.1 (0.78-5.58) 2.1 (0.86-5.07) 1.0
9 (9.5) 10 (2.9) 2 (3.6)
2.8 (0.59-13.58)* 0.7 (0.17-3.73)* 1.0
History of douching No Yes
162 338
13 (8.0) 82 (24.3)
1.0 3.7 (1.98-6.82)
4 (2.5) 17 (5.0)
1.0 2.1 (0.69-6.32)*
Type of douche used None Antiseptic solution Bath soap and water Salt and water solution Native preparation
162 118 173 22 25
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Maternal factors GBS colonization site Vagina Rectum Vagina and rectum
479 21
93 (34.5) 2 (28.6)
1.0 0·4 (0.10-1.91)*
19 (4.0) 2 (9.5)
1.0 2.5 (0.55-11.74)*
467 33
85 (18.2) 10 (30.2)
1·0 2.0 (0.90-4.26)
16 (3.4) 5 (15.2)
1.0 5.0 (1.72-14.74)*
407 18
73 (17.9) 3 (16.7)
1.0 0.9 (0.26-3.24)
16 (4.0) 3 (16.7)
1.0 4.9 (1.28-18.60)*
Abnormal vaginal discharge in the past 3 mos. No Yes
448 52
80 (17.) 15 (28.9)
1.0 1.9 (0.98-3.56)
13 (2.9) 8 (15.4)
1.0 6.1 (2.39-15.48)
Current pregnancy and neonatal factors Sex of neonate Female Male
261 239
36 (13.8) 59 (24.7)
1.0 2.0 (1.39-3.24)
10 (3.8) 11 (4.6)
1.0 1.2 (0.50-2.90)
Neonate colonized with GBS No Yes
405 95
-
12 (3.0) 9 (9.5)
1.0 3.4 (1.40-8.39)
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Prior baby with sepsis No Yes
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1.0 2.9 (1.42-6.02) 4.4 (2.28-8.49) 4.3 (1.44-12.86) 2.2 (0.65-7.32)*
Prior stillbirth or baby death No Yes
-
4 3 9 2 3
(2.5) (2.5) (5.2) (9.1) (12.0)
1.0 0.6 (0.15-2.79)* 0.8 (0.17-3.42)*
(8.0) (20.3) (27.8) (27.3) (16.0)
Antibiotic use in past 3 months No Yes
13 24 48 6 4
3 (10.7) 6 (7.2) 5 (8.3)
1.0 1.0 (0.23-4.69)* 2.2 (0.65-7.18)* 4.0 (0.68-22.96)* 5.4 (1.13-25.68)*
23
482 18
92 (19.1) 3 (16.7)
1.0 0.8 (0.24-2.99)*
18 (3.7) 3 (16.7)
1.0 5.2 (1.36, 19.42)*
No. of intrapartum vaginal exams 0-3 ≥4
428 72
56 (13.1) 39 (54.2)
1.0 7.9 (4.57-13.50)
15 (3.5) 6 (8.3)
1.0 2.5 (0.94-6.68)
Intrapartum fever No Yes
468 32
88 (18.8) 7 (21.9)
1.0 1.2 (0.51-2.88)
16 (3.4) 5 (15.6)
1.0 5.2 (1.8-15.35)*
Premature rupture of membranes (>1 hr) No Yes
470 30
83 (17.7) 12 (40.0)
1.0 3.1 (1.44-6.70)
18 (3.8) 3 (10.0)
1.0 2.8 (0.77-10.06)*
Prolonged rupture of membranes (>18 hrs) No Yes
483 17
85 (17.6) 10 (58.8)
17 (3.5) 4 (23.5)
1.0 8.4 (2.49-28.59)*
Prolonged labour (>18 hrs) No Yes
487 13
Gestational age at birth <37 weeks (preterm) ≥37 weeks (term)
71 429
Birth weight ≤ 2·5 kg > 2·5 kg
19 (3.9) 2 (15.4)
1.0 4.5 (0.93-21.63)*
20 (28.2) 75 (17.5)
1.9 (1.04-3.29) 1.0
10 (14.1) 11 (2.6)
6.2 (2.54-15.28) 1.0
99 401
22 (22.2) 73 (18.2)
1.3 (0.75-2.20) 1.0
10 (10.1) 11 (2.7)
4.0 (1.64-9.67) 1.0
443 57
80 (18.1) 15 (26.3)
1.0 1·6 (0.86-3.07)
10 (2.3) 11 (29.3)
1.0 10.4 (4.17-25.69)
86 (18.0) 9 (42.9)
1·0 3.4 (1.40-8.39)
479 21
-
-
AC C
413
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1.0 5.3 (1.74-16.12)
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Baby with sepsis within the 12 week follow up period No Yes
1.0 6.7 (2.48-18.07)
88 (18.1) 7 (53·9)
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Apgar score (5 mins) >7 ≤7
RI PT
Gestational Diabetes No Yes
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No. = Number; OR= odds ratio; 95% CI = 95% confidence interval; P = P-value
415
Note: Not all denominators are equivalent as some data were missing.
416
*Fisher’s exact test was used for variables with ≤ 5 in at least one cell.
24
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Table 3. Multivariate analysis of factors associated with neonatal colonization and the
418
development of sepsis in the 12 weeks following birth. Neonatal GBS
Sepsis signs or
colonization
development
OR
95% CI
OR
95% CI
Maternal rectal GBS colonization*
26.6
13.69-51.58
1.4
0.50-3.96
Maternal age ≤ 25 years*⊥
0.9
0.41-1.77
3.1
1.10-8.82
History of douching
2.1
0.98-4.58
1.7
0.52-5.91
Prior stillbirth or baby death
0.8
0.28-2.28
3.6
0.90-14.25
Abnormal vaginal discharge⊥
1.2
0.51-3.02
6.4
2.08-19.79
≥4 intrapartum vaginal exams*
3.0
1.52-5.97
1.3
0.40-4.44
Intrapartum fever
0.8
0.24-2.61
3.4
0.92-12.80
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Characteristic
Prolonged rupture of membranes (>18 hrs)*⊥
4.2
1.03-17.17
3.0
0.68-13.03
Premature rupture of membranes (>1 hr)*⊥
1.7
0.60-5.04
1.3
0.30-5.48
Preterm birth (< 37 weeks)⊥
1.0
0.44-2.08
4.9
1.64-14.55
1.0
0.42-2.55
8.2
3.00-22.30
1.3
0.73-2.42
0.9
0.31-2.47
Prolonged labour (>18 hrs)
1.5
0.31-6.37
1.6
0.21-11.67
Low birth weight (≤ 2.5 kg)⊥
0.8
0.41-1.68
0.6
0.14-2.97
-
1.8
0.62-5.47
0.50-6.67
-
Apgar score ≤ 7 (5 mins)⊥
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Male gender
Neonatal GBS colonization
EP
Baby developed sepsis within 12 weeks
1.8
-
OR= odds ratio; 95% CI = 95% confidence interval; P = P-value
420
Note: Rectally colonized women included those with rectal only and rectal plus vaginal
421
colonization. For both outcomes (neonatal GBS colonization* and sepsis⊥), each model was
422
adjusted for the significant variables identified by stepwise selection to comprise the base
423
model; the Hosmer and Lemeshow P-value was >0.05 in each model examined. All
424
additional variables were added separately to each base model and were adjusted for each of
425
the variables included. The adjusted ORs and Wald confidence intervals are noted.
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ACCEPTED MANUSCRIPT Figure 1. Neighbor-joining phylogeny illustrating the type of group B Streptoccocus strains
428
shared between 35 mothers and their newborns. The number of mother-baby pairs with
429
specific sequence types (STs) is noted as well as the previously defined clonal complex (CC)
430
and distribution of cps types representing each lineage. S=singleton.
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