Health Burden of Group B Streptococci Colonization in the Obstetric Population at University of Benin Teaching Hospital, Benin City, Nigeria

HEALTH BURDEN OF GROUP B STREPTOCOCCI COLONIZATION IN THE OBSTETRIC POPULATION AT UNIVERSITY OF BENIN TEACHING HOSPITAL, BENIN CITY, NIGERIA

A DISSERTATION SUBMITTED TO THE NATIONAL POSTGRADUATE MEDICAL COLLEGE OF NIGERIA IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE PART II FELLOWSHIP EXAMINATION OF THE COLLEGE

Edward Eromosele OKOSUN MBBS (A.B.U., ZARIA, 2003)

MAY, 2017

DECLARATION

It is hereby declared that this work is original unless otherwise acknowledge. The work has not been presented to any other College, Faculty or School for the award of a

Fellowship, Degree or Diploma; nor, has it been published or submitted elsewhere for publication.

………………………………………………………………….. Dr. Edward Eromosele OKOSUN MB; BS Department of Obstetrics and Gynaecology University of Benin Teaching Hospital Benin City.

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TABLE OF CONTENTS

TITLE PAGE ...... i

DECLARATION ...... ii TABLE OF CONTENTS ...... iv DEDICATION ...... viii ACKNOWLEDGEMENT ...... ix ABSTRACT ...... 1 CHAPTER 1 ...... 3 INTRODUCTION ...... 3

CHAPTER 2 ...... 9 LITERATURE REVIEW ...... 9

2.1 Epidemiology of GBS ...... 9

2.1.1 Prevalence of GBS Colonization and invasive GBS Disease 9 2.1.2 Risk Factors for GBS Colonization and Invasive GBS Disease 13 2.1.2A Obstetric Risk Factors ...... 13 2.1.2B Bio-Demographic Risk Factors ...... 16 2.1.2C Neonatal Risk Factors ...... 18 2.1.2D Risk Factors for Maternal Invasive GBS Disease ...... 18 2.2 Isolation and Identification of GBS ...... 18

2.2.1 Timing of GBS Specimen Collection ...... 18 2.2.2 Sites of GBS Specimen Collection ...... 20 2.2.3 Transportation of GBS specimens ...... 20 2.2.4 Isolation of GBS from specimens ...... 21 2.2.4A Enrichment, culture and identification of GBS from specimens ...... 21 2.2.4B Identification of GBS by newer methods: DNA probes; nucleic acid amplification test; optical immunoassays; and enzyme immunoassays ...... 22

2.3 Prevention of early-onset neonatal invasive GBS disease ...... 23

2.3.1 Risk-based strategy ...... 23 2.3.2 Culture-based strategy ...... 24 2.3.3 Appraisal of screening strategies ...... 25 2.4 Antibiotic susceptibility of GBS substrates ...... 26

2.5 Limitations of previous studies ...... 26

2.6 Effect of GBS on pregnant women ...... 27

2.7 Justification for the research ...... 27

CHAPTER 3 ...... 30 AIM AND OBJECTIVES ...... 30

3.0 Aim ...... 30

3.1 Objectives ...... 30

3.2 Hypothesis ...... 30

3.2.1 Null Hypothesis: ...... 30 3.2.2 Alternative Hypothesis ...... 30 CHAPTER 4 ...... 31 METHODOLOGY ...... 31

4.1 Study Setting ...... 31

4.2 Study Design ...... 32

4.3 Study Population ...... 32

4.4 Selection Criteria ...... 32

4.4.1 Inclusion Criteria ...... 32 4.4.2 Exclusion Criteria ...... 32 4.5 Sample Size Calculation ...... 33

4.6 Sampling Technique ...... 33

4.7 Data Management ...... 34

4.7.1 Data Collection Tools and Methods...... 34 4.7.2 Training of research assistants ...... 35 4.8 Quality control ...... 35

4.9 Specimen collection ...... 36

4.10 Transport of swabs and processing of specimens ...... 38

4.11 Presumptive identification of GBS...... 39

4.12 Confirmation of GBS ...... 40

4.13 Mother-infant pair follow up ...... 40

4.14 Data Analysis ...... 40

4.15 Definition of Terms ...... 41

4.16 Outcome Measure ...... 41

4.16.1 Primary outcome measure ...... 41 4.16.2 Secondary outcome measures: ...... 41 4.17 Ethical Considerations ...... 42

CHAPTER 5 ...... 44 RESULTS ...... 44

CHAPTER 6 ...... 63 DISCUSSION, CONCLUSION AND RECOMMENDATIONS ...... 63

6.1 Discussion ...... 63

6.1.1 Recommendations for Policy Development; Practice and Future Research ...... 77 6.2 Conclusion ...... 80

6.3 Recommendations ...... 80

REFERENCES ...... 82 APPENDICES ...... 104 APPENDIX I ...... 104

APPENDIX II ...... 106 vi

INFORMED CONSENT FORM ...... 106

APPENDIX IlI ...... 109

STANDARDIZED INTERVIEWER SCHEDULE ...... 109

APPENDIX IV ...... 112

ETHICAL APPROVAL ...... 112

APPENDIX V ...... 113

APPENDIX VI ...... 114

APPENDIX VII ...... 115

List of product specifications: ...... 115

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DEDICATION

I dedicate this work to God almighty, because he is the reason that I am; to Professor

Edward Okosun for being my wonderful and dependable Dad; to Lady Florence Okosun for being our matriarch; to both my parents for being an extremely synergistic confluence of my nature versus nurture; to my brother Odia and sister Rayle for standing the test of time and keeping our fold together; to my lovely, beautiful, feminine, delicate and resilient wife Obiajulu who has shown me that love truly endures (Dalu Ob’m); to our children

Ehimudiamen and Obehioye who light up our home and often say “but dad, you know I love you”; to Professor Alfred Ehigiegba of whom I often think “Oh! If I could cut, write, drive and work like you”; to Mrs. Roxanna Gaius-Obaseki for being a God sent mother to me; to Professor and Mrs. Eugene Okpere for watching over me; and to Professor Anibaba

Ande who welcomed me into an elite fold of Obstetrician Gynecologist’s at an early stage.

God, bless you all!

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ACKNOWLEDGEMENT

I acknowledge Professor Michael Ibadin for going the extra mile to ensure I reach the desired end; Professor Augustine Orhue, my teacher; Professor Olabisi Aisien, the matriarch of our Department; Professor Prosper Gharoro, a curious mind and a great teacher with lots of terabytes of cerebral storage; Professor Michael Aziken, the epitome of an astute, hardworking clinician and one who always has my back; Professor Joseph

Onakewhor who always said to me “ it is in the character of this department to ensure everyone finishes what they started before they leave us” (may his soul find eternal rest);

Professor Anthony Okonkwo for epitomizing teaching as a vocation; my other teachers at the Department of Obstetrics and Gynaecology, University of Benin Teaching

Hospital to whom I say truly! iron “sharpeneth” iron!; Matron Itohan Okunzuwa, for being a mother and teacher in and out of the Family Planning Unit; the entire staff of the

Department of Medical Microbiology, University of Benin Teaching Hospital; and the staff of the Department of Microbiology, University of Benin: both departments for the microbiology aspect of this work; for allowing me hands on participation at all stages of the laboratory aspects of this dissertation and my close observation to ensure strict adherence to this dissertation’s protocol. Thank you all!

ABSTRACT

Background: Streptococcus agalactiae or Group B streptococci (GBS), are gram-positive cocci which may be beta-haemolytic; coagulase negative and catalase negative. GBS have been reported as the leading cause of early-onset neonatal sepsis in the industrialized nations since the 1970’s. However, only recently has GBS been reported as an important cause of neonatal sepsis in sub-Saharan Africa. Thirty to fifty per cent of cases of early- onset GBS sepsis develop in neonates born to mothers without clinical risk factors. Women with recto-vaginal colonization by GBS usually have no symptoms of disease. A culture is therefore required to detect the presence of the organism in the vagina or rectum. There are still opportunities for simple preventive measures affecting maternal and child health.

Aim: To estimate the burden of GBS disease in the obstetric and neonatal population at

University of Benin Teaching Hospital (UBTH).

Methodology: A prospective cohort study. Swabs were taken from the parturient’s lower vagina and rectum. Four samples were also collected immediately after birth from the neonate’s throat; anterior nares; external ear canals; and base of the umbilical cord. Early- onset GBS neonatal sepsis was defined as confirmed isolation of GBS from blood and/or cerebro-spinal fluid cultures in neonates who presented with clinical features of sepsis.

Specimens were selectively enriched in Lim broth before being sub-cultured to supplemented blood agar plates. Suspected bacterial colonies were subjected to biochemical tests and then Group B latex agglutination tests. Data was coded into an IBM

SPSS spread sheet and analysed using IBM SPSS Statistics, version 22.0, Chicago, Illinois,

U.S.A.

Results: Thirty percent of mother-infant pairs in this study were colonized with GBS. Two percent of colonized mothers developed postpartum endometritis. The early onset invasive neonatal GBS disease among study participants was 7.8% with an attack rate of 25.7% among GBS positive mother-infant pairs, a stillbirth incidence of 1.0% and a case fatality rate of 15.4%.

Conclusion: Maternal recto-vaginal colonization with GBS is prevalent (intrapartum) in the obstetric population delivering at UBTH. It increases the risk of maternal postpartum complications; neonatal colonization with GBS; GBS early-onset neonatal disease

(sepsis); and perinatal mortality from GBS sepsis.

Recommendation: Antenatal mothers should receive health education on the significance of GBS screening. Maternal healthcare services should incorporate late antenatal screening

(as a surrogate for intrapartum screening) for recto-vaginal colonization by GBS and selective intrapartum antibiotic prophylaxis.

CHAPTER 1

INTRODUCTION

Millennium Development Goals (MDGs) four and five identified maternal and child health as priorities for international development.1 Their greatest unmet need was in sub-Saharan

Africa which accounts for one tenth of the world population but almost half of all maternal and child deaths worldwide.2,3 In all the countries reported in sub-Saharan Africa, except

Eritrea, insufficient or no progress in reducing child mortality was made between 1990 and

2015 to achieve the MDG four [a two-thirds reduction in under five mortality rates between

1990 and 2015].4 In 2005, the World Health organisation (WHO), estimated the maternal mortality ratio in sub-Saharan Africa to be one hundred times that in developed countries.2

The neonatal mortality rate in sub-Saharan Africa has also been estimated to be four times the rate in Europe and the Americas. Additionally, stillbirths are thought to equal the number of neonatal deaths worldwide.5 Of these neonatal deaths, three-quarters occur early

(within 7 days of birth).5 This suggests that some are related to antenatal and intrapartum events. Therefore, the Obstetrician and Neonatal Physician had crucial roles in ensuring the attainment of the MDG’s four and five.

Over a quarter of all neonatal deaths worldwide are estimated to be caused by neonatal sepsis [infection within the first 28 days of life].5 Group B streptococci (GBS) have been reported as the leading cause of early-onset [within the first week of life] neonatal sepsis and serious neonatal infections in North America and Europe since the 1970’s.6,7 However, only recently has GBS been reported as an important cause of neonatal sepsis in sub-

Saharan Africa. The primary risk factor for early-onset GBS neonatal infection is maternal recto-vaginal colonization intrapartum.8 The prevalence of GBS colonization varies for different regions of the world and different institutions partly because the isolation of GBS requires stringent microbiological methods. Prevalence of 10 - 30%, 6.5 - 36.0%, 17.8%,

19.0% and 20% have been reported in U.S.A., Europe, developing countries, sub-Saharan

Africa and Nigeria respectively.6,9,10 This suggests that the prevalence is quite high in

Nigeria and thus may be an important local cause of neonatal sepsis.

Streptococcus agalactiae or GBS, are gram-positive cocci which may be beta-haemolytic, catalase negative, and/or hippurate positive. They are part of the normal flora of the rectum and female urogenital tract. Their chief importance is that they cause invasive disease primarily in pregnant or postpartum women, young infants (less than 90 days old) and older adults with underlying medical conditions such as diabetes mellitus.11,12 GBS in pregnant women causes clinical infections; but, most women have no symptoms associated with genital tract colonization. Urinary tract infections caused by GBS complicate 2 - 4% of pregnancies.13,14 Pregnancy or the postpartum period may be complicated by amnionitis, premature delivery, low birth weight, stillbirth, endometritis, sepsis, or rarely meningitis caused by GBS.15,16 Fatality in this group is rare.

Neonatal GBS infections occurring within the first week of life (< 7 days) are designated early-onset disease. Late-onset infections occur in infants aged over one week (greater than

7 days), with most infections evident during the first 3 months of life.17 Almost 50% of all

GBS cases occur in new-borns and early onset GBS disease may account for as much as

80% of neonatal GBS infections; therefore, efforts to prevent GBS disease have been

concentrated on this group.18 Approximately 25% of cases of neonatal GBS disease occur in premature infants.18

Neonates may acquire GBS vertically or in extremely rare cases by the haematogenous route. Early-onset GBS infections are acquired vertically through exposure to GBS from the vagina of a colonized woman who is typically asymptomatic. They occur primarily when GBS ascends from the vagina to the amniotic fluid after onset of labour or rupture of membranes; GBS can also invade through intact membranes.19,20 GBS infected amniotic fluid can then be aspirated into the foetal lungs, leading to bacteraemia, stillbirth, neonatal pneumonia and other syndromes of invasive GBS disease. The foetus also can become infected with GBS during passage through the birth canal; neonates who were exposed to the organism through this route can become colonized at the mucous membrane sites in the gastrointestinal or respiratory tracts, but these colonized neonates commonly remain healthy.

Neonates with early-onset GBS disease generally present with respiratory distress, apnoea, or other signs of sepsis within the first 24 - 48 hours of life.21,22 In young infants, the most common clinical syndromes of invasive GBS disease are bacteraemia or pneumonia, and less often meningitis.17 Cellulitis, osteomyelitis and septic arthritis can also occur.

Meningitis is higher among those with late-onset infections.6 Infants who have GBS disease may require prolonged hospitalization and expensive supportive therapy, and survivors may suffer permanent disability (such as, hearing or visual loss or mental retardation).23 Of those who develop meningitis, 15 - 30% of survivors have long-term neurological sequelae. When neonatal infections caused by GBS appeared in the developed

world in the 1970’s, case fatality ratios were as high as 50%; however, their case fatality ratios in developed countries since the 1990’s have declined as low as 4% for both early- onset and late-onset GBS disease because of advances in neonatal care.24 Striking declines in the disease incidence coincided with increase prevention activities in the 1990’s24 and a further reduction occurred following universal culture based screening for GBS.25 Recent reports from sub-Saharan Africa have reported case fatality ratios as high as 33%.26 Few studies in sub-Saharan Africa differentiate between early and late-onset neonatal sepsis.

This distinction is important because early-onset neonatal sepsis is often acquired vertically; as such, their aetiologies are different and so are the means of their prevention.

The close relationship between mothers and their infants results in shared aetiologies and risk factors for infectious diseases. In developed countries, interventions such as risk-based antibiotic prophylaxis [based on culture or risk factors in pregnancy] have been quite effective in reducing both early-onset neonatal bacterial sepsis24 and puerperal sepsis.27 On the other hand, most developing countries have less robust strategies for the prevention of early-onset bacterial and puerperal sepsis. As a result, 15% of all neonatal deaths in countries such as USA and UK result from infection; but 34% of neonatal deaths in countries such as the Democratic Republic of Congo and Nigeria result from these causes.28

Thirty to fifty per cent of cases of early-onset GBS sepsis develop in neonates born to mothers without clinical risk factors.29 Colonized women usually have no symptoms of disease. A culture is therefore required to detect the presence of the organism in the vagina or rectum. Antenatal cultures have been found to be more effective than risk factors in identifying candidates for intrapartum antimicrobial chemoprophylaxis.30 Although the

exact duration of antibiotics needed to prevent vertical transmission of GBS has been debated,31,32 beta-lactam antibiotics for GBS prophylaxis administered for at least 4 hours before delivery have been found to be highly effective at preventing vertical transmission of GBS as measured by infant colonization33 and protection against early-onset GBS disease.34 The cost per case prevented by selective intrapartum chemoprophylaxis for GBS is like that associated with other maternal screening and intervention programmes for other perinatal diseases such as congenital syphilis.35 Evidence also suggests that treating GBS infection in new-borns is costlier than preventing the infection and that well implemented programmes will substantially reduce illness and death resulting from GBS disease.23

Currently, research in developed countries aim to reduce neonatal sepsis even more through the development of maternal vaccines against prevalent pathogens, such as group B streptococcus.36 Conversely, research in sub-Saharan Africa has focused on treating maternal and child infections rather than prevention; whereas there are still opportunities for simple preventive measures affecting maternal and child health.

Most data on GBS epidemiology over the years has come from Europe and North America.

In Africa, Zimbabwe and Malawi (mainly) have research programmes on GBS colonization and the burden of disease.37,38 Given the high prevalence of maternal and neonatal infection, their associated morbidity and mortality in sub-Saharan Africa and the effectiveness of simple interventions at preventing maternal and early-onset neonatal sepsis, there is need to evaluate the prevalence of maternal GBS colonization and its disease burden in Nigeria and at the University of Benin Teaching Hospital (UBTH) in particular.

Currently, a proportion of pregnant women delivering at UBTH have been observed to

present with obstetric risk factors for GBS sepsis (preterm delivery, prolonged rupture of membranes or intrapartum pyrexia); however, till date, there are no studies that have specifically addressed the burden of GBS in the obstetric and or early neonatal population at UBTH using appropriate microbiological methods.

Since the burden of GBS disease is mainly in pregnant and or postpartum women and their neonates in particular, this study focused on this group of subjects. The measures used to prevent early-onset GBS disease might also prevent some peri-partum maternal infections.24,39

This study evaluated the burden of GBS in our obstetric population and will facilitate the development of strategies to prevent maternal and early-onset neonatal GBS sepsis.

Furthermore, majority of the treatments for puerperal and neonatal sepsis are started empirically, therefore, this study will also enhance the treatment and control of infections caused by GBS by providing local epidemiological data on the antibiotic sensitivity profile of the strains involved for evidence based treatment.40

CHAPTER 2

LITERATURE REVIEW

2.1 Epidemiology of GBS

2.1.1 Prevalence of GBS Colonization and invasive GBS Disease

Maternal colonization by group B streptococci is the most important risk factor for invasive group B streptococcal disease in pregnant/postpartum women and their neonates.8

Approximately 10 - 30% of pregnant women are colonized in the vagina or rectum.6,41,42

The incidence of early-onset GBS infection is approximately 1 per 1,000 live births. In the absence of intervention, 1 - 2% of neonates born to colonized mothers develop early-onset

GBS infections.17,23,43

In the United States of America studies have reported prevalence of colonization in pregnancy of 26.5% in Hawaii44; 26% in Texas42; and 18.6% in New York.41 A systematic review of the prevalence of maternal group B streptococcal colonization in European countries from 1996 - 2006, reported that GBS colonization rate ranged from 6.5 - 36%, with one-third of studies reporting rates of 20% or greater.9 The regional carriage rates were as follows: Eastern Europe, 19.7 - 29.3%; Western Europe, 11.0 - 21.0%;

Scandinavia, 24.3 - 36.0%; and Southern Europe, 6.5 - 32.0%.9 In the United Kingdom, the prevalence has been estimated at 28.0%.45 The prevalence of group B Streptococcal colonization in pregnancy in Poland has been reported to be 19.7%46; although, cervical, vaginal and perianal specimens were obtained rather than vaginal and rectal specimens.46

In a systematic review of studies on GBS colonization among women living in developing countries10; the method of each report was considered adequate if specimens were collected from the vagina and if selective broth media were used. They identified 34 studies which reported cultures from 7,730 women; overall colonization rate was 12.7%. Among only those studies in which methods were considered adequate, 17.8% (675 of 3801) of women were identified as colonized.10 Studies with adequate methods found significantly higher colonization rates (RR, 2.3; 95% CI, 2.0 to 2.6) than those using inadequate methods. When analysis was restricted to reports with adequate methods, the prevalence of colonization by region was as follows: Middle East/North Africa, 22%; Asia/Pacific, 19%; sub-Saharan

Africa, 19%; Americas, 14%; and India/Pakistan, 12%.10 The same study reported the following colonization rates: Gambia 22%; Ivory Coast, 19%; Togo, 4%; Mozambique,

1%; and Nigeria, 20%. The above rates mostly refer to vaginal colonization only; although, the Togo and Gambia studies included rectal specimens.

A preliminary study on the prevalence of group B Streptococcus colonization in 97 antenatal women at Queen Elizabeth Central Hospital Blantyre, Malawi using lower vaginal and rectal specimens reported 16.5% of participants were GBS positive yielding.

Eighty-eight percent of colonized women had a previous bad pregnancy outcome38; however, in this study specimens were transported to the laboratory without transport media. A low recto-vaginal colonization report of 14% from Zaria was not surprising as specimens were vaginal and perianal rather than lower vaginal and rectal swabs.47 Genital colonization by group B streptococci at term pregnancy in Calabar, Nigeria was reported to be 9.0%48; however, specimens were taken from the lower vagina only and rectal

specimens were not taken. An earlier study from Ibadan, South-western Nigeria reported a maternal colonization rate of 19.5%.49 In a more recent study on the isolation and characterization of Group B Streptococci and other pathogens among pregnant women in

Ibadan, Nigeria, GBS colonization was 10.0% among 100 pregnant women.50 In this study, high vaginal swabs were obtained for culture; whereas, obtaining cultures from the lower vagina and rectum could increase yield. A recent report from Ile-Ife Nigeria (2012) states a maternal colonization rate of 11.3%; however, specimens were collected from the vagina only and rectal specimens were not collected.51

A more recent study from Obafemi Awolowo University Teaching Hospital found a late antenatal recto-vaginal GBS colonization rate of 18.2%52; however; sheep blood agar used was not supplemented with antibiotics. While that is standard practice, its supplementation with antibiotics would have enhanced its selectivity for Group B Streptococci even after selective broth enrichment (in Todd-Hewitt Broth supplemented with Colistin Sulphate 10

µg/ml and Nalidixic Acid 15 µg/ml).53 Also, a recent study from University College

Hospital, Ibadan reported a low prevalence of late antenatal recto-vaginal colonization of

9.6% despite using superior CHROMagarTMStrepB agar plates.54 Selective broth enrichment prior to sub-culture is the gold standard (see sections 2.2.4A and 2.2.4B). The yield in that study could have been higher if prior selective broth enrichment had been employed and more so, with use of the broth additive “LIM RambaQUICKTM StrepB”.

“LIM RambaQUICKTM StrepB” has been developed by the same manufacturer of

CHROMagarTMStrepB agar plates specifically for selective enrichment prior to plating on

CHROMagarTMStrepB agar plates. It has been demonstrated that “LIM RambaQUICKTM

StrepB” reduces overgrowth of other organisms on CHROMagarTMStrepB agar plates; thereby, making isolation of GBS easier from CHROMagarTMStrepB agar plates.55,56,57

This recent study from Ibadan that used CHROMagarTMStrepB agar plates also relied on presumptive diagnosis using the catalase test without confirmatory diagnosis by Group B

Streptococcus latex agglutination test due to the cost implications (see section 2.2.4A). The

Christie Atkins Munch-Petersen test when used alone or in addition to the catalase test would have been more sensitive than the catalase test alone (see section 2.2.4A). The catalase test used could have been more prone to false positive results (GBS negative results) if the colonies tested were contaminated by the sheep blood agar used during sub- culture; or, if a wire loop was used to pick up the colonies for the catalase test.58

Significant geographic variation exists in the proportion of women colonized with GBS; but, the range of colonization reported from developing countries is similar to that identified in populations studied in the United States and Europe.9,10,41,42,44 GBS is the leading infectious cause of early neonatal morbidity and mortality in the industrialized world6,7; however, despite similarities in maternal colonization rates between the industrialized and the developing countries, a World Health Organization multicentre study of the bacterial aetiology of serious infections in young infants less than 3 months of age reported that the “virtual absence of GBS was striking”.59 Until recently, GBS was infrequently reported in the developing world as an infectious cause of early neonatal morbidity and mortality.26 Specimen collection and microbiologic methods may account for some of the variations in prevalence reported.

It is not surprising that the evaluation of microbiology of neonatal septicaemia at the

University of Benin Teaching Hospital in 2010 did not find a single case of GBS sepsis as

Group B streptococcus is a fastidious organism and no attempt was made to support its growth.60

2.1.2 Risk Factors for GBS Colonization and Invasive GBS Disease

2.1.2A Obstetric Risk Factors

Studies have identified certain obstetric, maternal and neonatal risk factors for early-onset

GBS disease. In a cohort study of predictors of early-onset GBS disease during the 1980’s, neonates born to women who were identified by antenatal cultures as GBS carriers had 29 times greater risk of early-onset GBS disease than did neonates born to women whose antenatal cultures were negative.43 In the same study women with gestation less than 37 weeks, longer duration of membrane rupture (greater than 12 hours), or intrapartum pyrexia greater than 99.5°F (37.5°C) had 6.5 times greater risk of having a neonate with early-onset

GBS disease compared with women with none of those factors. Of note, women who had one of these risk factors but who had negative antenatal screening cultures were at relatively low risk for delivering neonates with early-onset GBS disease (attack rate 0.9 per 1,000 births) when compared with women who were colonized antenatal but had none of the risk factors (attack rate 5.1 per 1,000 births). In a preliminary study of the obstetric population in a community, the incidence of GBS disease in selected women colonized with GBS who experienced either preterm labour or preterm rupture of membranes (at less than 37 weeks’ gestation) or prolonged rupture of membranes (greater than 12 hours before

delivery) was 8-fold greater than among colonized women without any of these risk factors and 45-fold greater than among women with these risk factors who were not colonized.61,62

A multi-centre case control study also demonstrated the presence of preterm delivery, intrapartum fever or membrane rupture ≥ 18 hours in 49% of early-onset GBS disease.

Early-onset GBS disease was associated with intrapartum fever (matched OR, 4.1; CI, 1.2

- 13.4) and frequent vaginal exams (matched OR, 2.9; CI, 1.1 - 8.0).63 Another multi-centre case-control study for early-onset neonatal GBS sepsis in the late 1990’s reported prematurity (OR 10.4, 95% CI 3.9-27.6), rupture of membranes more than 18 hours before delivery (OR 25.8, 95% CI 10.2-64.8), rupture of membranes before onset of labour (OR

11.1, 95% CI 4.8-25.9), and intrapartum fever (OR 10.0, 95% CI 2.4-40.8) as significant risk factors for early-onset GBS infection.64

Multiple gestations have been postulated as a risk factor for early-onset GBS disease; but, few studies have shown increased risk independent of prematurity.65,66 Some large studies failed to detect increased risk associated with multiple gestations.67,68

Some obstetric procedures such as frequent vaginal examinations (greater than 5 or 6) after the onset of labour or membrane rupture63,69,70; intrauterine foetal monitoring71,72,73; and mechanical cervical ripening devices74 have been significantly associated with peri partum and perinatal infectious outcomes. Most studies have been limited by an inability to randomly allocate women to treatment groups and have yielded conflicting results as certain procedures might have been used more frequently in high-risk settings.75 A population-based case-control study for risk factors for early-onset GBS disease in neonates after adjusting for potential confounders showed intrauterine foetal monitoring 14

was associated with a more than 2-fold increased risk of neonatal GBS disease (OR 2.24,

95% CI 1.22-4.13).76 In the same study, case neonates were also more likely to have a mother with amnionitis (OR 15.03, 95% CI 5.58-41.89).76

A meta-analysis of available studies examining the use of membrane stripping among women of undetermined GBS colonization status found no significant increase in overall peri-partum or perinatal infection rates among women who underwent this procedure and their infants compared with those who did not.77 Although concerns have been raised about performing membrane stripping and mechanical and/or pharmacological cervical ripening on GBS-colonized women, available data are not sufficient to determine whether these procedures are associated with increased risk of early-onset disease.78,79

Although a risk does exist for transmission of GBS from a colonized mother to her neonate during a planned Caesarean delivery performed before onset of labour in a woman with intact membranes, a retrospective study at a single hospital,80 and a national population- based study from Sweden81 indicated that the risk of early-onset GBS among full term neonates delivered per abdomen is extremely low. Data regarding the risk for transmission to preterm neonates born via elective Caesarean section are limited; however, the risk for transmission is likely much lower than in the setting of vaginal delivery or Caesarean delivery following rupture of membranes or onset of labour.6 Patients expected to undergo planned Caesarean delivery should nonetheless still undergo routine vaginal and rectal screening for GBS at 35 - 37 weeks because onset of labour or rupture of membranes may occur before the planned Caesarean delivery.

Previous delivery of a neonate with invasive GBS disease may increase the risk of early- onset GBS disease in subsequent pregnancies and intrapartum antibiotic prophylaxis has been promoted for such women.30,82,83,84, However, because colonization is transient, a substantial proportion of women with GBS colonization in one pregnancy may no longer be colonized during a subsequent pregnancy.85,86 As a result, colonization with GBS in a previous pregnancy is not considered an indication for intrapartum prophylaxis in subsequent pregnancies; rather, women require evaluation for antenatal colonization in each pregnancy.

2.1.2B Bio-Demographic Risk Factors

The incidence of invasive early-onset GBS disease has also been reported to be higher in infants born to mothers who are less than 20 years of age,67,68 or of Black race or Hispanic ethnicity.18,67,87

Heavy colonization in genital cultures, defined as culture from direct plating rather than only from selective broth, is associated with higher risk for early-onset GBS disease.88

Studies have demonstrated that the odds of GBS infection were 2.54 times higher in infants born to heavily colonized mothers compared with lightly colonized mothers.89 GBS bacteriuria defined as GBS identified in clean-catch urine specimens is considered a surrogate for heavy maternal colonization and is also associated with higher risk for early- onset GBS disease.90 GBS bacteriuria is found in 2 - 7% of pregnant women.91,92 Infants born to mothers with GBS bacteriuria during pregnancy are more frequently and more heavily colonized with GBS.90,92,93 In addition, these infants are at increased risk for invasive GBS disease. A prospective study that provided data comparing attack rates of

GBS infection in infants born to mothers with or without GBS bacteriuria reported a 2.5% prevalence of GBS bacteriuria in women screened between 12 and 38 weeks’ gestation.

There were five cases (7.35%) of invasive GBS disease among 68 infants born to women with GBS bacteriuria compared with zero case among 2,677 women without GBS bacteriuria (P <0.001).91 Persson et al reported one infant with GBS disease among ten born to mothers with GBS bacteriuria levels of over 105 CFU/mL. Six of those women, excluding the mother of the infected infant, received antepartum antibiotics. Any GBS- positive urine culture is a marker for heavy genital tract colonization, therefore; a subsequent culture of the vagina or rectum is not recommended because a negative culture could be a false-negative. GBS bacteriuria is an indication for antibiotic prophylaxis for invasive GBS disease antenatally before onset of labour or membrane rupture.94 Antibiotics do not eliminate GBS from the genitourinary and gastrointestinal tracts, and recolonization after a course of antibiotics can occur95; thus, even after antenatal antibiotic prophylaxis for GBS bacteriuria, GBS bacteriuria at any time during pregnancy remains an indication for intrapartum antibiotic prophylaxis.23,94

The incidence of early-onset GBS disease is also increased with low maternal levels of anti-GBS capsular antibody.96 Sufficient amounts of GBS capsular polysaccharide type- specific serum IgG in mothers have been shown to protect against invasive disease in their infants. This is the basis for research into development of a vaccine against GBS; however, vaccines are yet to achieve licensure. 36,97,98

2.1.2C Neonatal Risk Factors

Neonatal risk factors for early-onset GBS disease include low birth weight and heavy surface colonization with GBS.88,99 Risk factors for late-onset disease are not well documented; however, some evidence suggests that this may be acquired by vertical or nosocomial transmission100,101,102; although acquisition through community sources is also possible.103

2.1.2D Risk Factors for Maternal Invasive GBS Disease

Factors that independently increase the risk for clinical amnionitis include: GBS colonization; duration of membrane rupture greater than 6 hours; duration of internal monitoring greater than 12 hours and more than 6 vaginal examinations.

2.2 Isolation and Identification of GBS

Several factors affect the isolation of GBS from cultures. Numerous studies have demonstrated that the accuracy of antenatal screening cultures in identifying intrapartum colonization with GBS can be enhanced by careful attention to the timing of cultures as close to delivery as feasible; the anatomic sites swabbed; and the precise microbiological methods used for culture and detection of organisms.

2.2.1 Timing of GBS Specimen Collection

Vaginal colonization is unusual in childhood but becomes more common in late adolescence.104 Colonization rates are similar for pregnant and non-pregnant women.

Colonization may be transient; chronic; or intermittent.103,105 Vertical transmission of GBS from the mother to foetus primarily occurs after onset of labour or rupture of foetal membranes. As such, colonization in early pregnancy is not predictive of neonatal sepsis.89 18

Culture screening of both the vagina and rectum for GBS during antenatal care late in pregnancy can detect women who are likely to be colonized with GBS at the time of delivery and are therefore at greater risk of perinatal transmission of the organism.106

In evaluation of a comprehensive GBS treatment strategy involving over 800 women the sensitivity of antenatal cultures 6 or more weeks before delivery for identifying colonization status at delivery was 43% and the specificity was 85%. When cultures were obtained between 1 and 5 weeks before delivery, the sensitivity was 87% and specificity was 97%. The negative predictive value of GBS cultures performed less than 5 weeks before delivery was 95-98%; therefore, the likelihood that a negative antenatal culture will become positive in the 5 weeks after was 5%. When cultures were obtained more than 5 weeks before delivery there is a much greater chance that results will not accurately predict colonization status intrapartum; thus, clinical utility decreases because negative predictive value declines.44 In an evaluation of the predictive value of antenatal culture, it was found that among 26 women whose antenatal cultures were collected less than 5 weeks before delivery, there was a 100% concordance with intrapartum culture status (no false-negative or false-positive antenatal cultures).106 Added benefits of late antenatal screening include: women who book late for antenatal care were eligible; reduced pressure on clinicians to treat GBS carriage antenatally; cultures may take up to 72 hours, so it is still feasible to start intrapartum antibiotics early based on antenatal screening results, thereby enabling adequate antibiotic levels to be reached in the amniotic fluid.23

2.2.2 Sites of GBS Specimen Collection

The gastro-intestinal tract is the primary human reservoir of GBS, with the genito-urinary tract being the most common site of secondary spread. Swabbing the lower vagina and rectum (inserting swab through the anal sphincter), increases the yield substantially compared with sampling the cervix or sampling the vagina without also swabbing the rectum.107108109 When both vaginal and rectal cultures are combined, recovery rate of GBS is increased by 31% or more.110 When clinicians do not culture both sites they miss at least one quarter of patients whose cultures are positive for GBS and who might benefit from antibiotic prophylaxis. Although a small number of studies have examined the ability of perianal or vaginal-perianal cultures to detect GBS colonization, there is limited data on their performance compared with recto-vaginal cultures.111,112 A speculum is not required to take the lower vaginal swabs. When women in the outpatient clinic setting collect their own screening specimens, with appropriate instruction, GBS yield is similar to when specimens are collected by a health-care provider.113,114,115 Although swabbing both sites is recommended and use of two swabs can be justified, both swabs can be placed in a single broth culture medium because the site of isolation is not important for clinical management and laboratory costs can thereby be minimized.94

2.2.3 Transportation of GBS specimens

The use of appropriate transport media can help sustain the viability of GBS in settings where immediate laboratory processing is not possible such as during shipment from satellite clinics to a central microbiology laboratory.116,117 GBS isolates can remain viable in transport media for several days at room temperature; however, the recovery of GBS

isolates declines during 1 - 4 days, particularly at high temperatures and this can lead to false results. Even when appropriate culture media are used, the sensitivity is greatest when the specimen is stored at 4°C before culture and processed within 24 hours of collection.81,118,119,120

2.2.4 Isolation of GBS from specimens

2.2.4A Enrichment, culture and identification of GBS from specimens

Because vaginal and rectal swabs are likely to yield diverse bacteria, use of selective enrichment broth is recommended to maximize the isolation of GBS and avoid overgrowth of other organisms. Many laboratories streak a blood agar plate with the swab, incubate it for 24 hrs and discard it if no growth of GBS is identified. However, the CDC guidelines recommend the use of selective broth media [enriched media supplemented with antimicrobial agents to inhibit competing organisms and enhance the growth of GBS better than agar media]; incubation of inoculated selective broth for 18 - 24 hours; sub-culturing the broth to a sheep blood agar plate; and if GBS is not identified after incubation for 18 -

24 hours, re-incubate and inspect at 48 hours to identify suspected organisms.23 When selective medium is used, there is a 50% higher rate of GBS isolation.110,121 Addition of

5% sheep blood to selective broths can increase the recovery of GBS.122

Selective enrichment broths also can contain chromogenic substrates that provide for a change in colour in the presence of beta-haemolytic GBS. Such broths can facilitate the identification of beta-haemolytic GBS; however non-haemolytic strains will not be detected by these broths alone.123,124,125,126,127,128 Following enrichment, the conventional means for identification of GBS is by isolation on sub-culture to blood agar plates and 21

presumptive identification by the CAMP test,129 or serologic identification using latex agglutination with GBS antisera.130 More recently chromogenic agars that undergo colour change in the presence of beta-haemolytic colonies of GBS have been developed.131,132

Like pigmented enrichment broths, these chromogenic agars can only facilitate detection of beta-haemolytic strains, while the majority will not detect non-haemolytic GBS strains.

2.2.4B Identification of GBS by newer methods: DNA probes; nucleic acid

amplification test; optical immunoassays; and enzyme immunoassays

More rapid tests for the detection of GBS directly from enrichment broths, or after sub- culture have been developed, including DNA probes133 and nucleic acid amplification test

(NAAT).134 The performance of commercially available NAAT on non-enriched samples have demonstrated varying sensitivities (62.5 - 98.5%) and specificities (64.5-99.6%) compared with the gold standard of enrichment followed by sub- culture.127,135,136,137,138,139,140,141,142,143 When comparing both intrapartum NAAT on non- enriched samples and late antenatal enriched culture results to intrapartum enriched culture; two studies found intrapartum NAAT to be slightly more sensitive (95.8% and 90.7% respectively) than late antenatal enriched culture (83.3% and 84.3% respectively); although, with widely overlapping confidence intervals.138,140 Another study reported a statistically significant increase in sensitivity of intrapartum NAAT on non-enriched samples (94.0%) compared with antenatal enriched culture (54.3%).135 The sensitivity of

NAAT for GBS can be increased to 92.5 - 100% with use of an enrichment step before testing the sample134,142; however, this lengthens the time taken to obtain results, thus, will only be useful in late antenatal testing.

Despite the availability of NAAT for GBS in resource rich settings, utility of such test in the intrapartum setting remains low. Although a highly sensitive specific and rapid test could be used to assess intrapartum GBS colonization in a parturient with unknown GBS colonization status who has no risk factors, thereby circumventing the need for antenatal screening, current data does not support their use in replacement of antenatal culture or risk based assessment of women with unknown GBS status on admission for labour because of several factors. These factors include the fact that: the additional time required for enrichment of samples makes it unsuitable for intrapartum testing; the sensitivity in the absence of enrichment is inadequate when compared to enriched culture; concerns about turnaround time during typical use; a delay in administration of antibiotics while waiting for results; lack of a substrate for susceptibility testing in penicillin-allergic women; the complexity of available NAAT is still moderate to high; availability of testing at all times; staffing requirements; inhibitory cost.6 Other rapid tests such as optical immunoassays and enzyme immunoassays when used on a sample without enrichment are also not sufficiently sensitive to detect GBS colonization in the intrapartum setting.136,144,145,146,147

2.3 Prevention of early-onset neonatal invasive GBS disease

2.3.1 Risk-based strategy

Initial CDC guidelines in the 1990s recommended the use of one of two strategies to identify women who should receive intrapartum antibiotic prophylaxis for GBS: a risk- based strategy or a culture-based screening strategy.23 Providers of antenatal care using the risk-based approach identified candidates for intrapartum chemoprophylaxis according to the presence of any of the following risk factors: delivery at less than 37 weeks’ gestation,

intrapartum temperature of 100.4°F (38.0°C) or more, or rupture of amniotic membranes for 18 hours or more. In an effort to avert neonatal infections, maternal fever alone in labour may be used as a sign of chorioamnionitis and hence an indication for antibiotic treatment, particularly among women with a significant risk factor for chorioamnionitis (prolonged labour or prolonged rupture of membranes).6 Because of the association between epidural labour analgesia and fever, chorioamnionitis may be over-diagnosed in women with epidural, leading to unnecessary diagnostic evaluation and intervention148; however, multistate surveillance data suggest that with common epidural use (67% of births), intrapartum temperature of 100.4°F [38.0°C] or more (3.3% of births) and physician diagnoses of chorioamnionitis (3.1% of births) remain relatively rare.149

2.3.2 Culture-based strategy

Providers using the culture-based screening approach screened all pregnant women for vaginal and rectal GBS colonization between 35 and 37 weeks’ gestation. Colonized women were offered intrapartum antibiotics at the time of onset of labour or rupture of membranes if before labour. Under both strategies, intrapartum antibiotic prophylaxis was offered to women with GBS bacteriuria at any time during the index pregnancy or for women who had given birth previously to an infant with invasive early-onset GBS disease.

The guideline also recommended that women with unknown GBS colonization status at the time of delivery be managed according to the presence of intrapartum risk factors.

2.3.3 Appraisal of screening strategies

Theoretical predictions based on population estimates of the proportion of early-onset GBS cases without obstetric risk factors (approximately 45%) suggests that a culture-based screening approach would lead to greater declines than a risk-based approach.29,150

A CDC-sponsored multistate study provided the first large-scale direct comparison of the two strategies. By incorporating population-based surveillance for early onset GBS disease into a sample survey of a population of over 600,000 live births, the CDC found that the screening-based (culture-based) approach was more than 50% more effective than the risk- based approach at preventing perinatal GBS disease.30 Maternal GBS colonization might increase clinical suspicion for early-onset GBS disease in an infant; however, in an era of universal screening, over 60% of early onset GBS cases have occurred among infants born to women who had a negative antenatal GBS culture screen.149,151,152 False-negative cases still occur because culture at 35 - 37 weeks’ gestation will fail to detect some women with intrapartum GBS colonization; however, suboptimal specimen collection timing, methods, transport and/or laboratory processing may be contributing factors. Signs of sepsis in a new-born can be indicative of early-onset GBS disease regardless of maternal colonization status, due to inherent limitations in screening strategies.6

Among neonates with signs of early-onset GBS, the detection of GBS can be increased by performing culture of both blood and cerebrospinal fluid (CSF). Blood cultures can be sterile in as much as 15 - 33% of newborns with meningitis153,154,155,156,157 and the management of neonates with abnormal CSF findings differs from that of an infant with normal CSF.6

2.4 Antibiotic susceptibility of GBS substrates

GBS isolates with confirmed resistance to penicillin or ampicillin have not been observed.158,159,160,161 Penicillin remains the agent of choice for intrapartum antibiotic prophylaxis because of its narrower spectrum of action making it less likely to select for resistant organisms. The efficacy of both penicillin162 and ampicillin163 as intrapartum agents for the prevention of early-onset neonatal GBS has been demonstrated in clinical trials. In contrast, the proportion of GBS isolates with in vitro resistance increases following implementation of neonatal GBS disease prevention protocols. The prevalence of GBS resistance among invasive GBS isolates in North America ranged between 7 - 25% for erythromycin and 3 - 15% for clindamycin.159,160,164 Therefore, women with risk factors for penicillin allergy/anaphylaxis require antibiotic susceptibility testing of GBS isolates.

2.5 Limitations of previous studies

Most studies from sub-Saharan Africa refer to vaginal or cervical colonization and often omitted collection of rectal specimens, the main reservoir. A significant number of studies did not focus on the optimal timing for specimen collection in relation to intrapartum colonization and used microbiological methods which do not favour the isolation of GBS.

In addition, a good proportion of these studies did not associate maternal GBS colonization with obstetric risk factors (past obstetric outcome or the development of intrapartum risk factors) nor did they evaluate the effects on maternal and neonatal health.

This study sought to circumvent these shortcomings by: taking both lower vaginal and rectal specimens intrapartum for cultures and immediately inoculating the specimens into enrichment media. This study used selective enrichment media to favour the growth of

GBS and incubated sub-cultured specimens for 36-48 hours before determining absence of

GBS. This study also evaluated the association between GBS colonization and obstetric risk factors; and observed the effects of recto-vaginal GBS colonization on maternal and perinatal outcome.

2.6 Effect of GBS on pregnant women

GBS are part of the normal flora of the rectum and female urogenital tract. Their chief importance is that they cause invasive disease primarily in pregnant or postpartum women, young infants (less than 90 days old) and older adults with underlying medical conditions such as diabetes mellitus.11,12 GBS in pregnant women cause clinical infections but most women have no symptoms associated with genital tract colonization. Urinary tract infections caused by GBS complicate 2 - 4% of pregnancies.13,14 Pregnancy or the postpartum period may be complicated by amnionitis, premature delivery, low birth weight, stillbirth, endometritis, sepsis, or rarely meningitis caused by GBS.15,16 Fatalities in this group are rare.

2.7 Justification for the research

Thirty to fifty per cent of cases of early-onset GBS sepsis develop in neonates born to mothers without clinical risk factors.29 Colonized women usually have no symptoms of disease. Therefore, a culture is required to detect the presence of the organism in the vagina or rectum. Antenatal cultures have been found to be a more effective than risk factors for identifying candidates for intrapartum antimicrobial chemoprophylaxis.30 Although the exact duration of antibiotics needed to prevent vertical transmission of GBS has been debated,31,32 beta-lactam antibiotics for GBS prophylaxis administered for at least 4 hours

before delivery have been found to be highly effective at preventing vertical transmission of GBS as measured by infant colonization33 and protection against early-onset GBS disease.34 The cost per case prevented by selective intrapartum chemoprophylaxis for GBS is like that associated with other maternal screening and intervention programmes for other perinatal diseases such as congenital syphilis.35 Evidence also suggest that treating GBS infection in new-borns is costlier than preventing the infection and that well implemented programmes will substantially reduce illness and death resulting from GBS disease.23

Currently, research in developed countries aim to reduce neonatal sepsis even more through the development of maternal vaccines against prevalent pathogens, such as group B streptococcus.36 Conversely, research in sub-Saharan Africa has focused on treating maternal and child infections rather than prevention; although, there are still opportunities for simple preventive measures affecting maternal and child health.

Most data on GBS epidemiology over the years has come from Europe and North America.

Only Zimbabwe and Malawi in Africa have active research programmes on GBS colonization and the burden of disease.37,38 Given the high prevalence of maternal and neonatal infections, their associated morbidity and mortality in sub-Saharan Africa and the effectiveness of simple interventions at preventing maternal and early-onset neonatal sepsis, there is need to evaluate the prevalence of maternal GBS colonization and its burden of disease in Nigeria and University of Benin Teaching Hospital (UBTH). Currently, a proportion of pregnant women delivering at UBTH have been observed to present with obstetric risk factors for GBS sepsis (preterm delivery, prolonged rupture of membranes or intrapartum pyrexia); but till date, there are no studies that have specifically addressed the burden of GBS in the obstetric and or early neonatal population at UBTH using appropriate microbiological methods.

Since the burden of GBS disease is mainly in pregnant and or postpartum women and their neonates, this study focused on this group of subjects. The measures used to prevent early- onset GBS disease might also prevent some peri-partum maternal infections.24,39

This study seeks to highlight the burden of GBS in our obstetric population and facilitate the development of strategies to prevent maternal and early-onset neonatal GBS sepsis.

Furthermore, since majority of the treatments for puerperal and neonatal sepsis are started empirically, this study will also enhance the treatment and control of infections caused by

GBS by providing local epidemiological data on the pattern of its clinical manifestations to enhance early detection and treatment.4

CHAPTER 3

AIM AND OBJECTIVES

3.0 Aim

This study aims to estimate the burden of GBS disease in the obstetric and neonatal population at the University of Benin Teaching Hospital.

3.1 Objectives

1. To determine the prevalence for recto-vaginal colonization with GBS in mothers

delivering at UBTH.

2. To determine the prevalence of neonatal colonization and early onset GBS sepsis

in neonates born to mothers delivered at the University of Benin Teaching Hospital.

3. To determine the effect of intrapartum GBS colonization of the recto-genital tract

on mothers during labour and the postpartum period as well as neonatal outcomes

at the University of Benin Teaching Hospital.

3.2 Hypothesis

3.2.1 Null Hypothesis:

Maternal recto-vaginal colonization with GBS intrapartum does not increase the risk of neonatal colonization with GBS, maternal or neonatal early onset GBS infection.

3.2.2 Alternative Hypothesis

Maternal recto-vaginal colonization with GBS intrapartum increases the risk of maternal complications; neonatal colonization with GBS and early onset GBS disease.

CHAPTER 4

METHODOLOGY

4.1 Study Setting

The study was conducted at the Department of Obstetrics and Gynaecology of the

University of Benin Teaching Hospital. The University of Benin Teaching Hospital

(UBTH), a tertiary health facility, came into being in 1973. As the sixth of the 1st generation Teaching Hospitals in Nigeria, it was established to complement her sister institution, University of Benin, and to provide secondary and tertiary care to Edo and Delta

States and their environs. This hospital serves as a major referral centre for Edo, Delta,

Ondo and Kogi States. Patients are usually referred from General Hospitals, government owned health centres, private medical centres and from other departments in the hospital.

It also provides the necessary facilities for the training of high and middle level manpower for the health industry and spearheads research opportunities for lecturers in the University and other interested persons with local morbidity burdens as research question(s). UBTH which is located along the Benin–Lagos Expressway has expanded her facilities tremendously over the years such that it/she now has facilities for over 700 in-patients.

The Antenatal Clinic; Labour Ward and its adjoining side laboratory; Labour Ward

Theatre; Lying-in Wards; Family Planning Unit; and Human Reproductive Research

Programme Unit all form part of the Obstetrics and Gynaecology Department Complex.

The antenatal clinic is run between 09:00 Hours and 15:00 Hours on Mondays, Tuesdays,

Thursdays and Fridays. An average of 162 clients are seen in the antenatal clinic per day.

The Labour Ward has an average of 3,100 deliveries yearly. The Lying-in Wards have a capacity of 84 beds. The neonatal unit is adjacent to the labour ward and the medical microbiology unit is part of the laboratory complex on UBTH premises. There are facilities for culture-based screening of antenatal clients and their neonates. These units all serve the

Department of Obstetrics and Gynaecology.

The Department of Obstetrics and Gynaecology is staffed with 17 Consultants (of which 9 are professors) and 41 Resident Doctors.

4.2 Study Design

This was a prospective cohort study.165

4.3 Study Population

The study population comprised of pregnant women who delivered at UBTH

Labour ward and their neonates.

4.4 Selection Criteria

4.4.1 Inclusion Criteria

i. Women in labour who gave written consent to participate in the study and

presented with their neonates for follow-up during the study.

4.4.2 Exclusion Criteria

i. Clients with premature labour

ii. Clients planned for elective Caesarean section, and

iii. Clients who had received antibiotics in the preceding two weeks.

4.5 Sample Size Calculation

The size of the sample required to detect a true difference of a given magnitude between proportions in two independent groups, with a given significance level; a given power or precision; and continuity correction was derived using formula 22 given by Sahai et al166 and obtained from a computer software called WINPEPI.167

Where A refers to exposed (neonates of colonized mothers); and

B refers to unexposed (neonates of un-colonized mothers).

Ratio of sample size B:A of 2.29 (Saura R O 1994).167,168

The known proportion in Sample B of 0.15 (15%).168

ODD Ratio of 2.25. (from a study by Saura R O 1994).168

The sample size was calculated using WINPEPI 8:1 and the above values

The sample size required for this study was estimated to be 332 mother- infant pairs.

Approx. 95% CI for difference between proportions (D) =D - 0.090 to D + 0.090

For further details on the derivation of sample size see appendix 1.

4.6 Sampling Technique

Systematic random sampling was used in recruiting intrapartum women who met the inclusion criteria until the minimum sample size was reached.

The labour ward has an average of 8 deliveries per day.

Sampling frame = 8 clients

8 Sampling interval = = 2 4 Clients were recruited during admission in labour ward in active phase labour. The data and specimens were collected by the principal investigator (E. E. O.) and two trained research assistants (Resident Doctors). The first client was selected through balloting.

Subsequently, every second client meeting the inclusion criteria was counselled and those who consented were recruited into the study until the required sample size was obtained.

The selected clients were counselled and interviewed and their specimens were collected by the principal investigator.

The sampling interval allowed recruitment of 4 mother-infant pairs per day; therefore, the period of client recruitment was estimated at 12 weeks but spanned 24 weeks. Client follow up was continued for approximately 7 days after recruitment making the total duration of the study 25 weeks. This study was conducted at University of Benin Teaching Hospital from January 1st, 2016 till July 7th, 2016.

4.7 Data Management

4.7.1 Data Collection Tools and Methods

The objectives and potential benefits of this study were explained to the clients and informed consent obtained from willing participants (See appendix II for informed consent form).

The clients’ demographic and obstetric variables were collected using a standardized interviewer schedule (see appendix III for standardized interviewer schedule). Other relevant data were collected from the client’s case records. 34

The methodology for collection of specimens, sepsis screening of neonates and microbiological processing for GBS isolation was discussed with the research team

(including my supervisors, the Neonatologist; and the Medical Microbiologist).

4.7.2 Training of research assistants

Two Resident Doctors in the Department of Obstetrics and Gynaecology were trained as research assistants. The Residents were trained on the proposed method of specimen collection and transportation using simulations with a mannequin available in the Family

Planning Unit. Their responsibility was to assist the principal investigator in collection of specimens.

4.8 Quality control

Streptococcus agalactiae American Type Culture Collection 12386, Kwik-Stik

Microbiologics Minnesota; Escherichia coli American Type Culture Collection 25922,

Kwik-Stik Microbiologics Minnesota; Staphylococcus aureus American Type Culture

Collection 25923, Kwik-Stik Microbiologics Minnesota; and Streptococcus pneumoniae

American Type Culture Collection 25922, Kwik-Stik Microbiologics Minnesota were used as control organisms and tested on each batch of Lim Broth and Columbia blood agar supplemented with 5% defibrinated sheep blood; Nalidixic Acid 15micogram per millilitre and Colistin Sulphate 10microgram per millilitre prepared.

Sterility Test of each batch of the Lim broth was by random sampling. Any four bottles of the broth were randomly selected per batch prepared. The selected bottles were incubated aerobically at 35 - 37oC for 7 days. The selected incubated bottles were examined for turbidity daily. Any bottles showing turbidity were sub-cultured on blood agar plates and 35

incubated aerobically at 37oC for 48 hours. The growths were then identified. The whole of the contaminated batch was discarded.

Sterility Test of each batch of Columbia blood agar base supplemented with 5% defibrinated sheep blood; Colistin Sulphate; and Nalidixic Acid was also by random sampling. Four blood agar plates were randomly selected per batch prepared. The selected plates were incubated aerobically at 35 - 37oC for 48 hours. The incubated plates were examined for growth daily. Any plate showing growth was examined fully and the growth was identified. The whole of the contaminated batch was discarded.

A swab stick from each new batch of sterile swab sticks was also inoculated in Lim Broth which was then subjected to the same sterility test as the un-inoculated Lim Broth.

The procedure for collecting and processing clinical specimens for isolation of GBS was according to the CDC 2010 guidelines as outlined below:

4.9 Specimen collection

Intrapartum culture specimens were collected when recruited participants were admitted in active phase labour. The client’s case files were marked with a coloured sticker so that they were identified from the case files and the principal investigator was notified when they were in second stage of labour.

Vaginal swab culture specimens were collected without using a vaginal speculum by, inserting one swab 2 - 3cm into the vagina, sweeping the swab circumferentially across the mucosa of the lower one-third of the vagina (vaginal introitus) and leaving it in the vagina for approximately 5 seconds. Then, a second swab was used to collect the rectal swab

culture specimen by carefully inserting the swab approximately 2.5cm beyond the anal sphincter and gently rotating it to touch the anal crypts. No antiseptic preparation of the perineum or vulva was carried out before taking the swabs. Cultures were collected in the labour ward setting by the principal investigator or either of the research assistants as earlier stated.

A total of four samples were collected from each neonate immediately after birth (before the neonate was cleaned) viz: the external ear canals swab; anterior nares swab; throat swab and base of the umbilical cord swab.

The swab specimens were immediately taken to the laminar air flow cabinet at the laboratory on the labour ward. This laboratory is also adjacent to the Neonatal Special Care

Baby Unit. In the laminar air flow cabinet, each swab was reopened for inoculation of each swab into a separate McCartney bottle containing 5 ml of selective enrichment broth medium: Todd-Hewitt broth supplemented with Colistin Sulphate (10 μg/ml) and Nalidixic

Acid (15 μg/ml) [Lim broth].

Neonatal cord blood specimens were taken from neonates with perinatal asphyxia or those born to mothers with obstetric risk factors for GBS colonization (prolonged rupture of membranes or intrapartum pyrexia).

Neonatal blood culture specimens were also taken from all neonates that manifested features suggestive of early onset neonatal GBS disease. Cerebro-spinal fluid specimens and pustule swabs were taken when clinically indicated.

Blood culture specimens were collected using a strict aseptic technique. Wearing gloves, the venepuncture sites were thoroughly disinfected using 2% tincture of iodine and a circular action, to swab the area beginning at the point where the needle would enter the vein. The iodine was then allowed to dry for at least 1 minute. Venepunctures were then performed.

Blood culture specimens were inoculated with an aseptic technique by, cleaning the black rubber stopper in the middle of the bottle cover with methylated spirit/ethanol-ether swabs following which the black rubber stopper was then pierced with a sterile needle to instil 1 mill of blood into 10 ml of the same selective enrichment broth medium above. The bottle covers were never opened to inoculate them with the blood specimens. To increase the chances of isolating GBS from blood, two specimens (collected at different times) were cultured.

4.10 Transport of swabs and processing of specimens

Specimen requisitions indicated clearly that specimens were for Group B streptococcal testing. The dates and times of collection of each of the specimens were clearly indicated on each specimen.

Specimens were transported after having been inoculated into selective enrichment broth.

Inoculated selective enrichment broth specimens were kept at room temperature for no longer than an hour. All inoculated selective enrichment broth specimens including intrapartum specimens collected at night were transported to the main Medical Microbiology

Laboratory at UBTH for immediate incubation within an hour of collection.

Inoculated Lim broth bottles were incubated in ambient air at 37.00C for 18 - 24 hours.

After 18 - 24 hours incubation, the inoculated Lim broths were examined for growths as evident by turbidity; in the absence of which, the inoculated Lim broths were re-incubated for another 18 - 24 hours.

After 18 - 24 hours’ incubation (or up to 48 hours when indicated) the inoculated selective enrichment broths were then sub-cultured onto another selective medium: Columbia blood agar base supplemented with 5% defibrinated sheep blood, Colistin Sulphate (10 μg/ml) and Nalidixic Acid (15 μg/ml). Inoculated blood agar Petri dishes were then incubated in ambient air at 37.00C for 18-24 hours.

4.11 Presumptive identification of GBS

After 18 - 24 hours’ incubation the inoculated blood agar plates were inspected for identification of colonies suggestive of GBS (a narrow zone of beta haemolysis on blood agar, and gram-positive cocci). Haemolysis was often difficult to observe; so, when beta haemolysis was not observed, colonies were removed from the blood agar plates and the blood agar plates were re-examined before conclusively determining the absence of beta haemolysis (i.e. presence of gamma haemolysis). Typical colonies without haemolysis were also further tested. If GBS was not identified after incubation for 18 – 24 hours, the plates were re-incubated overnight and re-examined at 36 - 48 hours for suspicion of GBS colonies. Suspicious colonies were subjected to biochemical tests for presumptive diagnosis vis: Gram staining (crystal violet, lugol iodine, acetone and safranin); Catalase test, Coagulase test; and Christie Atkins Munch-Petersen (CAMP) test. Beta haemolysis was often demonstrable only after removing the colonies from the blood agar plates.

4.12 Confirmation of GBS

The streptococcal grouping latex agglutination test was used for specific identification of

GBS colonies.

Following definitive isolation of GBS, antimicrobial sensitivities were performed according to the Clinical and Laboratory Standard’s Institute-Recommended methods for disk susceptibility testing

4.13 Mother-infant pair follow up

Study participants were reviewed 24 hours and 48 hours postpartum before they were discharged home. Mothers were given a dedicated mobile phone number via which to reach the principal investigator if they noticed any problems; features of postpartum endometritis; or features of neonatal sepsis. The study participants had mobile phone call backs on the 6th day postpartum to enquire of wellness and request them to return for evaluation on the 7th day postpartum. To minimize loss of cases, the Professor Jackson

Omene Special Care Baby Unit (neonatal unit) was also scouted daily in case any of the discharged study participants were readmitted there.

4.14 Data Analysis

Data was coded into an IBM SPSS spread sheet and analysed using IBM SPSS Statistics, version 22.0, Chicago, Illinois, U.S.A. and WINPEPI updated 2011 version 8.1.

Categorical variables were expressed as percentages. Significance of associations were determined using Chi-square test (*) with Yate’s continuity correction (§) where appropriate; Fisher’s exact test (ǂ); or, Chi-Square for Linear Trend (Ʊ). Numerical

variables were expressed as mean ± standard deviation and compared using student’s t- test. All tests of association were considered significant at a P-value of less than 0.05.

Degree of association was expressed as relative risk with their 95% confidence interval.

Social class was assigned using the social class stratification by Olusanyan et al.169

4.15 Definition of Terms

Culture positive mothers were defined as confirmed isolation of GBS from lower vaginal and or rectal cultures intrapartum.

Neonatal colonization was defined as confirmed isolation of GBS from any of the designated culture sites from neonates at birth.

For multiple gestations, neonatal colonization was defined as confirmed isolation of GBS from any of the designated culture sites from one or more neonates at birth.

Early-onset GBS neonatal sepsis was defined as confirmed isolation of GBS from blood and or cerebro-spinal fluid cultures in neonates who present with clinical features of sepsis within the first 48hours after birth (prior to the usual time of discharge at 3 days of age).

4.16 Outcome Measure

4.16.1 Primary outcome measure

Proportion of the obstetric population with GBS positive or negative cultures.

4.16.2 Secondary outcome measures: i) Proportion of neonates with GBS positive or negative cultures. ii) Proportion of mothers with maternal complications.

a. Proportion of mothers with GBS positive cultures having prolonged labour.

b. Proportion of mothers with GBS positive cultures having prolonged rupture

of membranes.

c. Proportion of mothers with GBS positive cultures having fever in labour iii) Proportion of neonates who have clinical neonatal sepsis. iv) Proportion of neonates with sepsis and GBS positive cultures. v) Proportion of neonates with sepsis and GBS negative culture. vi) Neonatal Care Unit admission rate. vii) Duration of hospital admission. viii) Mode of delivery ix) Foetal outcome:

a. Apgar sores at 1st and 5th minutes.

b. Birth weight. x) Duration of labour

4.17 Ethical Considerations

Ethical clearance was obtained from the Research and Ethical Committee of the University of Benin Teaching Hospital.

Written informed consent was obtained from willing participants.

Specimens were collected in the presence of a chaperon.

Privacy was maintained during contact with participants.

Client confidentiality was maintained.

All clients recruited into the study were subjected to the same procedures.

This study did not interfere with patient care.

Clients received health education and were told the possible benefit of the study.

Tests were performed free.

The findings of the study would assist in estimating the burden of GBS disease in the obstetric population; identifying the proportion of the obstetric population that would require treatment; and help in subsequent development of policies about care of similar populations.

CHAPTER 5

RESULTS

A total of 335 mother-infant pairs were studied from January 1st, 2016 till July 7th, 2016.

Mean age was 30.0 ±4.6 years (range 16 - 41 years) and majority (69.2%) of the participants were between 25-34 years. There was no statistically significant difference in the mean ages of mothers with intrapartum recto-vaginal colonization by GBS when compared to mothers without colonization (30.1 ± 4.6 years; 29.9 ± 4.6 years, t = 0.387, P

= 0.699).

More than three-quarters (76.1%) of the parturients had tertiary education. The predominant tribes in both cohorts were Benin and Esan (62.7%). The predominant religion among the participants was Christianity (98.5%). Most participants were married and social class two was the most prevalent. Two clients were single and one client was divorced; as such, 3 clients did not have the full complement for social class stratification. No linear trend was observed between the social classes in this study. Socio-demographic characteristics and GBS colonization of respondents are depicted in Table I. Intrapartum recto-vaginal colonization with GBS did not show any significant association with the socio-demographic variables except for tribe.

The prevalence of maternal recto-vaginal colonization with GBS in this study was 30.1%.

All the GBS colonized mother-infant pairs had GBS isolated from their maternal recti.

Majority (88.1%) of the participants who were GBS colonized were both rectal and vaginal carriers. There were no participants who were only vaginal GBS carriers without

corresponding rectal carriage. The prevalence of neonatal colonization with GBS in this study was also 30.1% (See Table II).

Table III shows that there was a 100% vertical transmission (colonization) rate of GBS from the 101 GBS colonized mothers to their 101 neonates. Two neonates (0.9%) born to

GBS culture negative mothers were initially not colonized; but, were found to be colonized at follow-up 7 days postnatum. These finding were statistically significant (P, less than [<]

0.001; relative risk [RR], 117.000; 95% confidence interval [CI], 29.436 – 465.049).

Table IV shows that 26 of the 101 neonates born to mothers with intrapartum recto-vaginal colonization by GBS developed early onset neonatal invasive GBS disease indicating an attack rate of 25.7%. None of the neonates born to mothers without recto-vaginal colonization by GBS developed early onset neonatal invasive GBS disease. The association between maternal intrapartum recto-vaginal colonization by GBS and development of early onset neonatal invasive GBS disease was statistically significant. Because one cell had zero counts, all cells in the contingency table were adjusted by adding 1 count to each cell to allow for calculation of relative risks and confidence intervals (P, <0.001; RR, 61.864; CI,

8.521 – 449.171).

Tables Va and Vb show the associations between maternal intrapartum recto-vaginal colonization with GBS and obstetric variables of participants. Maternal intrapartum recto- vaginal colonization with GBS did not show any significant association with parity; a history of previous spontaneous miscarriage; previous preterm birth; previous preterm premature rupture of membranes; and previous premature rupture of membranes. Mothers with intrapartum recto-vaginal colonization with GBS had slightly higher relative risks for 45

previous stillbirths; previous early neonatal deaths; and previous neonatal care unit admissions for early onset neonatal sepsis but none of these observations were statistically significant. The association between intrapartum recto-vaginal colonization by GBS and previous perinatal death was not statistically significant (P, 0.144; RR, 1.405; 95% CI,

0.915 – 2.157). Thirty nine percent of mothers had a history of a previous bad obstetric outcome. There was no significant association between intrapartum recto-vaginal colonization by GBS and mothers having had a history of any previous bad obstetric outcome comprising of a computation of any: previous spontaneous miscarriage; previous preterm birth; previous preterm premature rupture of membranes; previous premature rupture of membranes; previous stillbirth; previous early neonatal death; and previous infant with early onset neonatal sepsis (P, 0.786; RR, 1.047; 95% CI, 0.751 – 1.460).

There was no significant association between maternal intrapartum recto-vaginal colonization with GBS and a history of vaginal discharge in the index pregnancy. The relative risk associated with having had a previous urinary tract infection in the index pregnancy was more in GBS colonized mothers but was not statistically significant (P,

0.106; RR, 1.563; 95% CI, 0.958 – 2.550). Maternal intrapartum recto-vaginal colonization by GBS was significantly associated with duration of rupture of membranes to delivery of

6 hours or more in the index pregnancy (P, 0.045; RR, 1.365; CI, 1.015 – 1.834); but, this was not significantly associated with adverse maternal nor perinatal outcomes. Recto- vaginal colonization by GBS also showed an increase risk of duration of rupture of membranes to delivery of 12 hours; but, this was not statistically significant (P, 0.080; RR,

1.569; CI, 0.950 – 2.594). Duration of rupture of membranes to delivery of 18 or 24 hours or more showed no significant associations with GBS status.

Intrapartum pyrexia showed no significant association with maternal GBS carriage (36.6°C

± 0.4°C; 36.6°C ± 0.4°C, t = 0.172, P = 0.864). However, maternal GBS carriage was associated with an increased relative risk of intrapartum pyrexia; although, this finding was not statistically significant (P, 1.000; RR, 2.317; CI, 0.146 – 36.678). Over half (68.7%) of the confinements in this study occurred at full term (39 weeks 0 days to 40 weeks and 6 days of gestation) or late term (41 weeks 0 days to 41 weeks and 6 days of gestation). Less than a third (31.3%) of the confinements were early term deliveries (37 weeks 0 days to 38 weeks and 6 days of gestation). There was a statistically significant difference in the mean gestational ages at confinement among mothers with recto-vaginal colonization by GBS when compared to mothers who were not GBS carriers. (279.6 ± 9.4 days; 275.9 ± 8.7 days, respectively; t, 3.509; P, 0.001).

There was no case of chorioamnionitis among GBS culture positive mothers. There were

3 cases of chorioamnionitis all in mothers without intrapartum recto-vaginal colonization with GBS. These 3 parturients had rupture of their foetal membranes 18, 10 and 3 hours before delivery; had active phase labour durations of 1, 5 and 6 hours; and intrapartum temperatures of 38.3, 36.2 and 36.7 respectively. This observation was not statistically significant (P, 0.609; RR, 0.573; 95% CI, 0.065 – 5.062). There were two cases of postpartum endometritis in this study. Both were in mothers colonized with GBS. The first occurred in a retroviral disease positive parturient on highly active antiretroviral therapy with recto-vaginal colonization by GBS who had an active phase labour duration of 4

hours, spontaneously ruptured her foetal membranes an hour before delivery and shortly after delivery developed uterine tenderness and malodourous lochia. She had good recovery following administration of Amoxycillin/Clavulanic Acid. Her neonate also developed early onset GBS sepsis. The second case occurred in another parturient with intrapartum recto-vaginal GBS colonization who presented in advanced second stage of labour, had artificial rupture of foetal membranes and immediately had a spontaneous vaginal delivery. This finding of postpartum endometritis among GBS colonized mothers was not statistically significant (P, 0.166; RR, 6.874; 95% CI, 0.724 – 65.302). There were no parturients with penicillin allergies.

There were statistically significant associations between neonatal colonization with GBS and neonatal sepsis (P, <0.001; RR, 3.089; 95% CI, 1.649 – 5.785); neonatal care unit admissions (P, 0.002; RR, 2.439; 95% CI, 1.361 – 4.369); and duration of neonatal care unit admissions (Chi-Square for Linear Trend P, 0.001; RR, 8.667 for neonatal admission duration of 7 days or more among neonates with GBS colonization in comparison to neonates without GBS colonization). GBS colonized neonate’s first minute APGAR scores were significantly lower than those of GBS non-colonized neonates (Chi-Square for Linear

Trend P, 0.002; RR, 12.907 for APGAR scores of 3 or less). In this study, at the fifth minute postnatum, neonates colonized with GBS were significantly more prone to perinatal asphyxia (Chi-Square for Linear Trend P, 0.011; RR, 2.457 for mild birth asphyxia). The occurrence of one fresh stillbirth in a GBS colonized foetus (with GBS bacteraemia) born to a GBS colonized mother was not found to be statistically significant (P, 0.665; RR,

4.583; 95% CI, 0.420 - 49.975). Presence of a live foetus was considered as an inclusion

criterion as such no macerated stillbirth was observed. GBS colonized neonates showed an increased probability of suffering early neonatal death; however, this was not found to be statistically significant (P, 0.050; RR, 9.267; 95% CI, 1.049 – 81.887). One GBS colonized neonate with GBS bacteraemia was born in shock with perinatal asphyxia and suffered early neonatal death before the fifth minute postnatum. Another GBS colonized neonate with GBS bacteraemia also born in shock with perinatal asphyxia was admitted to the neonatal care unit with hypothermia (temperature was 35.0oC), the neonate developed hyperpyrexia [temperature 40.1oC] 10 hours postnatum with severe dyspnoea and suffered early neonatal death 13 hours postnatum. When stillbirths and early neonatal deaths were viewed holistically from a standpoint of perinatal deaths there was a statistically significant association with GBS colonized neonates (P, 0.016; RR, 11.584; 95% CI, 1.371 – 97.901).

There was no significant association between neonatal GBS colonization status and neonatal sex; neonatal birthweight; number of vaginal examinations in labour; or state of the umbilical cord. There were statistically significant associations between neonatal GBS colonization status and abnormal neonatal temperature; respiratory rates; and heart rates.

Colonized neonates were more likely to develop pneumonia than non-colonized neonates, but this was not statistically significant. There was no case of neonatal meningitis (see

Tables VIa and VIb).

There were statistically significant associations between early onset invasive neonatal GBS disease and neonatal sepsis (P, <0.001; RR, 12.584; 95% CI, 7.419 – 21.345); cord sepsis

(P, <0.001; RR, 17.827; 95% CI, 3.117–101.959); neonatal pneumonia (P, <0.001; RR,

15.846; 95% CI, 3.745 – 67.046) and Hypoxic ischaemic encephalopathy (P, 0.025; RR,

11.885; 95% CI, 1.745 – 80.956) (see Table VII). The GBS attack rate was 25.7% among

GBS positive mother-infant pairs with a case fatality rate of 15.4%. The median duration for onset of features of early onset GBS disease in this study was 18 hours ± 12 (range 0-

144), with 76% manifesting within 48 hours postnatum. The mean cost of neonatal unit admission per case of early onset invasive GBS disease was 22,440 naira. Not all cases of early onset neonatal GBS disease had neonatal unit admission as: one was stillborn; one suffered early neonatal death before the fifth minute postnatum; two mothers declined neonatal unit admission and discharged their neonates home against medical advice; one was discharged home and presented with features of neonatal sepsis on the 6th day postnatum and was successfully managed as an outpatient with full recovery on follow up; and one GBS colonized neonate who had a single spike of pyrexia of 37.70C remained normal during subsequent observation and antibiotic treatment.

Table VIIIa and VIIIb show a subgroup analysis of GBS colonized neonates that did or did not develop early onset GBS disease in relation to obstetric and neonatal parameters. There was no significant association between neonates that developed early onset GBS and their mothers having had: 6 or more vaginal examinations intrapartum; prolonged durations of active phase labour.; or intrapartum temp over 37.5°C (36.5°C ± 0.4°C; 36.6°C ± 0.4°C; t

= -1.621; P = 0.108). Although the associations were not statistically significant and the outcomes of interest were rare, there were increased relative risks of developing early onset neonatal GBS disease among colonized neonates of colonized mothers who were observed to have: intrapartum temperature below 36.6 or above 37.2 (P, 0.047; RR, 2.206; 95% CI,

1.057 – 4.604); prolonged premature rupture of membranes of 24 hours or more (P,1.000;

RR,1.980; 95% CI, 0.475 – 8.247); prolonged rupture of membranes before delivery of 24 50

hours or more (P, 0.823; RR,1.600; 95% CI, 0.518 – 4.943); uterine tenderness (P, 0.577;

RR, 4.000; 95% CI, 2.849 – 5.617); malodourous lochia (P, 0.577; RR, 4.000; 95% CI,

2.849 – 5.617); and postpartum endometritis (P, 1.000; RR, 1.980; 95% CI, 0.475 – 8.247).

GBS colonized neonates who developed early onset GBS disease showed statistically significant associations with perinatal asphyxia; neonatal shock and perinatal death (see

Table VIIIb).

Table I: Socio-Demographic variables and GBS colonization of participants

Socio-Demographic Variable GBS positive GBS negative Total n=335 P value culture n = culture n= 234 [n (%)] 101 [n (%)] [n (%)] Age group (years) <15 0 (0.0) 0 (0.0) 0 (0.0) 0.624Ʊ 15-19 1 (1.0) 3 (1.3) 4 (1.2) 20-24 10 (9.9) 23 (9.8) 33 (9.9) 25-29 35 (34.7) 88 (37.6) 123 (36.7) 30-34 33 (32.7) 76 (32.5) 109 (32.5) 35-39 20 (19.8) 39 (16.5) 59 (17.6) 40-44 2 (2.0) 5 (2.1) 7 (2.1) >44 0 (0.0) 0 (0.0) 0 (0.0)

Highest level of education No formal 0 (0.0) 0 (0.0) 0 (0.0) 0.447Ʊ Primary 7 (6.9) 15 (6.4) 22 (6.6) Secondary 20 (19.8) 38 (16.2) 58 (17.3) Tertiary 74 (73.3) 181 (77.4) 255 (76.1) Others 0 (0.0) 0 (0.0) 0 (0.0)

Tribe Benin 44 (43.6) 111 (47.4) 155 (46.3) <0.001‡ Esan 19 (18.8) 36 (15.4) 55 (16.4) Etsakor 11 (10.9) 17 (7.3) 28 (8.4) Urhobo 5 (5.0) 4 (1.7) 9 (2.7) Ika 13 (5.6) 0 (0.0) 13 (3.9) Yoruba 1 (1.0) 6 (2.6) 7 (2.1) Ibo 13 (12.9) 31 (13.2) 44 (13.1) Hausa 2 (2.0) 3 (1.3) 5 (1.5) Others 6 (6.0) 13 (5.6) 19 (5.7)

Religion Christianity 100 (99.0) 230 (98.3) 330 (98.5) 1.000‡ Islam 1 (1.0) 3 (1.3) 4 (1.2) Traditional African Religion 0 (0.0) 1 (0.4) 1 (0.3)

Marital status Married 86 (85.1) 207 (88.5) 293 (87.5) 0.503‡ Single 1 (1.0) 2 (0.9) 3 (0.9) Divorced 0 (0.0) 0 (0.0) 0 (0.0) Separated 1 (1.0) 0 (0.0) 1 (0.3) Widowed 0 (0.0) 1 (0.4) 1 (0.3) Common Law 13 (12.9) 24 (10.3) 37 (11.0)

Socioeconomic Class* 1 8 (8.0) 18 (7.8) 26 (7.8) 0.920Ʊ 2 56 (56.0) 139 (59.9) 195 (58.7) 3 28 (28.0) 51 (22.0) 79 (23.8) 4 7 (7.0) 23 (9.9) 30 (9.0) 5 1 (1.0) 1 (0.4) 2 (0.6)

Cigarette smoking Yes 0 (0.0)) 3 (1.3) 332 (99.1) 0.609§ No 101 (100) 231 (98.7) 3 (0.9) * Pearson Chi-Square § Yate’s Continuity Correction ‡ Fisher’s Exact Test Ʊ Chi-Square for Linear Trend

Table II: Prevalence of GBS isolates from culture specimens in the participants

Frequency Percentage (%) (n = 335) Mother-infant pair GBS status Positive 101 30.1 Negative 234 69.9

All mothers and baby’s specimen positive Yes 85 24.5 No 250 74.6

Intra-partum recto-vaginal GBS cultures Positive 101 30.1 Negative 234 69.9

Maternal GBS colonized site Rectum only 12 3.6 Both rectum and vaginal 89 26.6 None 234 69.9

Neonatal cultures for GBS colonization Positive 101 30.1 Negative 234 69.9

All initial neonatal result All positive 87 26.0 Some positive 14 4.2 All negative 234 69.9

Neonatal throat swab Positive 96 28.7 Negative 239 71.3

Neonatal nostril swab Positive 90 26.9 Negative 245 73.1

Neonatal ears swab Positive 90 26.9 Negative 245 73.1

Neonatal umbilical swab Positive 98 29.3 Negative 237 70.7

Neonatal blood culture Positive 24 7.2 Negative 36 10.7 Not done 275 82.1

Neonatal pustules swab Positive 2 0.6 Not done 333 99.4

Early-onset neonatal GBS disease (sepsis) Positive 26 7.8 Negative 309 92.2

Table III: GBS colonization intra-partum versus the colonization in early neonatal period.

Neonates Neonates not Relative P value 95% Confidence colonized with colonized with Risk Interval GBS n=101 GBS n=234 [n (%)] [n (%)]

Mothers 101 (100.0) 0 (0.0) 117.00 <0.0001* 29.436 - 465.049 colonized with GBS intra-partum

Mothers not 2 (0.9) 232 (99.1) colonized with GBS intra-partum

* Pearson Chi-Square

Table IV: Maternal intra-partum GBS colonization and neonatal early-onset invasive GBS disease

Neonates with Neonates without Relative P value 95% Confidence invasive early- invasive early- Risk Interval onset GBS onset GBS disease disease n=26 [n n=309 (%)] [n (%)]

Mothers 26 (25.7%) 75 (74.3%) 61.864 <0.001* 8.521 – 449.171 colonized with GBS intra-partum

Mothers not 0 (0.0%) 234 (100%) colonized with GBS intra-partum

* Pearson Chi-Square

Table Va: Obstetric variables among participants

Obstetric variable GBS-Positive GBS negative Relative P value 95% culture n=101 culture n=234 risk Confidence [n(%)] [n(%)] Interval Parity 1-4 90 (89.1) 216 (92.3) 1.290 0.339* 0.785 - 2.120 >4 11 (10.9) 18 (7.7)

Previous Spontaneous miscarriage Yes 19 (18.8) 47 (20.1) 0.944 0.788* 0.620 - 1.438 No 82 (81.2) 187 (79.9)

Previous preterm deliveries Yes 3 (3.0) 13 (5.6) 0.610 0.460§ 0.217 - 1.715 No 98 (97.0) 221 (94.4)

Previous preterm premature rupture of membranes Yes 1 (1.0) 3 (1.3) 0.828 1.000§ 0.150 - 4.555 No 100 (99) 231 (98.7)

Previous premature rupture of membranes Yes 12 (11.9) 29 (12.4) 0.967 0.896* 0.583 - 1.604 No 89 (88.1) 205 (87.6)

Previous Stillbirth Yes 9 (8.9) 14 (6.0) 1.327 0.331* 0.775 - 2.273 No 92 (91.1) 220 (94.0)

Previous early neonatal death Yes 6 (5.9) 11 (4.7) 1.181 0.635* 0.607 – 2.298 No 95 (94.1) 223 (95.3)

Previous infant with neonatal sepsis Yes 9 (8.9) 13 (5.6) 1.392 0.255* 0.819 – 2.367 No 92 (91.1) 221 (94.4)

Vaginal Discharge in index pregnancy Yes 9 (8.9) 20 (8.5) 1.032 0.913* 0.584 – 1.823 No 92 (91.1) 214 (91.5)

Urinary tract infection in index pregnancy Yes 10 (9.9) 12 (5.1) 1.563 0.106* 0.958 – 2.550 No 91 (90.1) 222 (94.9)

Human Immunodeficiency Virus status in index pregnancy Positive 7 (6.9) 6 (2.6) 1.845 0.112§ 1.084 – 3.138 Negative 94 (93.1) 228 (97.4) * Pearson Chi-Square § Yate’s Continuity Correction

Table Vb: Obstetric variables among participants continued

Obstetric variable GBS-Positive GBS negative Relative P value 95% culture n=101 culture n=234 risk Confidence [n(%)] [n(%)] Interval Preterm labour in index pregnancy Yes 1 (1.0) 2 (0.9) 1.158 1.000§ 0.106 –12.631 No 100 (99.0) 232 (99.1)

Preterm premature rupture of membranes Yes 1 (1.0) 2 (0.9) 1.158 1.000§ 0.106 –12.631 No 100 (99.0) 232 (99.1)

Membrane Sweeping in index pregnancy Yes 2 (2.0) 7 (3.0) 0.732 0.875§ 0.213 – 2.512 No 99 (98.0) 227 (97.0)

Prolonged rupture of membranes in index pregnancy (24 hours) Yes 5 (5.0) 17 (7.3) 0.681 0.433* 0.258 - 1.797 No 96 (95.0) 217 (92.7)

Intra-partum fever >37.5° in index pregnancy Yes 1 (1.0) 1 (0.4)) 2.317 1.000§ 0.146 –36.678 No 100 (99.0) 231 (99.6)

Duration of active phase labour ≥12 8 (7.9) 19 (8.1) 0.976 0.951* 0.442 - 2.155 <12 93 (92.1) 215 (91.9)

Gestational age Early term 20 (19.8) 85 (36.3) 0.545 0.003* 0.355 - 0.836 Full and Late term 81 (80.2) 149 (63.7)

Body mass index Overweight/Obese 94 (93.1) 212 (90.6) 1.273 0.460* 0.653 –2.479 Normal 7 (6.9) 22 (9.4)

Use of Oxytocics Yes 12 (11.9) 37 (15.8) 0.787 0.350* 0.467 - 1.325 No 89 (88.1) 197 (84.2)

Foul smelling purulent liquor Present Absent 0 (0.0) 3 (1.3) 0.573 0.609§ 0.065 - 5.062 101 (100.0) 231 (98.7)

Uterine tenderness Present 1 (1.0) 0 (0.0) 4.583 0.665§ 0.420 –49.975 Absent 100 (99.0) 234 (100.0)

Postpartum endometritis Yes 2 (2.0) 0 (0.0) 6.874 0.166§ 0.724 –65.302 No 99 (98.0) 234 (100.0) * Pearson Chi-Square § Yate’s Continuity Correction

Table VIa: Neonatal variables and outcomes

Variable GBS-Positive GBS negative Relative P value 95% culture n=101 culture n=234 risk Confidence [n(%)] [n(%)] Interval Neonatal Sepsis NNS 20(19.8) 15(6.4) 3.089 <0.001* 1.649 – 5.785 None 81(80.2) 219(93.6)

Neonatal Care Unit admission Yes 20(19.8) 19(8.1) 2.439 0.002* 1.361 – 4.369 No 81(80.2) 215(91.9)

Duration of admission (Days) <7 89(89.9) 231(98.7) 7.879 <0.001§ 2.216 – 28.017 ≥7 10(10.1) 3(1.3)

Mode of delivery Vaginal 101(100.0) 233(99.5) 1.098 1.000§ 0.220 – 5.485 Instrumental 0(0.0) 1(0.4)

APGAR score at 1 minute ≥7 86(86.0) 222(94.9) 2.730 0.006* 1.310 – 5.691 <7 14(14.0) 12(5.1)

APGAR score at 5 minutes ≥7 94(94.0) 231(98.7) 4.680 0.038§ 1.194 – 18.344 <7 6(6.0) 3(1.3)

Stillbirth Fresh stillbirth 1(1.0) 0(0.0) 4.583 0.665§ 0.420 – 49.975 No stillbirth 100(99.9) 234(100.0)

Early neonatal death Yes 4(4.0) 1(0.4) 9.267 0.050§ 1.049 – 81.887 No 97(96.0) 233(99.6)

Perinatal death Yes 5(5.0) 1(0.4) 11.584 0.016§ 1.371 – 97.901 No 96(95.0) 233(99.6)

Sex Male 54(54.0) 113(51.8) 1.061 0.720* 0.766 – 1.470 Female 46(46.0) 105(48.2)

Birth weight Low 2(2.0) 6(2.6) 0.826 1.000§ 0.246 – 2.773 Normal/High 99(98.0) 228(97.4) * Pearson Chi-Square § Yate’s Continuity Correction

Table VIb: Neonatal variables and outcomes continued

Variable GBS-Positive GBS negative Relative P 95% culture n=101 culture n=234 risk value Confidence [n(%)] [n(%)] Interval

Number of vaginal examination ≥6 16(15.8) 38(16.2) 0.980 0.928* 0.626 – 1.533 <6 85(84.2) 196(83.8)

Neonates Temperature <36.2 or > 37.5 19(18.8) 15(6.4) 2.935 0.001* 1.554 – 5.540 36.2 – 37.5 82(81.2) 219(93.6)

Neonates Respiratory rate <40 or >60 10(9.9) 7(3.0) 1.100 0.008* 1.296 – 8.450 40 - 60 91(90.1) 227(97.0)

Neonates Heart rate <100 or >150 14(13.9) 13(5.6) 2.495 0.010* 0.217 – 5.115 100 - 150 87(86.1) 221(94.4)

State of umbilical cord Septic 3(3.0) 2(0.9) 3.475 0.330§ 0.590 – 20.483 Wet and clean 98(97.0) 232(99.1)

Hypoxic ischaemic encephalopathy Yes 2(2.0) 2(0.9) 2.317 0.747§ 0.331 – 16.220 No 99(98.0) 232(99.1)

Pneumonia Yes 4(4.0) 3(1.3) 3.089 0.247§ 0.704 – 13.552 No 97(96.0) 231(98.7)

* Pearson Chi-Square § Yate’s Continuity Correction NNS - Neonatal sepsis

Table VII: Early onset GBS in relation to obstetric and neonatal parameters

Variable Yes No Relative P value 95% n=26 n=309 risk Confidence Interval Sex of baby Male 18 (10.2) 159(89.8) 2.008 0.081* 0.898 – 4.491 Female 8 (5.1) 150(94.9)

Number of vaginal examination ≥6 3 (5.6) 51 (94.4) 0.679 0.701§ 0.211 – 2.181 <6 23 (8.2) 258(91.8)

Highest temperature >37.5 ≤37.5 20 (76.9) 21(6.8) 11.319 <0.001§ 7.120 – 17.992 6 (23.1) 288(93.2)

Respiratory rate <60 8 (30.8) 9 (2.9) 10.564 <0.001§ 4.452 – 25.070 ≥60 18 (69.2) 300 (97.1)

Heart rate <100 4 (15.4) 0 (0.0) 55.535 <0.001§ 6.720 – 458.952 ≥100 22 (84.6) 309(100.0)

State of umbilical cord Septic 3 (11.5) 2 (0.6) 17.827 <0.001§ 3.117 – 101.959 Clean 23 (88.5) 307 (99.4)

Hypoxic ischaemic encephalopathy Yes 2 (7.7) 2 (0.6) 11.885 0.025§ 1.745 – 80.956 No 24 (92.3) 307 (99.4)

Sepsis Yes 18 (69.2) 17 (5.5) 12.584 <0.001§ 7.419 – 21.345 No 8 (30.8) 292 (94.5)

Pneumonia Yes 4 (15.4) 3 (1.0) 15.846 <0.001§ 3.745 – 67.046 No 22 (84.6) 306 (99.0)

Prolonged rupture of membranes ≥24 hours 2 (7.7) 20 (6.5) 1.188 1.000§ 0.294 – 4.807 <24 hours 24 (92.3) 289 (93.5) * Pearson Chi-Square § Yate’s Continuity Correction

Table VIIIa: Subgroup analysis of GBS colonized neonates that did versus those that did not develop early onset GBS in relation to obstetric and neonatal parameters

Variable Yes No Relative P value 95% n=26 n=75 risk Confidence Interval Previous premature rupture of membranes ≥2 2 (7.7) 1 (1.3) 2.722 0.329§ 1.138 – 6.513 <2 24 (92.3) 74 (98.7)

Previous early onset neonatal sepsis ≥2 <2 1 (3.8) 0 (0.0) 4.000 0.577§ 2.849 – 5.617 25 (96.2) 75(100.0)

Any previous bad obstetric outcome Yes 14 (53.8) 26 (34.7) 1.779 0.085§ 0.920 - 3.442 No 12 (46.2) 49 (65.3)

Number of vaginal examination ≥6 23 (88.5) 62 (82.7) 1.443 0.700§ 0.491 - 4.241 <6 3 (11.5) 13 (17.3)

Intra-partum fever >37.5° in index pregnancy Yes 0 (0.0) 1 (1.3) 1.259 1.000§ 0.246 - 6.444 No 26 (100.0) 74 (98.7)

Active phase labour (hours) ≥12 2 (7.7) 6 (8.0) 0.969 1.000§ 0.278 - 3.377 <12 24 (92.3) 69 (92.0)

Intrapartum features of sepsis Yes 1 (3.8) 2 (2.7) 1.307 1.000§ 0.255 - 6.707 No 25 (96.2) 73 (97.3)

Uterine tenderness Yes 1 (3.8) 0 (0.0) 4.000 0.577§ 2.849 – 5.617 No 25 (96.2) 75(100.0) * Pearson Chi-Square § Yate’s Continuity Correction

Table VIIIb: Subgroup analysis of GBS colonized neonates that did versus those that did not develop early onset GBS in relation to obstetric and neonatal parameters

Variable Yes No Relative P value 95% n=26 n=75 risk Confidence Interval Foul smelling purulent lochia Yes 1 (3.8) 0 (0.0) 4.000 0.577§ 2.849 – 5.617 No 25 (96.2) 75(100.0)

Postpartum endometritis Yes 1 (3.8) 1 (1.3) 1.980 1.000§ 0.475 – 8.247 No 25 (96.2) 74 (98.7)

APGAR score at 1 minute <7 8 (32.0) 6 (8.0) 4.000 0.008§ 1.536 – 10.414 ≥7 17 (68.0) 69 (92.0)

APGAR score at 5 minutes <7 6 (24.0) 0 (0.0) 19.963 <0.001§ 2.573 – 154.903 ≥7 19 (76.0) 75(100.0)

Neonatal shock Yes 4 (15.4) 0 (0.0) 13.750 <0.004§ 1.679 – 112.624 No 22 (84.6) 75(100.0)

Stillbirth in index pregnancy Yes 1 (3.8) 0 (0.0) 5.500 <0.577§ 0.519 – 58.318 No 25 (96.2) 75(100.0)

Early neonatal Death in index pregnancy Yes No 4 (15.4) 0 (0.0) 13.750 <0.004§ 1.679 – 112.624 22 (84.6) 75(100.0)

Perinatal death in index pregnancy Yes 5 (19.2) 0 (0.0) 16.500 <0.001§ 2.077 – 131.067 No 21 (80.8) 75(100.0) * Pearson Chi-Square § Yate’s Continuity Correction

CHAPTER 6

DISCUSSION, CONCLUSION AND RECOMMENDATIONS

6.1 Discussion

This study showed a prevalence of intrapartum recto-vaginal colonization by GBS of

30.1%. In addition, rates of rectal and vaginal colonization by GBS of 30.1% and 26.2% respectively were demonstrated. In mothers with rectovaginal colonization by GBS, the bacteria were found in 100% of rectal and 88% of vaginal swabs. As such, it was demonstrated that as much as 12% of mothers colonized by GBS would have been missed with the use of only vaginal specimens. The neonatal colonization rate in this study was also 30.1%. There was a 100% vertical transmission rate of GBS from colonized mothers to their new-born neonates.

The prevalence of early onset GBS disease was 7.8% (78 per 1000 births). All cases of neonatal GBS sepsis were confirmed by neonatal GBS bacteraemia. Seventy six percent of the cases of early onset neonatal GBS disease in this study manifested within 48 hours postnatum. The GBS attack rate was 25.7% among GBS positive mother-infant pairs with a case fatality rate of 15.4%. It was also observed that GBS colonized mothers had a marginally increased risk of postpartum endometritis. Maternal recto-vaginal colonization by GBS was significantly associated with rupture of membranes of 6 hours or more. GBS colonization was not significantly affected by; nor, did it significantly affect outcomes such as prolonged rupture of foetal membranes (greater than 24 hours before delivery); prolonged labour (active phase labour duration greater than 12 hours); low birth weight

(less than 2.5 kg) and maternal parity. However; there was a significantly increased predisposition of neonates with GBS colonization to perinatal asphyxia; neonatal sepsis; and perinatal deaths.

Majority (62.7%) of the mothers in both cohorts were Benin and Esan which are tribes indigenous to the state where this study was carried out. The predominant religion in the study location is Christianity and this was reflected in this study’s findings (98.5%).

The prevalence of intrapartum recto-vaginal colonization by GBS in this study is in keeping with those reported from Jordan (30.4%)170; Sweden (30.0%)171; and the Czech Republic

(29.4%).172 It is also comparable with; though, slightly higher than reports from United

Kingdom (28.0%)45; Hawaii (26.5%)44; Texas (26.0%)42; Georgia U.S.A. (26.0%)16; and

Gambia (22.0%).168 Like this study, most of the studies mentioned above employed maternal cultures from multiple sites and further enhanced isolation of GBS by using selective enrichment media. The prevalence of maternal colonization by GBS in this report is much higher than the findings in many reports from this sub region. These include reports from Mozambique (1%); Togo (4%); Malawi (16.5%); Ivory Coast (19%); and Nigeria

(20%).10,38 This study’s finding was also higher than reports of other studies from Nigeria.

These include a report from Zaria in Northern Nigeria (14%)47 and recent reports from

Western Nigeria (18.2%52 and 9.6%54). While these latter mentioned studies with low prevalence attempted to utilise methods that could help isolate GBS, they all fell short in terms of completeness in adherence to a full complement of steps that would ensure optimal yield of GBS. In contradistinction to this study, one or more steps omitted by the latter studies mentioned above include: sampling both the recti and vagina of mothers110; use of

transport media or direct inoculation of GBS into culture media116,117; use of selective enrichment broth media110,121; use of selective enriched blood agar plates; use of more sensitive test for presumptive diagnosis such as the Christie Atkins Munch-Peterson test129; and use of a definitive diagnostic (GBS latex agglutination) test for confirmation of isolation of GBS.

The culture sites were an important aspect of this work. This work’s finding of a higher rectal than vaginal colonization by GBS is comparable to the prevalence of anorectal and vaginal colonization by GBS of 24.4% and 21.7% respectively in the Czech Republic.172

This preponderance to rectal carriage of GBS is also seen in a report from Ethiopia of prevalence of carriage of GBS in the rectum and vagina of 14.3% and 10.4% respectively.173 Though disparity between the prevalence of rectal and vaginal carriage of

GBS in this work is smaller (4%), the finding of a higher prevalence of rectal than vaginal colonization by GBS in this study is in tandem with the observation of a 14% higher rectal than vaginal carriage of GBS at Alabama U.S.A.174

This study’s observation of a higher detection of maternal colonization by GBS in rectal than vaginal swabs though with a smaller difference (100% and 88% i.e. a 12% difference) is also slightly similar to findings by Philipson et al who reported rectal and vaginal detection rates of GBS of 89.7% and 58.6% respectively among GBS carriers.110 However, this study contrasts that from Gambia where GBS detection in rectal and vaginal swabs were 50% and 93% respectively.168 In addition, this study’s findings were also distinctly different than findings of the recent study at University College Hospital Ibadan which reported detection of GBS in the recti and vagina of colonized mothers of 30.4% and 60.9%

respectively.54 These differences were observed because a selective enrichment broth medium was used in this study prior to sub-culturing onto a selective enriched agar medium.110,121

The observation that all mothers colonized by GBS had GBS isolated from their recti contrasted with all the other studies mentioned above. This is because this study used two sequential selective enrichment media; as such, the sub-culture from a selective enrichment broth medium onto a selective enriched agar medium further suppressed the growth of other resistant bacteria in the rectal swabs allowing for easier isolation of GBS from the rectal swabs and detection of low inoculums of GBS. This study and the former mentioned above which showed a tendency to rectal carriage support the fact that the gastrointestinal tract is the main reservoir for GBS and that omitting rectal specimens could lead to significant decrease in reported prevalence.

The neonatal colonization rate in this study is comparable to the 34.5% observed in a pilot study from Poland even though mothers colonized by GBS had been given intrapartum antibiotic prophylaxis in the Polish study.46 This work’s finding is also comparable; though higher than the 23% neonatal colonization reported from Gambia.168 However, the prevalence of neonatal colonization in this report is much higher than those from Alabama

(12%)100; Turkey (1.6%)175; Tanzania (8.9%)176; and a report in the 1980’s from University

College Hospital Ibadan in Western Nigeria (8.5%).49 The low prevalence in the latter mentioned studies may be in part due to their relatively low maternal rectovaginal colonization by GBS when compared with this work’s findings. The detection of relatively high GBS colonization rates of neonates in this study may be accounted for in part by the

sampling of more than one body site in the neonates. Had only one site been used; the detection rates would have been 95.1% from the throat; 89.1% from the nostrils; 89.1% from the external auditory canals; and 97.0% from the umbilicus when compared with the detection rates using a combination of all four sites. The use of selective enrichment media also allowed for the detection of GBS from lightly colonized neonates.

The 100% vertical transmission rate of GBS from mothers with intrapartum recto-vaginal colonization by GBS to their new-born neonates in this work; though high, is like the 93% reported from Zaria.47 However, it is much higher than vertical transmission of GBS reported from Poland (34.5%)46; Turkey (15.2%)175; and Gambia (33%).168 This work’s vertical transmission rate is similar to the findings of other works which have reported vertical transmission rates between 29 - 85% with a mean rate of 50%.177 The higher vertical transmission rate found here may likely be due to sampling from multiple sites at delivery before the neonates were cleaned. The finding that two neonates born to mothers without recto-vaginal colonization eventually became colonized was not surprising and has been previously documented to occur in as often as 5% to 15% of neonates born to culture- negative mothers.168,177,178

GBS colonization of neonates born to GBS culture negative mothers may occur by nosocomial transmission or may be community acquired.100,101,102,103 Using molecular typing of the GBS strains isolated from mothers and their new neonates, a Polish study has been able to suggest the possibility of horizontal transmission of GBS from the hospital environment to neonates.179 This may account for the finding of neonates of GBS culture negative mothers eventually being colonized.

The prevalence of early onset neonatal GBS disease found in this study is much higher than reports from surveillance at 9 hospitals in the U.S.A. (3.21 per 1000 births) before the issuance of the U.S.A. CDC’s 1996 consensus guidelines for prevention of early onset GBS disease. This work’s observed prevalence of early onset neonatal GBS infection is also much higher than that reported from multistate surveillance in the U.S.A. (1.8 per 1000 births) after the issuance of the CDC’s 1996 guideline.180 Currently the prevalence of early onset GBS in the U.S.A. is 0.34 – 0.37 per 1000 births. This work’s findings are also much higher than data from United Kingdom (0.57 per 1000 births)181; South Africa (2.06 per

1000 births)182; and Malawi (0.92 per 1000 births).26

The contrast is less when this work’s high prevalence is considered in comparison to the prevalence of early onset GBS among neonates born to mothers with rectovaginal colonization by GBS intrapartum at Maryland U.S.A. (16 per 1000 births; but, 0.4 per 1000 live births in GBS culture negative women at delivery).89 The prevalence of early onset

GBS in this study is comparable with though higher than the attack rate among neonates with heavy colonization by GBS in an 80% black population at Alabama U.S.A. (50 per

1000 births). This study’s attack rate is also more in keeping with a composite attack rate in women with significant GBS bacteriuria extrapolated from 3 studies reporting on the prevalence of early onset GBS disease among mothers with significant bacteriuria in their index pregnancies (76 per 1000 births).13,14,91 However, generalization of the findings of these studies is limited by the small sample size of 92 mother-infant pairs. It remains unclear why GBS causes colonization in some women and their neonates, while it leads to infections in other mother-infant pairs. Colonization density, obstetric and midwifery

practices, selective intrapartum and neonatal antibiotic prophylaxis, racial predispositions, geographic variations, virulence variability among GBS clones and infection susceptibility of the host, play roles in the development and manifestation of invasive GBS infection.

GBS bacteriuria is a surrogate for heavy maternal colonization by GBS. Infants born to mothers with GBS bacteriuria are more frequently and more heavily colonized by GBS.131

The high attack rates of invasive GBS infections reported in mothers with heavy colonization by GBS and mothers with GBS bacteriuria suggest that mothers and their neonates in this study were more frequently heavily colonized by GBS.93,183,184,185,186

Exclusion of mothers with a history of use of antibiotics in the antecedent two weeks to delivery, including the exclusion of mothers who had intrapartum antibiotics has likely contributed to the high prevalence of GBS in this study.

Current evidence suggests that maternal rectovaginal colonization by GBS is commoner and heavier in African women as is the prevalence of early onset GBS infections in their neonates.87,100 The prevalence of early onset neonatal GBS infections has been observed by previous studies to be up to 12 times higher in heavily colonized neonates when compared to neonates who are lightly colonized.100 This may have contributed to the high prevalence of early onset GBS disease as observed in this study.100 (rectovaginal colonization in Blacks versus whites OR 1.742; adjusted RR 2.187). Maternal type specific immunoglobulins to colonizing serotypes of GBS and the virulence of the colonizing strains of GBS are also variables worth considering as they may have impacted the high prevalence of early onset GBS in this study. Baker et al reported that women whose infants remained colonized but well had normal antibody levels in their sera (73%) more often

than those whose infants developed symptomatic infection (17%).96 Forty eight percent of colonized women had low capsular polysaccharide-specific IgG levels [less than 0.5 microg/ml] in their delivery sera in a report from Texas.42 A preponderance towards more virulent strains of GBS such as serotype III over less virulent strains like serotype V will be associated with a higher prevalence of neonatal early onset GBS infection. However, this study was not designed to explore these possibilities. Studies are currently underway to investigate these latter 2 possibilities.

As earlier stated in the methodology of this study, U.B.T.H. which is the facility where this study was done serves as a major referral centre for Edo, Delta, Ondo and Kogi States. It provides structures and infrastructure for high and middle level manpower and is equipped for the provision of comprehensive obstetric health care services. It also has a level 4 neonatal health care unit. The import of these is that U.B.T.H. receives a health burden which is skewed in favour of complicated obstetric cases. A study from Poland found that neonates born to colonized mothers with a complicated pregnancy were more often colonized with GBS than those born to mothers with a normal pregnancy (35 % versus

26.7 %).179 This suggests that deliveries taken at UBTH which were more likely to be from complicated pregnancies would be more likely to be GBS colonized and more predisposed to adverse outcomes associated with GBS colonization and infections.

This study’s higher detection rate for early onset GBS disease may also in part be attributable to this study’s immediate inoculation of the specimens into enrichment broth to preserve their viability. Also, other attributable factors are: where beta haemolysis was difficult to observe, removing the suspected colonies from the blood agar plates then re-

examining the blood agar plates before conclusively determining the absence of beta haemolysis (i.e. presence of gamma haemolysis); and quite importantly subjecting typical colonies without beta haemolysis to further biochemical and diagnostic tests as up to 2.3% of GBS can be non-haemolytic.26,187

The duration to onset of symptoms and signs found in this study is comparable with; but, slightly longer than the findings from Texas. which reported an onset of illness within 24 hours of 61% and within 48 hours of 93%.87 The neonates in that study would most likely have had closer monitoring as in this study some of the cases were detected by the principal investigator (E. E. O.) who subsequently notified the appropriate medical personnel.

The observation in this study of no significant association between rectovaginal colonization by GBS and risk for postpartum endometritis is in line with findings by other studies.87,170 This work did not find any invasive GBS syndromes among its parturients.

The finding that GBS colonization was not significantly affected by; nor, did it significantly affect maternal or neonatal outcomes such as prolonged rupture of foetal membranes; prolonged labour; low birth weight and maternal parity were in agreements with the findings of the Gambian study.168 This study’s findings are different than the findings of some studies which reported on predictors of early-onset GBS disease conducted during the 1980’s.8,100 This study’s lack of observed associations may be related to the relatively modest birth population in which this study attempted to detect significant trends over time. Moreover, most studies that demonstrated significant associations between GBS and preterm labour; preterm premature rupture of membranes; prolonged rupture of membranes; and low birth weight included preterm neonates. This fact may also

account for why these risk factors were found to be contributory to early onset neonatal

GBS disease in other reports.

The increased predisposition of GBS colonized neonates in this study to perinatal asphyxia; neonatal sepsis; and perinatal deaths has been well documented by other workers.177 The case fatality observed in this study is like reports from Texas (16%)87 and Canada

(16.2%)192. Both these studies were done in the 1990’s before implementation of prevention guidelines; however, case fatality in these countries is currently 4 – 6% because of advances in perinatal care. The case fatality in this study is also similar to the case fatality reports from Uganda (18.1%)188; and South Africa (19.8%)189; but, much lower than that from Malawi (38%).26 The observation of a perinatal mortality of the two neonates born in shock agrees with findings from South Africa that the presence of septic shock when compared to the absence of shock in neonatal early onset GBS disease is associated with an 82% versus 1.9% risk for perinatal mortality.189 However, in this work the associations between these adverse perinatal outcomes and invasive GBS disease were not made by the attendant medical personnel. Hopefully this data shall provide a contemporary assessment of the place of GBS in peripartum and perinatal morbidities and mortality.

The strengths of this study are: this dissertation sought to meet its outlined objectives for which it was adequately equipped. The sample size for this study was calculated using a formula required to detect a true difference of a given magnitude between proportions in two independent samples, with a significance level (α) set at 5% and a power (1-β) of 80%.

Thus, this study sample is likely representative of the general population; and its findings likely reproducible in the general population due to the use of an adequate sample size.

This study used internationally accepted protocols and materials as such its finding should be reproducible (see appendix VII for a list of product specifications).

Like other cohort studies, this study allowed for thorough quality control in the measurement of the study variables. The standardized interviewer schedules were all administered by the principal investigator (E.E.O.); all client specimens were taken by the principal investigator and 2 research assistants and the samples were all processed by the same microbiologist all in a bid to limit inter-observer errors. Prospective data collection allowed contamination and co-intervention to be measured in a rigorous fashion. An example of such an interaction is the addition of supplemental selective antibiotics to blood agar which improved culture yield in this study.190

This was a well-planned prospective cohort study. Well planned cohort studies are often validated by other well-planned prospective studies for example planned randomized control trials; as such, the findings of this study will likely be scientifically validated and of scientific value.

Several outcomes were studied at once to provide a complete description of experiences after exposure including rates of progression and natural history of disease. Prevalence rates of the disease outcomes can be calculated in the exposed and unexposed mother- infant pair sets.190

It was observed during the preceding pilot study that it was desirable to inoculate the Lim

Broth directly on the Labour Ward immediately after collecting the swab specimens. This may have enhanced culture yield. Care was taken to minimize contamination of the broth

during inoculation. This was done by using a lamina air flow cabinet in the Side Laboratory on the Labour Ward.

Challenges experienced during this study include that: it was extremely difficult to procure gentamicin in good time for preparation of selective enrichment broth [TransVag broth] for the selective enrichment of specimens to enhance isolation of GBS. This was circumvented by switching to the United States of America’s Centre for Disease Control’s other first line recommendation for selective broth enrichment with Lim broth where

Gentamicin was substituted for with Colistin Sulphate which had the benefits that Nalidixic

Acid and Colistin Sulphate are available as a fixed dose formulation “Staph/Strep selective

[CNA] supplement SR0070E” Lot 1710904 from Oxoid and Remel, Oxoid Ltd,

Basingstoke, Hampshire, England; and unlike with TransVag broth, with Lim broth supplementation with 5% defibrinated sheep blood is not mandatory.

Group B Streptococcus testing is not routinely requested for at the University of Benin

Teaching Hospital. Prior to the onset of this study, a pilot study was conducted on 72 mother-infant pairs. During the pilot study, it was observed that the Tryptic soy blood agar plates were often overcrowded by growth of resistant organisms despite selective enrichment of specimens in Lim broth before sub-culturing onto the blood agar. This may often be the case, particularly with respect to the rectal swabs. Because of this, it was opted that enriched samples be sub-cultured onto another selective medium Columbia blood agar base supplemented with 5% defibrinated sheep blood, Nalidixic Acid and Colistin Sulphate which is also a first line optional recommendation by the Centre for Disease Control. This proved effective in curbing the growth of resistant organisms on the blood agar sub-

cultures.191 This modification was accompanied by an increase in isolation of GBS from

14% at the beginning of the pilot study to 30% at the end of the pilot study.

Limitations of this dissertation’s study protocol include: following up the mother-infant pairs in this study only until discharge home at the second or third day postpartum as outlined by my dissertation protocol would be insufficient to explore the full impact of intrapartum recto-vaginal colonization with GBS on postpartum women; neonatal colonization with GBS; and the incidence of early onset GBS sepsis (onset before 7 days postnatum). As such clients were given a dedicated mobile phone number via which to reach the principal investigator if they noticed any problems; features of postpartum endometritis; or features of neonatal sepsis. Clients were reviewed 24 hours and 48 hours postpartum and had call backs on the 6th day postpartum to enquire of wellness and remind them to return for evaluation and repeat sample collection on the 7th day postpartum. To minimize loss of cases the Professor Jackson Omene Special Care Baby Unit (neonatal care unit) was also scouted daily in case any of the discharged study clients were readmitted there.

Secondly, this was a cohort study; as such, was slightly prone to the limitations of cohort studies vis: A large sample size was needed for this study with expected rare outcome variables; however, the outcomes were found to be much commoner than expected. Cohort studies often require potentially long durations for follow up and maintaining follow up can be difficult. In this study, some clients were lost to follow up as their lines were inadvertently barred due to incomplete line registrations during the recently conducted mobile network carrier client information update. These clients were sought out at the

postnatal clinics. Some clients felt weary when called back as they feared something must be amiss for them to have been called back despite being pre-informed and consenting to call backs. This problem was tackled by reassuring the clients about the fact that they were not being called back because of any detected problems and reminding them of the long- term benefits to them and to posterity.

Large cohort studies can be relatively expensive in time, money and personnel.190 The streptococcus latex agglutination test kit is quite expensive; as such, the gram stain test; catalase test, coagulase test and the highly sensitive Christie Atkins Munch-Petersen

(CAMP) tests were first performed for presumptive diagnosis before proceeding to confirmatory diagnosis with the Group B Streptococcus Latex Agglutination Test only in presumptively positive specimens; thereby, reducing expenditure on the number of latex agglutination test kits required. These limitations notwithstanding, the observations made based on this internationally acceptable protocol for the identification of GBS colonization remains a major strength of this study.

Blinding to interventions could not be achieved with resulting potential for bias; however, the investigator did not interfere with standard protocols. On occasions when the principal investigator detected complaints; or features suggestive of sepsis, the appropriate personnel were informed.

GBS is not the only causative organism for postpartum endometritis or neonatal sepsis.

Other bacteria may be confounding variables as such a profile of other isolates from the stipulated sites would have been desirable. However, GBS is a fastidious organism and efficient isolation is hinged on selective and enrichment media which suppress the growth 76

of other bacteria (particularly the gram negatives and rods) to allow cultivation and isolation of GBS. This invariably impairs a true profiling of all the bacterial isolates in the specimens collected. Separate non-selective media were not inoculated because of cost considerations; however, notes were taken of the other bacteria that grew despite using a selective enrichment medium. A report of this finding has been included in the appendix of this dissertation as appendix VI Table X.

An antibiogram would be helpful for empirical treatment in suspected cases of GBS colonization or sepsis. This was not part of the approved proposal for this dissertation; however, the development of an antibiogram adds value to advising clinicians on the choice of antibiotics in suspected cases of GBS sepsis. (see appendix V Table IX).

6.1.1 Recommendations for Policy Development; Practice and Future Research

GBS has been demonstrated to be important in postpartum and early neonatal morbidity and mortality. GBS emerged as the leading cause of neonatal morbidity and mortality in

North America and the United Kingdom since the 1970’s. The implementation of the 2002 consensus guideline to prevent early onset GBS disease in neonates through universal antenatal culture based screening at 35 - 37 weeks’ gestation and selective intrapartum antibiotic prophylaxis has been associated with a substantial decline in the incidence of neonatal early onset GBS infections.24 Whereas in North America and the United Kingdom it has been sought to reduce further the scourge of GBS and potential vaccine testing has been achieved; in our environment, simple preventive measures such as the already tested and trusted antenatal screening and selective intrapartum antibiotic prophylaxis still remain unexplored options.

The summed cost for the 39 neonatal unit admissions for neonates in this study was

1,013,580 Naira [N]. Of this N588,480 was accounted for by 20 early onset GBS disease cases (19.8% of GBS positive neonates) as against N425,100 accounted for by 19 (8.1%)

GBS negative neonates. This is excluding the 1 stillbirth and 1 early neonatal death that occurred before the fifth minute postnatum both in GBS positive neonates with GBS bacteraemia. The estimated cost for universal late antenatal culture screening for the 335 mothers in this study at an estimated cost per capita of N1,000 (as both the vaginal and rectal swabs can be inoculated into a single selective enrichment broth) would come to

N335,000 (which is more than the cost for analysing 335 specimens in this study). GBS selective intrapartum antibiotic prophylaxis is best done with Penicillin G which shows highest sensitivity on the antibiogram and is at least risk of GBS antibiotic resistance.

Penicillin G is also a narrow spectrum antibiotic; hence, its use is least likely to select for the emergence of resistant strains of other bacteria not targeted. Penicillin G is also a very cheap antibiotic being routinely sold for N50 for 1,000,000 units (1 mega unit). The recommended dose for selective intrapartum antibiotic prophylaxis is a loading dose of 5 mega units which would cost N250 (N50 x 5). The CDC consensus guidelines advocates a minimum of 4 - 6 hours of selective intrapartum antibiotic prophylaxis with a 4-hour dosing interval with a maintenance dose of 3 mega units to accrue minimum benefit at a cost of

N400 (N250 loading dose + N150 maintenance dose [N50 x 3]). This would bring the cost of selective intrapartum antibiotic prophylaxis for the 101 GBS positive mothers in this study to N50,500 (N400 x 101parturients plus N100 x 101parturients for sterile water for injection and syringes). Thus, the estimated total cost for a simple program for prevention of vertical transmission of GBS by late antenatal culture based screening of all the 335

participants in this study (N335,000) followed by selective intrapartum antibiotic prophylaxis with Penicillin G for the 101 GBS colonized mothers (N50,500) would sum up to N385,500 only as against a neonatal unit admission cost of N588,480 for neonates with early onset GBS disease. This cost did not consider the tragedy of the stillbirth and early neonatal death who never got admitted and the increased risk of perinatal asphyxia associated with early onset GBS disease thereby impacting the output of our next generation. In the United States of America between 1993 and 2007 universal antenatal culture based screening at 35 - 37 weeks’ gestation and selective intrapartum antibiotic prophylaxis caused an 80% decline in early onset GBS disease from 1.7 to 0.34 per thousand livebirths.11,24 It should be acknowledged that the consensus guidelines prevention program left a residual 20% of expected cases. The 80% decline in early onset

GBS disease would seem reason enough to encourage the institution of policies for universal antenatal culture based screening at 35 - 37 weeks’ gestation and selective intrapartum antibiotic prophylaxis as this has long term benefits of a healthier neonatal population, with less perinatal asphyxia. It should be a priority to enact policies that will protect our new-borns from prolonged hospitalization; expensive supportive therapy; and permanent disabilities such as hearing or visual or mental deficits which are well documented complications of early onset GBS disease.

This study established that maternal recto-vaginal colonization with GBS intrapartum increases the risk of neonatal colonization with GBS; maternal and or neonatal early onset

GBS infection. GBS colonization is prevalent in our environment; as such, the role of the obstetrician is important in preventing neonatal morbidity and mortality resulting from

early onset GBS disease. GBS though an uncommon problem in postpartum women should still be considered a cause of postpartum complications such as postpartum endometritis.

The implication of this study on future research is the need for the development of an antibiogram adapted to our local environment to enhance the efficiency of interim empirical treatment that has an almost uniformly good outcome with early antimicrobial therapy for this otherwise fulminant infection.

It would also be pertinent to explore the serotype distribution of GBS in this environment to check for their clinical correlates in a bid to find out why only some of the colonized clients manifest morbidity and mortality.

6.2 Conclusion

The prevalence of maternal recto-vaginal colonization with Group B Streptococcus is

30.1% intrapartum in the obstetric population delivering at the University of Benin

Teaching Hospital. It increases the risk of maternal postpartum complications; neonatal colonization with Group B Streptococcus; Group B Streptococcal early-onset neonatal disease (sepsis) and perinatal mortality from Group B Streptococcus sepsis.

6.3 Recommendations

1) Group B streptococcus should be considered a significant cause of postpartum

complications and neonatal morbidity and mortality in our environment; thus,

protocols and policies aimed at its control should be instituted.

2) Antenatal mothers should receive health education on the significance of GBS

screening. Maternal healthcare services should incorporate late antenatal screening

(as a surrogate for intrapartum screening) for recto-vaginal colonization by GBS

and selective intrapartum antibiotic prophylaxis.

3) Local antibiogram and culture sensitivity profiles should be established for GBS to

enhance empirical treatment of suspected GBS infections while awaiting isolation

and antibiotic sensitivity testing.

4) Other well planned prospective randomized studies are desired to validate the

findings of this study and the efficacy in our peculiar environment of solutions

already being used elsewhere.

5) Studies screening all neonatal unit admissions for GBS should be encouraged as

they will provide complementary information regarding the burden of GBS among

children born outside the hospital setting and its overall implications on neonatal

unit admissions and expenditure.

6) Studies are required to look at the survivors of neonatal GBS disease to quantify in

our local environment the magnitude of permanent disabilities they suffer; such as,

hearing deficit, visual loss and mental deficits on a backdrop of what has already

been characterized in the literature from other parts of the world.

REFERENCES

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APPENDICES

APPENDIX I

To determine the increased risk of neonatal colonization by GBS in neonates of colonized mothers at University of Benin Teaching Hospital. The sizes of the samples required to detect a true difference of a given magnitude between proportions in two independent samples, with a given significance level; a given power or precision; and continuity correction is derived using formula 22 given by Sahai et al.166 and obtained from a computer software called WINPEPI

2 ′ 푛1 2(푘 + 1) 푛1 = [√( 1 + ′ )] 4 푘훿푛1

′ Where 푛1 is derived from an Asymptotic normal method.

Cohen recommends an allowable Type II error (beta) of about four times alpha. Thus, for an alpha of 5% (0.05), beta might be set at, say, 0.20 (power = 80%).167

Using the above formula; continuity correction; and the following values for a one tailed test:

Significance level (α) of 5%

Power (1 – β) of 80%

Non-inclusion of 10% of subjects

Ratio of sample size B:A of 2.29 (Saura R O 1994)168

The known proportion in Sample B of 0.15 (15%)167 104

ODD Ratio of 2.25. (from a study by Saura O R 1994)167,168

Where A refers to exposed (neonates of colonized mothers); and

B refers to unexposed (neonates of un-colonized mothers).

The sample sizes were calculated using WINPEPI 8:1 and the above values

The sample size for this study was 332 mother-infant pairs.

Approx. 95% CI for difference between proportions (D) =D - 0.090 to D + 0.090

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APPENDIX II

INFORMED CONSENT FORM

TITLE OF STUDY: HEALTH BURDEN OF GROUP B STREPTOCOCCI COLONIZATION IN THE OBSTETRIC POPULATION AT UNIVERSITY OF BENIN TEACHING HOSPITAL, BENIN CITY, NIGERIA. INVESTIGATOR: DR OKOSUN EDWARD EROMOSELE in Partial Fulfilment of the Requirement for the Part II Fellowship Examination of the National Postgraduate Medical College of Nigeria.

SUPERVISORS: Prof. Ande A.B.A. and Prof. Okonkwo C.A. (Department of Obstetrics and Gynaecology, University of Benin Teaching Hospital).

FINANCIAL SPONSORSHIP: This research project is self-sponsored.

PURPOSE OF RESEARCH: It is important you read and understand the following explanation of the proposed study procedures before agreeing to participate. This information describes the purpose, procedures, benefits, discomforts, risks and precautions associated with this study. It also describes your right to refuse to participate or withdraw from the study at any time. In order to decide whether you wish to participate in this research study, you should understand enough about its risks if any and benefits to be able to make an informed decision. This is known as the informed consent process. Please ask that any word(s) you do not understand be explained to you before signing this consent form. Make sure all your questions have been answered to your satisfaction before signing this document.

About the study

Currently, research in developed countries aim to reduce neonatal sepsis even more through the development of maternal vaccines against prevalent pathogens, such as GBS. Conversely, research in sub-Saharan Africa has focused on treating maternal and child infections rather than prevention; whereas, there are still opportunities for simple preventive measures affecting maternal and child health. Given the high prevalence of maternal and neonatal infection, their associated morbidity and mortality in sub-Saharan Africa and the effectiveness of simple interventions at preventing maternal and early-onset neonatal sepsis, there is a need to evaluate the prevalence of maternal GBS colonization and its burden of disease in Nigeria and University of Benin Teaching Hospital (UBTH) in particular. Currently, a proportion of pregnant women delivering at UBTH have been observed to present with obstetric risk factors for GBS sepsis (preterm delivery, prolonged rupture of membranes or intrapartum pyrexia); but, till date, there are no studies that have specifically addressed the burden of GBS in the obstetric/early neonatal population at UBTH using appropriate microbiological methods.

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This study aims to estimate the burden of GBS disease in the obstetric population at University of Benin Teaching Hospital; identify the proportion of the obstetric population that will require treatment; and also help in subsequent development of policies about care of similar populations. PROCEDURES INVOLVED IN THE STUDY: You are politely approached to respond to this self-administered questionnaire. Subsequently swabs would be taken from your lower vagina (vaginal introitus), followed by the rectum at admission in labour. Also a total of four samples will be collected from your neonate immediately after birth viz. external ear swab, nasal swab, throat swab and umbilical swab.

COMPENSATION: There will be no financial compensation for participating in this study.

VOLUNTARY PARTICIPATION: Please note that your participation in this study is entirely voluntary and no form of force will be used nor any form of discrimination meted out against you. In the event that you decide to stop participating, you are free to withdraw even if you had earlier agreed to participate.

RISKS: There is no form of physical pain or harm associated with participating in this study, however, we will sincerely appreciate your participation in the study.

BENEFITS: Findings from this research will be used to proffer solutions as to ways of improving obstetric outcome.

CONFIDENTIALITY: All information obtained in the course of this study will be treated confidentially. The names of the participants will not be written on the questionnaire. All the information obtained will be encoded in a file on my personal computer and pass worded. The questionnaires will afterwards be shelved and locked up in my personal documents cabinet.

CONTACT INFORMATION:

Dr. Okosun Edward Eromosele Department of Obstetrics and Gynaecology, University of Benin Teaching Hospital, Benin City, Phone Number: +2348126846318 Email: [email protected].

Chairman, Ethics and Research Committee, University of Benin Teaching Hospital Benin City. Phone Number: 08181940459 Telegram: UNITECHOS, BENIN Telex: 41120 NG 107

CERTIFICATE OF CONSENT I have read the above information (or it has been read to me). I have had the opportunity to ask questions about it and any questions that I have asked have been answered to my satisfaction.

(A) I consent voluntarily to take part as a participant in this research. (B) I do not consent to participate in this research Name of Participant:

Signature of Participant:

Date:

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APPENDIX IlI

STANDARDIZED INTERVIEWER SCHEDULE

1. Serial number:

2. Initials:

3. Hospital number:

4. Telephone number (optional):

5. Age (in years as at last birthday):…...a) <15 [ ] b)15-19 [ ] c) 20-24 [ ] d) 25-29 [ ] e) 30-34 [ ] f) 35-39 [ ] g)40-44 [ ] h) >44

6. Tribe: a) Bini [ ] b) Esan [ ] c) Etsakor [ ] d) Urhobo [ ] e) Ika [ ] f) Yoruba [ ] g) Ibo [ ] h) Hausa [ ] i) Others ……..

7. Religion: a)Christian[ ] b) Muslim[ ] c)Traditional worship[ ] d Others

8. Highest level of education: a) No formal education [ ] b) Primary [ ] c) Secondary [ ] d) Tertiary[ ] e) Others ………….

9. Marital status: a) Married [ ] b) Single [ ] c) Divorced [ ] d) Separated [ ] e) Widowed [ ].

10. Husbands occupation: …………………………………………

11. Average household income per month…………………………….

12. Use of vaginal pesseries? a) Yes [ ] b) No [ ]

13. Vaginal douching? a) Yes [ ] b) No [ ]

14. History of cigarette smoking? a) Yes [ ] b) No [ ]

PAST OBSTETRIC HISTORY

15. Parity: ……a) 0 [ ] b) 1-4 [ ] c) >4

16. Number of previous miscarriages:……..

17. Number of previous preterm deliveries:……..

18. Number of previous pregnancies complicated by preterm premature rupture of membranes:

109

19. Number of previous pregnancies complicated by premature rupture of membranes:

20. Number of previous Stillbirths:

21. Number of previous deliveries complicated by early-onset neonatal sepsis.

CONCERNING THE INDEX PREGNANCY

22. Urinary tract infections in index pregnancy? a) Yes [ ] b) No [ ] c) Don’t know [ ]

23. GBS bacteriuria in index pregnancy? a) Yes [ ]b) No [ ]c) Don’t know [ ]

24. Glycosuria in index pregnancy? a) Yes [ ] b) No [ ] c) don’t know [ ]

25. Human Immunodeficiency Virus status in index pregnancy?

a) negative [ ] b) positive [ ] c) don’t know [ ] d) decline to answer [ ]

PAST MEDICAL AND DRUG HISTORY

26. Use of antibiotics in the last two weeks?

a) Yes [ ] b) No [ ] c) don’t know [ ]

27. History of penicillin allergy? a) Yes [ ] b) No [ ] c) don’t know [ ]

28. Steroid therapy? a) Yes [ ] b) No [ ] c) don’t know [ ]

29. Known diabetic? a) Yes [ ] b) No [ ] c) don’t know [ ]

30. Other underlying chronic medical conditions?

INTRAPARTUM DATA

31. Membrane sweeping before onset of labour? a) Yes [ ] b) No [ ]

32. Onset of labour before 37 weeks? a) Yes [ ] b) No [ ]

33. Membranes rupture before 37 weeks? a) Yes [ ] b) No [ ]

34. Membranes rupture before maternal specimen collection?

a) Yes [ ] b) No [ ]

35. Duration of rupture of membranes before onset labour (Hours)?......

36. Duration of rupture of membranes before delivery (hours)? ……….. 110

37. Duration of active phase labour (hours)?......

38. Intrapartum temperature reading (°C)? …………………………..

39. Uterine tenderness? a) present b) absent

40. Foetal heart rate (bpm)? ……………………………………….

41. Maternal pulse rate (bpm)? …………………………………….

42. Number of vaginal examinations?……………………………..

43. Foul smelling or purulent amniotic fluid? a) present b) absent

44. Epidural analgesia in labour? a) Yes [ ] b) No [ ]

45. Intrapartum antibiotics? a) Yes [ ] b) No [ ]

46. Gestational age at delivery (weeks and days)? …………………

47. Birth weight of neonate?…………………………………………..

48. APGAR score at 1st minute……………………………………….

49. APGAR score at 5th minute……………………………………….

50. Admission into neonatal special care unit? a) Yes [ ] b) No [ ]

51. Developed features of neonatal sepsis? a) Yes [ ] b) No [ ]

LABORATORY DATA

52. Intrapartum GBS culture: a) Negative [ ] b) positive [ ].

If answer to question 52 is positive what is the degree of colonization? i) Light [ ] ii) Heavy [ ]

53. Neonatal GBS culture at birth: a) Negative [ ] b) positive [ ].

If answer to question 54 is positive what is the degree of colonization? i) Light [ ] ii) Heavy [ ]

Thank you

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APPENDIX IV

ETHICAL APPROVAL

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APPENDIX V

Table IX: Antibiotic sensitivity profile of the GBS isolates

Sensitivity Percentage profile (%) Overall antibiotics sensitivity Sensitive to all 2 1.9 Sensitive to some 102 98.1 Penicillin Sensitive 95 91.3 Resistant 9 8.7 Ampicillin Sensitive 61 58.7 Resistant 43 41.3 Erythromycin Sensitive 24 23.1 Resistant 80 76.9 Cephazolin Sensitive 46 44.2 Resistant 58 55.8 Clindamycin Sensitive 51 49.0 Resistant 53 51.0 Vancomycin Sensitive 73 70.2 Resistant 31 29.8 Erythromycin induceable clindamycin resistant Yes 29 27.9 No 74 71.2

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APPENDIX VI

Table X: Other microorganism isolated from different maternal and neonatal sites

Sites Microorganism Maternal Maternal Neonates Neonate Neonates Neonates Neonates vaginal rectal throat nostrils ears umbilicus blood No growth 108 (32.8) 123 (36.7) 131 (39.2) 124 (37.0) 121 (36.1) 120 (35.8) 30 (9.0) Staphylococcus 0 (0.0) 1 (0.3) 0 (0.0) 0 (0.0) 0 (0.0) 3 (0.9) 0 (0.0) aureus Staphylococcus 194 (57.9) 195 (58.2) 193 (57.6) 207 (61.8) 211 (63.0) 210 (62.7) 5 (1.5) epidermidis Streptococcus 0 (0.0) 0 (0.0) 8 (2.4) 4 (1.2) 0 (0.0) 0 (0.0) 0 (0.0) pneumoniae Streptococcus 2 (0.6) 5 (1.5) 1 (0.3) 0 (0.0) 0 (0.0) 1 (0.3) 0 (0.0) feacalis Enterococcus 2 (2 (0.6) 5 (1.5) 0 (0.0) 0 (0.0) 2 (0.6) 0 (0.0) 0 (0.0) feacalis Streptococcus 0 (0.0) 0 (0.0) 2 (0.6) 0 (0.0) 0 (0.0) 0 (0.0) 1 (0.3) viridans Yeast 9 (2.7) 1 (0.3) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Yeast and 18 (5.4) 1 (0.3) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Streptococcus epidermidis Streptococcus 1 (0.3) 1 (0.3) 0 (0.0) 0 (0.0) 1 (0.3) 1 (0.3) 0 (0.0) agalactiae and Staphylococcus epidermidis Streptococcus 1 (0.3) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) agalactiae and Enterococcus feacalis Staphylococcus 0 (0.0) 3 (0.9) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) epidermidis and Streptococcus feacalis Not applicable 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 275 (82.1)

One of the two neonates colonized with Streptococcus viridans had a septicaemia with isolation of Streptococcus viridans from blood culture eight hours postnatum. This neonate developed a pyrexia of 38.2oC, dyspnea, tachypnea and suffered early neonatal death 11 hours postnatum.

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APPENDIX VII

List of product specifications:

Amies Transport Medium (CM0425 Oxoid Ltd England)

Todd-Hewitt Broth (CM0189 Oxoid Ltd England)

Staph./Strep. Selective [CNA] Supplement (SR0070E Oxoid Ltd England)

Columbia Blood Agar Base (CM0331 Oxoid Ltd England)

Tryptone Soya Agar (CM0131 Oxoid Ltd England)

Sheep Blood Defibrinated (SR0051B Oxoid Ltd England)

PathoDxtra Strep Grouping Reagent Set (DR0710M Oxoid Ltd England)

PathoDxtra Strep Group B Latex (DR0702G Oxoid Ltd England)

Streptococcus agalactiae (ATCC 12386 Kwik-Stik Microbiologics Minnesota)

Escherichia coli (ATCC 25922 Kwik-Stik Microbiologics Minnesota)

Staphylococcus aureus (ATCC 25923 Kwik-Stik Microbiologics Minnesota)

Streptococcus pneumoniae (ATCC 49619 Kwik-Stik Microbiologics Minnesota)

Mueller-Hinton Agar (CM0337 Oxoid Ltd England)

Antimicrobial Susceptibility Test Discs:

Penicillin G P 10iu (CT0043B Oxoid Ltd England)

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Ampicillin AMP 10mcg (CT0003B Oxoid Ltd England)

Erythromycin E 15mcg (CT0020B Oxoid Ltd England)

Cephazolin KZ 30mcg (CT0011B Oxoid Ltd England)

Clindamycin DA 2mcg (CT0064B Oxoid Ltd England)

Vancomycin VA 30mcg (CT0058B Oxoid Ltd England)

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