a study of group b streptococcus in brisbane ...eprints.qut.edu.au/16462/1/karen_taylor_thesis.pdfa...

A STUDY OF GROUP B STREPTOCOCCUS IN BRISBANE: THE EPIDEMIOLOGY, DETECTION BY PCR

ASSAY & SEROVAR PREVALENCE

By Karen Leigh Taylor BASc Dip Ed

A thesis submitted for the degree of Master of Applied Research

March 2006

Infectious Diseases Programme School of Life Science

Queensland University of Technology

A study of Group B Streptococcus in Brisbane; the epidemiology, Page 2 detection by PCR assay and serovar prevalence

For Larah Jayne


ABSTRACT The neonate is still at risk of acquiring Group B Streptococcus (GBS) infection upon delivery even with the implementation of early onset GBS neonatal disease preventative protocols. GBS was reported as the most prevalent organism causing neonatal morbidity and mortality in the USA and Australia in the 1990s. GBS is also known to cause disease in children, women, the immunocompromised adult and the elderly, but it is the preterm neonates who are at greatest risk of GBS neonatal disease. The aim of this study was to determine the prevalence of lower genital tract (LGT) colonisation with GBS in Brisbane women of child bearing age. We also aimed: (i) to compare the GBS LGT prevalence rate of Indigenous and non Indigenous women; (ii) to determine whether previously reported risk factors for LGT colonisation with GBS were also risk factors associated with GBS colonisation of women in this study; (iii) to further develop and optimise a rapid PCR assay that could detect maternal LGT GBS colonisation; and (iv) to serotype the GBS strains that were isolated from pregnant and non pregnant women who participated in this study. This study recruited 374 women of childbearing age attending public medical providers and found an overall GBS prevalence of 98/374 (26.2%) for these Brisbane women, a rate higher than previously reported in Australia. When the GBS prevalence for pregnant women (25.6%) was compared to non pregnant women (27.2%) they were similar. We also compared the GBS LGT colonisation rate of women attending different medical providers. The GBS LGT prevalence rate for pregnant women attending the Mater was 36/118 (30.5%), whilst those women attending the Redlands Hospital antenatal clinic had a LGT GBS prevalence rate of only 7/53 (13.2%). By comparison, the LGT GBS prevalence rate for non pregnant women attending Biala Sexual Health clinic was 21/69 (30.4%) and 34/127 (26.8%) of women attending the Brisbane Family Planning Queensland were also GBS positive. The seven women recruited from Inala community centre tested negative for GBS LGT colonisation. The LGT GBS prevalence of Australian Aboriginal women was 5/22 (22.7%), a rate which was not significantly different from non-Aboriginal women 78/288 (27.1%). Established early onset GBS neonatal disease preventative policies have been recently revised. The CDC now recommends that all pregnant women are screened for LGT GBS colonisation during late gestation, and that any GBS isolates be tested for resistance to antibiotics if the GBS positive women have an allergy to penicillin. Queensland’s Department of Health recommend that Queensland medical agencies implement a non screening based preventative protocol, where clinicians monitor: women prior to labour for reported risk factors associated with maternal GBS colonisation: women in labour for ‘obstetric risk factors’. A number of risk factors have previously been reported in association with GBS LGT colonisation. However, in this current study we found that only one risk factor was significantly associated with current GBS: previous reported LGT GBS colonisation was significantly associated with maternal LGT GBS colonisation reported in this study. Women who previously tested positive for GBS were significantly more likely to be GBS positive in subsequent tests (OR 4.7; 95%CI, 1.8-12.5) compared to women with no previous history of GBS colonisation. An assessment of adverse pregnancy outcomes, preterm deliveries, and GBS colonisation data was made. It was established that 30 women had previously given birth to one or more preterm neonates and of these 30 women, nine (30%) of them tested positive for GBS in this current study. Of the 71 women who had given birth to neonates and who had suffered an adverse pregnancy outcome 25.3% also tested positive for GBS in this current study. GBS was


identified in up to 30% of all mothers who had delivered their neonate preterm, 27.4% of women who had previously suffered miscarriages and 16.7% of women who had previously had stillbirths. In this study we found that Australian Aboriginal women also had a greater risk of delivering neonates who suffered from an adverse pregnancy outcome in comparison to all other women. Twenty one of the 22 Aboriginal women had previously been pregnant at least once, and nine (42.9%) of these women had at least one prior adverse pregnancy outcome while seven (33.3%) of these women had previously delivered at least one neonate preterm. Of the 21 Aboriginal women who had a previous pregnancy more than half the total number of Aboriginal women (11/21) had either delivered one or more neonates preterm or had suffered from one or more adverse pregnancy outcomes. When the incidence of adverse pregnancy outcomes was compared for Aboriginal and all other women the results were surprising. Overall, this study found 216 women including Aboriginal women had previously been pregnant and of these women 71 (32.8%) of them suffered an adverse pregnancy outcome. By comparison, only 62 of 195 (31.8%) non Aboriginal women but nine out of 21 (41.9%) Australian Aboriginal women suffered from a previous adverse pregnancy outcome. The clinical LGT GBS isolates found in this study of Brisbane women were typed and all nine GBS serotypes plus non typeable GBS serotypes were detected. Seventy women tested GBS culture positive and vaginal and/or perianal samples obtained from these women were evaluated. GBS serotype III was the serotype most frequently isolated from this total population, from 47.4% of pregnant women and 51.7% of non pregnant women. From some women only a single GBS serotype was isolated: in these women we found that GBS serotype III (50%) was the predominant isolate, followed by GBS serotype Ia isolated from 16.7% women. In addition 4.2% of women were colonised with GBS serotypes; Ib, II and V, whilst GBS serotypes IV and VII were isolated from 2.1% women. Non typeable GBS strains confirmed by latex agglutination tests accounted for 11.9% of all strains isolated from these Brisbane women. This study identified multiple serovars in 15 clinical samples and found that 22 (31.4%) women were colonised with mixed GBS serotypes in samples collected from both vaginal and perianal regions. In five women the combination of serotypes III/Ia were identified and in other women combinations of serotypes III/II, III/IV, III/V, III/VIII, Ia/IV and Ib/NT were also detected. In two instances three serotype combinations were detected in samples from one woman and these included serotypes III/Ib/II and III/Ia/Ib. Isolates were also typed for women who were colonised in both vaginal and perianal regions and it was found that only 10 participants had identical isolates in both regions. GBS serotype III was the predominant serotype detected in women tested in this study and this is the serotype that has previously been associated with invasive infections in neonates. GBS neonatal disease is a world wide economic, health and social burden affecting different ethnic groups and is preventable. Currently no vaccine technology is available for the prevention of GBS neonatal disease and the most effective EOGND preventative protocol would be to test for maternal GBS colonisation during labour, or screen women for GBS at ≥36 weeks’ gestation and administer intrapartum antibiotic prophylaxis (IAP) to all women who tested positive for GBS. In this current study we utilised a rapid bsp PCR assay to detect LGT GBS colonisation in women of child bearing age. The PCR assay identified 62.5% of all vaginal and perianal positive culture GBS samples. The specificity of the PCR assay was 89% while the positive and negative predictive values were 56.8% and 91.1% respectively. This PCR assay using the current parameters is not an effective GBS detection assay but could be further optimised in the near future. This PCR assay could be an effective test in the future with the development of an alternative DNA extraction method to InstaGene (BioRad). However, this PCR assay if used in conjunction with the current culture method is able to detect a further 8.9% of women colonised with asymptomatic GBS.


Brisbane women aged between 26 to 35 years who are pregnant and who are attending public health care agencies are at greatest risk of being colonised with GBS. No disparity was identified when ethnicity or social standing were assessed. The overall results of this study demonstrate that the LGT GBS prevalence rate in Brisbane women is 26.2% but this rate was higher at 30.5% for women attending a Brisbane sexual health clinic and for pregnant women attending the Mater Mothers’ antenatal clinic. GBS serovar III has been identified as the dominant serovar in our population group and this strain has been reported as the major cause of GBS disease in neonates and infants aged to three months. Disparity (11.1%) was reported when the incidence of adverse pregnancy outcomes amongst Aboriginal women was compared to non Aboriginal women. From the outcomes of this study it has been suggested that Queensland adopt a screening based GBS preventative protocol. It has also been suggested that an Australian wide GBS prevention strategy may further reduce the incidence of neonatal disease.


TABLE OF CONTENTS Page CHAPTER 1: GROUP B STREPTOCOCCUS 1 1.1 Introduction 1 1.1.1 History of Group B Streptococcus (GBS) 2 1.1.2 Morphology of GBS 3 1.1.3 GBS Serotyping 4 1.2 GBS Disease in Neonates 4 1.2.1 Early Onset GBS Neonatal Disease (EOGND) 4 Signs, Symptoms, Treatment of EOGND 6

The EOGND Incidence 7 The Australian EOGND Incidence 8 EOGND Mortality Rates 10 1.2.2 Late Onset GBS Disease (LOGD) 12 Signs, Symptoms, Treatment of LOGD 13 The Australian LOGD Incidence Compared to Other Countries 13 Mortality and Morbidity of LOGD 14 1.3 GBS Disease in the General Population 14 1.3.1 GBS Disease in Pregnant Women 14 1.3.2 GBD Disease in the Foetus 15 1.3.3 GBS Diseases in Infants & Children 15 1.3.4 GBS Disease in Adults 16 1.3.5 Treatment of GBS Disease in the General Population 17 1.4 Prevention of GBS Neonatal Infections 17 1.4.1 Prevalence of GBS in Adult Populations 18 Prevalence of GBS in Indigenous Populations 20 1.4.2 Prevention through Treatment of the Colonised Mother 20 1.4.3 Prevention through Treatment of the Neonate 22 1.4.4 Prevention through Treatment of Sexual Partners 22 1.4.5 EOGND Prevention Protocols 22 Current Prevention Protocol (CDC and ACOG) 23 Evaluation of Previous EOGND Prevention Protocols 23 Current Culture Based Method for GBS Detection 25 Prevention Protocols Utilised within Australia 25 Assessment of Queensland’s EOGND Prevention Policy 26 1.4.6 Signs and Symptoms of GBS Colonisation 28 Gestational & Intrapartum GBS Colonisation 28 Demographic Factors Associated with GBS Colonisation 29 1.4.7 Prevention of LOGD 30 Risk Factors Associated with LOGD 30


1.5 Queensland & GBS Neonatal Disease (19 EOGND Cases Annually) 30 1.6 Vaccine Technology 31 Table of Contents continued Page 1.7 GBS Detection Assays 32 Rapid Culture Method 32 Urine & Gram Staining Methods 33 Immunoassay Detection Assays 33 Polymerase Chain Reaction (PCR) Assays 34 Real-Time PCR Assays 35 1.8 GBS in the Future 38 1.8.1 Resistance to Antibiotics 38 1.8.2 GBS: The New Causative Agent for Known Diseases 40 1.8.3 GBS Serotype Distribution & Prevalence 41 1.9 Project Assessment 45 Study Summary & Hypotheses 46 Overall Objectives of the Study 47 CHAPTER 2: ESTABLISHMENT OF THE STUDY, ETHICS APPROVAL, COMMUNITY CONSULTATION & PROCEDURAL STRATEGIES 48 2.1 Establishment of the Study Prior to Ethic Submission 48 2.2 Ethics Approval Sought after Establishment of the Study 49 2.3 Community Consultation 50 2.4 Recruitment Numbers & the Power of the Study 51 2.5 Population 51 2.6 Recruitment & Requirements of the Participant 51 2.7 Collection of Clinical Specimens 52 The Collection Package 52 Type of Swabs Used 53 Self or Professional Collected Samples 53 Transportation 54


2.8 Initial Processing of Collection Packages 54


Table of Contents continued Page CHAPTER 3: GBS: THE PREVALENCE, PREGNANCY, ASSOCIATED DEMOGRAPHIC, RISK, & SOCIAL FACTORS. THE METHODOLOGY & RESULTS 55 3.1 GBS: The Prevalence, Pregnancy, Associated Demographic, Risk, & Social Factors - Methodology 55 3.1.1 Statistical Analysis 55 3.2 GBS: The Prevalence, Pregnancy, Associated Demographic, Risk, & Social Factors - Results 56 3.2.1 GBS Prevalence 56 Population 56 Pregnant, Non Pregnant Women 56 Medical Agencies 56 Pregnant Women & Gestational Age 57 3.2.2 Demographic Factors Associated with GBS Colonisation 58

Ethnicity 58 Age 59 Education and Occupation 59

3.2.3 Risk Factors Associated with GBS LGT Colonisation 62 Previous Maternal GBS 62 Previous Pregnancy & the Number of Previous Pregnancies 62 Number of Pregnancies 63 Other Risk Factors: Contraception & Maternal UTIs 63 Summary of Risk Factors Associated with GBS LGT Colonisation 63

3.2.4 GBS Colonisation, Preterm Delivery & Adverse Pregnancy Outcomes 64 3.2.5 Indigenous Non Indigenous Population Comparison 66

Indigenous Women & the Ethnicity of their Partner 67 Indigenous Women & Age 68 Indigenous Women & Partner by Age 69 Aboriginal Women, GBS & Adverse Pregnancy Outcomes 70

CHAPTER 4: PCR ASSAY METHODOLOGY & RESULTS 73 4.1 PCR Assay Methodology 73 4.1.1 Population 74 4.1.2 Microbiological Testing & Storage of Isolates 75 Storage of GBS Isolates 76 4.1.3 The Developmental History of the Rapid bsp PCR Assay 76 The Bsp Gene and its Specificity 76 The Primers 76


Table of Contents continued Page

4.1.4 Development & Optimisation of the PCR Assay 77 The Swab 77 PCR Laboratory Minimal Detection Rates 77 Optimisation of DNA Sample Preparation 77

4.1.5 Detection of the PCR Product 79 4.1.6 Optimisation of the Rapid bsp PCR Assay 79

PCR Cycling Conditions 79 Optimisation of the Reaction Mix 80 The Final Reaction Mix 80

4.1.7 Inhibition Testing 81 4.1.8 Statistical Analysis 81 4.2 Rapid Bsp PCR Assay Results 81 4.2.1 GBS Culture/PCR Assay Comparison 82 4.2.2 PCR Assay Evaluation & Culture Type 83 4.2.3 PCR Assay’s Specificity Sensitivity & Predictive Values 83 4.2.4 PCR Assay’s Overall Performance 84 CHAPTER 5: GBS SEROTYPING METHODOLOGY & RESULTS 87 5.1 GBS Serotyping Methodology 87 5.1.1 Population for Serotyping Distribution & Prevalence 87 Single & Multiple Serovar Distribution 87 Serovars in Pregnant and non Pregnant Women 88 Detection of Serovars by bsp PCR Assay 88 5.1.2 Serotype Testing Kit 88 Interpretation of Assay Results 88 Controls 89 5.2 GBS Serotyping Results 89 5.2.1 Brisbane GBS Serotype Distribution 89

Pregnant Non Pregnant Women GBS Serotype Distribution 91 5.2.2 Rapid PCR Assay Detection of GBS Serotypes 92 5.2.3 Multiple GBS Serovars & Detection by PCR Assay 93 2

CHAPTER 6: DISCUSSION 95

Epidemiology: The GBS Prevalence 95


Screening Non Screening Protocols 96 Demographics & Other Risk Factors Associated with GBS Colonisation 99 Adverse Pregnancy Outcomes History & GBS Colonisation 103 Indigenous Women, GBS Prevalence & Adverse Pregnancy Outcomes 104 Rapid GBS Detection Test & Vaccine Technology 107 Serotyping of GBS Clinical Isolates 109 Conclusion 111

APPENDIX LIST OF FIGURES Page CHAPTER 4 Figure 1: Primers used to isolate the Bsp fragment for implication 76 Figure 2: Bsp gene fragment beginning at position 1127 and finishing at position1238 77 LIST OF TABLES Page CHAPTER 1 Table 1: The Australian EOGNS incidence during the 1900’s (Isaacs & Royle, 1999) 9 Table 2: Mortality rates associated with EOGND 11 Table 3: Maternal & neonatal GBS prevalence 19 Table 4: Protocols used to prevent GBS neonatal disease 24 Table 5: Comparisons of screening protocols within Australia, USA & UK 26 Table 6: Evaluation of GBS detection assays 37 Table 7: GBS resistance to antibiotics 40 Table 8: GBS Serovar Distribution and Frequency from Early 1970’s to 2005 44 CHAPTER 3 Table 9: Prevalence of GBS amongst women of the Brisbane region 57 Table 10: Demographic factors associated with GBS LGT colonisation 61 Table 11: Risk factors associated with GBS colonisation 64 Table 12: Adverse pregnancy outcomes 66 Table 13: GBS colonisation comparison between Aboriginal/Australasian Indigenous & Non Indigenous women 66 Table 14: GBS colonisation comparison of Aboriginal/Australasian Indigenous women including partners & Non Indigenous women including partners 67 Table 15: GBS colonisation of Aboriginal/Australasian Indigenous & Non Indigenous women by age 69 Table 16: GBS colonisation comparison of Aboriginal/Australasian Indigenous women including partners & Non Indigenous women including partners by age 70 Table 17: GBS colonisation & Aboriginal women 72


CHAPTER 4 Table 18: Vaginal, perianal, culture and PCR comparisons 83 Table 19: PCR sensitivity: direct, subculture GBS detection & PCR assay comparison 84 Table 20: PCR culture test comparison 85 CHAPTER 5 Table 21: Distribution of GBS serotypes including a vaginal & perianal comparison 91 Table 22: GBS serotype distribution of pregnant non pregnant women 92 Table 23: Rapid PCR assay detection of GBS serotypes 93 Table 24: Multiple serotypes isolated from 15 vaginal & perianal samples 94


DECLARATION

The work present in this thesis has not been previously submitted for a degree at this or any other university. To the best of my knowledge and belief, this thesis contains no material previously published or written by any other person except where due reference is made. Karen Leigh Taylor 4 October 2006


ACKNOWLEDGEMENTS Completion of a thesis is never a solo journey. There are a number of people whom I would like to personally acknowledge and thank them for their help, support and guidance. Firstly I would like to thank Dr C Knox for her continual support and encouragement. I am extremely grateful. People at QIMR have assisted this project in so many ways. I wish to thank Dr Michael Good, Dr Sri Sriparkash, Bacteriology Pathogenesis laboratory staff; Amm Nonlark, Dr Michael Batzloff, Ms Melina Georgousakis, Ms Jo Shera, Ms Catherine Denham, Mr John Hartas, Dr to be Mark Davies, Mr Hsien Kuo Sun, Mr Michael Blinks, Mr Thanh Tran, and Dr David McMillian for their extensive help, their kindness and especially their sense of humour. Thank you QIMR Epidemiology Department, Prof Adelle Green, Mrs Lyn Green, Ms Barbara Ranieri, Mrs Val Logan, Dr Patricia Valery, Mrs Maricel Hughes, Mrs Nirmala Pandeya, the many Sues and the many others for their encouragement and support. I would also like to thank so many more people for their valuable assistance Ms Narelle George and Ms Jan Bodmin from Qld Health Microbiology Pathology Laboratory, Royal Brisbane Hospital, Ms Sue Gill from QUT, Dr Wendy Munchoff PA Hospital, Dr Caroline Harvey and Ms Cynthia Pollard from Family Planning Qld, Mrs Denise Watego, and the many staff members of Queensland Health Agencies who have helped recruit patients and collect swabs for this project. I would like to thank Mr Mick Gooda, director of The Cooperative Research Centre for Aboriginal and Tropical Health (CRCATH), Queensland Institute of Medical Research (QIMR), Prince Alfred Hospital, and Australian Centre for International Tropical Health and Nutrition (ACITHN) who funded my project and awarded a PhD scholarship for which I am very grateful. I would also like to thank two very special people, my daughter Larah Jayne who has been there with me every step of the way and my dear friend Ms Mary Tidey, thank you, thank you, thank you. LIST OF ABBREVIATIONS


AAP American Academy of Pediatrics ACOG American College of Obstetricians and Gynecologists AICHS Aboriginal and Islander Community Health Services Brisbane Ltd ACITHN Australian Centre for International Tropical Health and Nutrition Biala Biala Sexual Health Clinic bp Base Pairs BSA Bovine Serum Albumin CAMP Christie, Atkins, and Munch-Petersen test CDC Centre for Disease Control cfu Colony Forming Units CPS Capsular Polysaccharide Structures dNTP Deoxynucleotide Triphosphates EIA Enzyme Immunoassay EOGND Early Onset GBS Neonatal Disease EOGNS Early Onset GBS Neonatal Sepsis FPQ Family Planning Queensland GAS Group A Streptococcus GBS Group B Streptococcus GIT Gastrointestinal Tract GUT Genitourinary Tract IAP Intrapartum Antibiotic Prophylaxis IgA Immunoglobulin A IgG Immunoglobulin G Inala Inala Community Health Centre IUD Intrauterine Device Latex Latex Agglutination Test LGT Lower Genital Tract LOGD Late Onset GBS Disease Mater Mater Women’s Hospital NICU Neonatal Intensive Care Unit OIA Optical Immunoassay PAH The Prince Alfred Hospital PCR Polymerase Chain Reaction PROM Preterm Rupture of Membranes QEII Queen Elizabeth II Hospital QH Queensland Health QIMR Queensland Institute of Medical Research QLD Queensland QUT Queensland University of Technology Redland Redland Hospital RWH The Royal Women’s Hospital SIDS Sudden Infant Death Syndrome STI Sexually Transmitted Infection THB Todd Hewitt Broth UGT Upper Genital Tract USA United States of America


UTI Urinary Tract Infection


CHAPTER 1: GROUP B STREPTOCOCCUS

1.1 Introduction Streptococcus agalactiae, also known as Group B streptococcus (GBS) is one of the leading causes of neonatal

morbidity and mortality throughout the world (Isaacs & Royle, 1999; Mehr et al., 2002; Mullaney, 2001; Pinar,

2004). In Australia, in the 1990’s, GBS was reported as the most prevalent organism causing neonatal disease

(Garland & Fliegner, 1991; Isaacs et al., 1995; Isaacs & Royle, 1999). Similarly within the United States of America

(USA) this microorganism has been the leading cause of neonatal disease since the 1970’s (CDC, 2004a; Dermer et

al., 2004; Mullaney, 2001). This bacterium is also known to cause disease in children, women, the

immunocompromised adult and the elderly (Amaya et al., 2004; Palazzi et al., 2004; Schuchat, 1998).

The neonate is at greatest risk of GBS infection upon delivery, and premature babies are at the greatest risk of death

and disease (Mullaney, 2001; Schimmel et al., 1998; Schuchat & Wenger, 1994). Most frequently the neonate

becomes colonised with GBS during labour through vertical transmission from the GBS colonised mother. Vertical

transmission of GBS from colonised mothers can result in 50 to 75% of their neonates becoming colonised with GBS

(Boyer et al., 1983; Dillon et al., 1987; Jeffery & Royal, 2002). During labour a healthy mother colonised with GBS

will not always show signs or symptoms of colonisation and, therefore, vertical transmission from mother to neonate

during delivery may occur unnoticed and result in neonatal disease. Maternal detection of GBS during pregnancy and

administration of treatment to the mother during labour will lead to a decreased incidence of neonatal GBS

colonisation and subsequently a reduction in the incidence of neonatal GBS disease.

Neonates in utero, can also become colonised with GBS. GBS can ascend into the upper genital tract (UGT) and pass

through placental membranes into the amniotic fluid where the GBS infected amniotic fluid may be aspirated by the

neonate causing miscarriages, preterm delivery and stillbirths (Garland, 1991; Katz & Bowes, 1988; Nizet, 2002).

Neonates can also acquire GBS, although less frequently than previously described; from infected breast milk

(Mullaney, 2001); through nosocomial (Mullaney, 2001) or community acquisition (Baker & Kasper, 1977); or as a

recurrent infection in a new site after antimicrobial treatment (Schuchat, 1998). Alternatively, GBS infection may

occur when neonates are treated for an illness and administered antibiotics. This alters the baby’s normal flora and

facilitates invasive GBS disease (Schuchat, 1998).


1.1.1 History of Group B Streptococcus GBS was first identified in 1887 as a pathogen of bovines; a cause of bovine mastitis (Schuchat & Wenger, 1994) and

was first reported as a human pathogen in 1953 (Schuchat, 1998). GBS has been categorised as normal human

regional flora asymptomatically colonising the gastrointestinal and genitourinary tracts of both men and women

(Jeffery & Royal, 2002; Schuchat, 1995). This organism has also been isolated from the throat and respiratory tract of

humans (James, 2001; Schuchat & Wenger, 1994). It has been suggested that the gastrointestinal tract may be the

reservoir for GBS in both humans and other animals which asymptomatically carry pathogenic GBS (Sneath, 1986a)

allowing this opportunistic organism to transiently colonise the lower genital tract (LGT) of women and as a result

this bacterium may be present at one point in time but subsequently not detected even a few days or weeks later (Bliss

et al., 2002; Chua et al., 1995b; Jeffery & Royal, 2002). This bacterium is a sexually transmitted infection (STI) as it

has been found in 31 to 65% of male urethras (Bliss et al., 2002; Centre, 1996; James, 2001). For these reasons the

current method for detecting GBS in pregnant women is not a true indicator of maternal GBS colonisation during

labour and the neonatal disease potential. Other organisms that also commonly asymptomatically colonise the female

LGT which may result in an adverse pregnancy outcome include; Ureaplasma parvum, U. urealyticum, Mycoplasma

hominis, other sexually transmitted and pathogenic bacteria (Knox, 1997) but of these organisms, GBS poses the

greatest risk to neonates (Isaacs & Royle, 1999).

1.1.2 Morphology of GBS GBS is a beta haemolytic facultative Gram positive diplococcus which can often be found growing in very long

chains of paired streptococci (Schuchat & Wenger, 1994). GBS cells are spherical or ovoid from 0.6 to 1.2µm in

diameter; however, colonies grown on blood agar plates range in size from 3 to 4mm, they are greyish-white in

colour, flat and appear mucoid (Stevens & Kaplan, 2000). GBS produces a distinctive narrow β–haemolytic zone

which sometimes may only be observed when the colony has been removed from the blood agar plate (Stevens &

Kaplan, 2000). non haemolytic or γ-haemolytic strains account for 1 to 2% of GBS isolates and α-haemolytic or

double zone GBS strains are rare (Stevens & Kaplan, 2000). Usually β–haemolysis is caused by the production of

haemolysins O and S which diffuse into the media and causes haemolytic activity. However, some strains produce a

characteristic opaque β-haemolytic zone which is different from the zones produced due to hemolysin O and S. It has


been suggested that opaque β-haemolytic zone may be due to a soluble haemolysin which has a low haemolytic

activity (Sneath, 1986a).

The pathogenic mechanisms of GBS are not fully understood. Several virulence factors have been identified and

include: capsular polysaccharides which avoids the hosts’ defences; enzymes such as C5a peptidases and

hyaluronidases which spread and destroy the host; beta haemolysins/cytolysin toxins, lipotechoic acid and surface

protein antigens which are recognised in human infections (Doran et al., 2003; Sneath, 1986a).

In order to initiate an infection the GBS microorganism must be able to adhere to many different cell types within the

human body and avoid the body’s immune defence systems. Firstly the microorganism must be able to adhere to the

cells of the mucous membranes, the epithelial cells lining the vagina and the rectum. Once attached to the body’s

surface areas and avoiding the body’s immune defences GBS can be vertically transmitted to the neonate during

delivery causing invasive disease soon after birth which may result in death within 48hours. Alternatively, once GBS

colonisation of the vagina is established, GBS can invade the placental membranes and enter the amniotic fluid

causing adverse pregnancy outcomes (Katz & Bowes, 1988). From a focus of infection in the lungs of the foetus or

neonate GBS can gain access to the circulatory system causing a rapid bacterial systemic infection and at birth can

result in a serious postnatal fatality or prior to birth result in stillbirth or miscarriage (Katz & Bowes, 1988; Nizet et

al., 2000).

1.1.3 GBS Serotyping All GBS serovars are capable of causing neonatal disease and possess a polysaccharide cell wall antigen composed of

rhaminose, N-acetylglucosamine and galactose (Sneath, 1986a). Differences within these antigens are used to

distinguish eight antigenic GBS serovars (I–VIII) (Schuchat, 1998). Type I has been divided into three subtypes Ia,

Ib, (Schuchat, 1998) and Ia/c (Mullaney, 2001). In addition, a small number of strains do not react with hyperimmune

sera; therefore, they are classified in a separate group termed non typeable (Schuchat, 1998). At present, both human

and other animal GBS species are classified together, as taxonomical differences between them have not been

identified. However, recently the human serovar III has been classified into four distinct phylogenetic lineages which

suggests that the human serovar III is largely unrelated to the bovine serovar III (Bohnsack et al., 2004).

1.2 GBS Disease in Neonates


Neonatal disease has been classified as Early Onset GBS Neonatal Disease (EOGND) and Late Onset GBS Disease

(LOGD). EOGND occurs within the first 72 hours after birth to one week of age while LOGD occurs beyond the

initial seven days after birth to three months of age.

1.2.1 Early Onset GBS Neonatal Disease (EOGND) Early onset GBS neonatal disease (EOGND) is caused by GBS invading and infecting the neonate’s lungs, causing

pneumonia, then subsequently GBS may disseminate systemically via the neonate’s circulatory system causing

septicemia (sepsis) and meningitis (Fletcher & Gordon, 1990; Mullaney, 2001). Prior to 2001, 15 to 50% of neonates

systemically infected with GBS die annually (Fletcher & Gordon, 1990; Mullaney, 2001). The mortality rate

associated with EOGND has been as high as 50% (Anthony & Okada, 1977); however, current data suggests a

neonatal mortality rate of 5 to 10% (Dermer et al., 2004; Fletcher & Gordon, 1990; Isaacs & Royle, 1999; Mullaney,

2001). Of those neonates with EOGND approximately half who survive can suffer from long term sequelae (Fletcher

& Gordon, 1990).

GBS causes early onset GBS neonatal septicemia (EOGNS) in approximately 80% of GBS infected neonates whilst

other manifestations of EOGND include pneumonia and meningitis which occur in 7 and 6% of infected neonates;

respectively (Fletcher & Gordon, 1990; Mullaney, 2001). Complications of GBS infections have also been reported

in 89% of GBS infected infants and children who suffered from GBS sepsis with or without pneumonia (CDC 1997).

Those neonates with EOGND who survive can suffer from neurodevelopmental defects such as psychomotor

retardation, spasticity, hemiparesis and seizures (Fletcher & Gordon, 1990). They can also suffer from hearing or

visual loss and an increased incidence of bronchopulmonary dysplasia (Fletcher & Gordon, 1990).

Weisman et al. (1992) and Schuchat (1998) found that up to 83% of all GBS infected neonates became symptomatic

very rapidly within the first 72 hours of life (Schuchat, 1998; Weisman et al., 1992). EOGND is not limited to

mothers who deliver their babies naturally (vaginal birth). Weisman et al (1992) studied the records of 245 neonates

with GBS bacteremia from nine hospitals across the USA and found that EOGND occurred in 37/149 (24.8%) of term

caesarean delivered babies and 29/96 (30.2%) of preterm caesarean delivered babies (Weisman et al., 1992).

GBS causes 60% of all sepsis in preterm neonates and sepsis is the second most common cause of death in preterm

babies (behind lethal malformations) (Doyle et al., 1999; Mehr et al., 2002). In Australia GBS mortality in very

preterm infants increased from 14% in 1980s to 44% in 1990s (Doyle et al., 1999; Mehr et al., 2002). Preterm


newborns infected with GBS are also at risk of long term sequelae such as the development of patent ductus

arteriousus, intraventricular hemorrhage, and periventricular leukomalacia (Baker and Edward 1994, (Schuchat, 1998;

Stoll et al., 1996). Complications can include lack of oxygen to the brain and vital organs of the body including the

heart causing irreversible brain and other organ damage.

An Australian study in 1999, identified microorganisms that were collectively responsible for 50% of all early onset

neonatal sepsis disease cases are other Streptococcal species such as Streptococcus pneumoniae, and Enterococcus

faecalis as well as Escherichia coli, Haemophilus influenzae type B, Listeria monocytogenes, other Gram-negative

bacilli, anaerobes, Staphylococcus aureus and Candida spp. (Isaacs & Royle, 1999). This Australian study also found

that GBS alone was responsible for 50% of all EOGNS cases (Isaacs & Royle, 1999).

Signs, Symptoms, Treatment of EOGND

Within 48 hours after birth all neonates infected with GBS will express signs and symptoms of early GBS invasive

neonatal infection, while 78% of all cases will show signs and symptoms in the first 24 hours after birth (Towers et

al., 1999) which suggests intrapartum pathogenesis. Weisman et al., (1992) found approximately 22% of term

neonates, despite being infected with GBS did not show any symptoms of infection within the first 24 hours (Towers

et al., 1999; Weisman et al., 1992). A study of African American neonates also found that these neonates did not

always show signs of EOGND infection (Towers et al., 1999; Zaleznik et al., 2000). In contrast 100% of neonates

who delivered preterm expressed early symptoms of EOGND (Weisman et al., 1992).

The most common sign of EOGND is respiratory distress (Mullaney, 2001), which may be verified by x-ray and

confirmed by GBS culture. Respiratory distress occurs in 80% of all EOGNS cases (Jeffery & Royal, 2002). Other

signs and symptoms include cyanosis, poor perfusion, hypotension, lethargy, poor muscle tone as well as poor

feeding, temperature instability and glucose instability (Mullaney, 2001; Weisman et al., 1992). Irritability and

hyperthermia (>37.2oC) were reported more frequently in term babies while lethargy or poor muscle tone or both,

neutropenia, and hypothermia (


10 to 14 days and for 14 to 21 days for infants with meningitis (Fletcher & Gordon, 1990). GBS infected neonates

require intense medical care involving maintenance of the infants’ body temperature and pH balance, and providing

cardiovascular support including fluid management and, if needed, respiratory support (Mullaney, 2001).

Asymptomatic neonates are sometimes diagnosed with EOGND based on maternal risk factors being expressed

during labour prior to infection but verified 24 hours later by neonatal blood cultures and these neonates should be

monitored and administered antibiotics if required (Weisman et al., 1992). Some neonates presented with EOGND

even after their mother had been treated with intrapartum antibiotic prophylaxis (IAP) (Gotoff, 2000a; Jeffery &

Royal, 2002). To date debate still exists whether to administer antibiotics immediately to neonates presenting with

EOGNS if the mother had received IAP.

The EOGND Incidence

Evidence indicates that the GBS maternal colonisation rate has not changed over time; however, in some countries the

incidence of neonatal GBS infection has altered significantly over the years (CDC., 1997; Gotoff, 2000a; Mullaney,

2001). Garland reported in 1995 that 2 to 5% of mothers are carriers of GBS (Garland & Kelly, 1995) and a current

EOGND incidence of 1.1 per 1000 live births has been reported in the USA (Boyer et al., 1983; Chen et al., 2001;

Isaacs & Royle, 1999; Janek et al., 2004; Jeffery & Royal, 2002; Morales et al., 1986). In 1998 and 2000 the USA

EOGND incidence was reported as low as 0.6 per 1000 live births (Dillon et al., 1987; Fletcher & Gordon, 1990;

Schrag et al., 2000). In 1999, Canada reported an incidence of 0.25 per 1000 (Davies et al., 2001).

The UK/Ireland EOGND incidence was reported at 0.48 per 1000 live births in 2002 (Heath et al., 2004) and

previously a two year study concluding in 2000 reported an incidence of 0.57 per 1000 live births (Oddie &

Embleton, 2002). Both incidences are double the current Australian EOGNS rate (CDC, 1997; Isaacs & Royle, 1999;

Zangwill et al., 1992). If antenatal screening was widely practiced throughout the UK and Ireland then the current

EOGND incidence may decrease further (Kenyon et al., 2005). The UK rate is low in comparison to an EOGNS

incidence of 0.7 to 1.0 per 1000 live births reported from the Czech Republic in 2004 (Strakova & Motlova, 2004)

and 0.76 per 1000 live births reported as the Danish mean annual rate also reported in 2004 (Andersen et al., 2004).

In 1999 the USA EOGND rate reported was 0.6 per 1000 live births, just 0.1 per 1000 higher than the Australian

EOGNS rate for the same time period (CDC, 1997; Isaacs & Royle, 1999; Zangwill et al., 1992). Prior to 1996 the

USA EOGND varied between one to four cases per 1000 live births (Jeffery & Royal, 2002). Despite the overall


decrease in EOGND within the USA some American populations are still at a greater risk of EOGND. During the

past 10 years neonates of African American descent have a higher rate of EOGND compared to other American

population groups (Isaacs, 1998). African American neonates are at an even greater risk if their mothers’ age is less

than 20 (Isaacs, 1998).

The Australian EOGND Incidence

In Australia the EOGNS incidence ranged from 0.1 to 0.2 per 1000 live births (Australasian, 1995; Isaacs et al., 1995;

Isaacs & Royle, 1999; Jeffery & Royal, 2002). In 2001 data from a combined assessment of obstetric hospitals of

Australia and New Zealand over a ten year period evaluating 298,319 live births also reported a incidence up to 0.2

per 1000 live births (Daley et al., 2004). A year earlier the Australian and New Zealand EOGNS incidence was 0.25

per 1000 live births (Daley et al., 2004) and an earlier nine month Australian study conducted in Brisbane,

Queensland, in 1999, reported an EOGND incidence of


rates were reported for the total Australian population and separately for the Indigenous population as 0.5/1000 and

1.8/1000 per live births; respectively (Table 1) (Isaacs & Royle, 1999).

Table 1: The Australian EOGNS Incidence during the 1900’s (Isaacs & Royle, 1999)

From 1991 to 1993 GBS caused 67% of the neonatal early onset infections, from 1993 to 1995, 61% of the cases and

from 1995 to 1997 only 52% of all cases (Isaacs & Royle, 1999). Isaacs and Royle (1999) also reported “the

reduction in incidence applies to all cities and to both Aboriginal and non Aboriginal babies” (Isaacs & Royle, 1999).

Although there has been a significant decrease in EOGNS rates over this six year period, Indigenous neonates still

have a 4.3 (95% CI 2.9-6.2) times greater chance of developing EOGNS (Isaacs & Royle, 1999).

After the implementation of preventative protocols, reports from selected hospitals also confirmed the reduction in the

EOGNS rate over time. Prior to 1988 the incidence of EOGNS at the Royal Prince Alfred Hospital in Sydney was 1.4

per 1000 live births. From 1988 until 2001 the hospital saw reductions in the incidence over time evaluating 36,342

live births. At the end of 2001 the incidence was significantly reduced to 0.26 per 1000 live births (Jeffery & Royal,

2002).

EOGND Mortality Rates

EOGND acquired by vertical transmission from the mother at birth can be rapidly fatal. In the early 2000s, USA

reported an EOGND mortality rate ranging from 2.7% and as high as 14% (Table 2) (Lukacs et al., 2004; Weisman et

al., 1992). In Australia, the EOGNS mortality rate prior to 2000 ranged from 6 to 15% (Table 2) (Connellan &

Wallace, 2000; Garland & Kelly, 1995; Isaacs et al., 1995; Isaacs, 1998; Isaacs & Royle, 1999; McLaughlin &

Crowther, 2000). In Melbourne a 10 year study from 1979 to 1988 was conducted at the Royal Women’s Hospital

which resulted in 29/104 (28%) deaths (Garland, 1991). In 2004, the UK and Ireland reported a rate of 9.7% while

Canada from 1993 to 1997 reported a rate of 13.6% which are consistent with USA and Australian data (Table 2)

(Heath et al., 2004).

hallaThis table is not available online. Please consult the hardcopy thesis available from the QUT Library


Even though mortality rates were comparable between the USA, Australia, Canada and UK/Ireland, the mortality

rates differ between full term and preterm delivered babies and preterm babies are at a much greater risk of death

(Towers et al., 1999). In 2004, the USA reported a decrease in mortality rates at 6.5 and 22.7% for term babies and

preterm babies; respectively (CDC, 2004b). Since 1992, the reported USA mortality rates for full term and preterm

babies were at 2 and 28%; respectively (Weisman et al., 1992).

All these studies have shown that there has in fact been a decrease in the mortality rates for all neonates including

neonates who were delivered at term or preterm in the USA since 1992 (Table 2). In the USA the EOGNS mortality

rates dramatically reduced from 55% in 1977 to 10 to 15% by the 1980’s then decreased slightly further to 14% by the

2000s, but during the 1990’s the EOGNS mortality rate increased to 28% (Anthony & Okada, 1977; CDC, 1997;

Dermer et al., 2004; Lukacs et al., 2004; Schuchat et al., 1990; Weisman et al., 1992; Zangwill et al., 1992). It was

estimated in 1973 in Australia, that the EOGNS mortality rate was 10% which is equivalent to the death of 30 out of

the 425 neonates who developed EOGNS (calculations are based on a sepsis rate of 1.7 per 1000 live births and a

birth rate of 250 000 per annum) (Table 2) (Isaacs & Royle, 1999). During the same period the EOGNS mortality rate

in the USA was 6% (Isaacs & Royle, 1999).

Table 2: Mortality Rates Associated with EOGND

Year Ref Study Size

1970’s 1980’s 1990’s 2000’s

USA 6-55% 10-15% 2-18% 2.7-14%


Benitz et al., (1999) stated the maternal GBS colonisation rate has not altered over the years (Benitz et al., 1999a);

even though Australian and USA studies have reported deceases in the GBS neonatal mortality rate (Isaacs & Royle,

1999; Mullaney, 2001). These reductions in GBS neonatal mortality rates may be attributed to one or all three of the

following reasons: (i) the introduction of protocols to prevent the transmission of GBS from the mother to the

neonate which incorporated the use of intrapartum antibiotic treatment; (ii) improvements in medical intervention for

both the mother and the neonate; and (iii) advances in medical technology including neonatal monitoring equipment

and the improvement in management of neonates in intensive care (Garland & Kelly, 1995; Mullaney, 2001;

Weisman et al., 1992).


1.2.2 Late Onset GBS Disease The source, the risk factors, and the routes of transmission associated with LOGD are poorly understood and as a

consequence the true incidence of LOGD has not been established (Schuchat, 1998). This neonatal disease can cause

similar illnesses to EOGND though most commonly LOGD manifests as meningitis (Fletcher & Gordon, 1990;

Schuchat, 1995). It has been reported that 20% of all neonates who have contracted LOGD and who survive may

suffer from long term illnesses such as severe neurological sequelae and other comparable sequelae caused from

EOGND (Beardsall et al., 2000; Towers et al., 1999). A German study in 2003 reported neonates with LOGD

meningitis have a neurological sequelae rate of 40%, a rate double that previously reported (Haase et al., 2003).

Neonatal LOGD can also cause septic arthritis, osteomyelitis, cellulitis-adenitis, pneumonia, pleural empyema,

endocarditis, urinary tract infection (UTI’s) and endophthalmitis (Fletcher & Gordon, 1990; Schuchat, 1995).

Additionally there were other reports of LOGD manifesting as neonatal lymphadenitis (Fluegge et al., 2003) and

necrotizing fasciitis (Lang et al., 2003).

GBS serovar III has been reported in USA, England/Wales, and western Sweden from 1988 to 2004 as the most

frequently isolated serotype causing LOGD (Baker, 1980; Bliss et al., 2002; Martinez et al., 2004; Mullaney, 2001;

Persson et al., 2004; Weisner et al., 2004). In 1988 in the USA serovar III was then the most frequently isolated

subtype causing 71% of all LOGD cases (Mullaney, 2001; Schuchat, 1998).

The major source of GBS infection in LOGD cases is thought to be the mother, because up to 50% of neonates with

LOGD had identical GBS serovars to their mothers (Towers et al., 1999). GBS responsible for LOGD may have been

acquired in utero or at time of delivery; however, the infant may have had some immunity (perhaps maternal)

resulting in the delayed development of the disease (Boyer 1992). GBS infected breast milk has also been recorded as

a source of LOGD (Kotiw et al., 2003). Kotiw used a PCR subtyping test and demonstrated serotypes obtained from

the mothers’ breast milk and the infant were identical (Kotiw et al., 2003). It is not understood how GBS infection of

the breast milk occurs (Schuchat, 1998). Other sources for LOGD could include nosocomial transmission (Jeffery &

Royal, 2002; Schuchat, 1998) and community transmission (Bingen et al. 1992). LOGD has also been reported as a

recurrence of EOGND (Kotiw et al., 2003; Schuchat, 1998). It has also been reported that LOGD may be the result of

a reinfection and the rate for LOGD infection of previously treated infants was approximately 1% (Mullaney, 2001;

Schuchat, 1998). It was also established that the infection site was new in 50% of re-infected neonates (Schuchat,

1998).


Signs, Symptoms, Treatment of Neonatal LOGD

Symptoms of LOGD generally appear after seven days of life and can include lethargy, poor feeding or irritability and

fever (Mullaney, 2001). Neonates with GBS meningitis may present with upper respiratory tract infection, otitis

media, facial cellulitis, septic arthritis and osteoarthritis (Mullaney, 2001). More serious LOGD “symptoms such as

apnea, seizures, leukopenia or neutropenia are more likely to have a fatal outcome” (Mullaney, 2001). Thirty to 40%

of neonates with LOGD will develop meningitis and a large number of these neonates will suffer from permanent

neurologic sequelae (Schuchat, 1998). Similar to EOGND meningitis sufferers, LOGD meningitis symptoms include

frequent seizures (Weisman et al., 1992) and the infected neonates require intense medical care and treatment.

The Australian LOGD Incidence Compared to Other Countries

In Australian the LOGD incidence is not known. In 2002, a LOGD incidence of 0.5 per 1000 neonates was reported

at the Royal Prince Alfred Hospital (Boyer et al., 1983; Dillon et al., 1987; Jeffery & Royal, 2002). A study

conducted in the UK and Ireland for a period of one year and concluded in 2001 reported a LOGD incidence of 0.24

per 1000 live births (Heath et al., 2004). Edwards and Baker (1999) reported that the incidence of LOGD remained

constant even with the introduction of maternal IAP at 0.3 to 0.5 cases per 1000 live births (Edwards & Baker, 2003).

Other USA studies from 1999 to 2001 have reported a much higher LOGD rate ranging from 2.1 to 2.4 per 1000 live

births (Dillon et al., 1987; Fletcher & Gordon, 1990; Schrag et al., 2000). A USA study, in 1999, which evaluated

46,959 patients over a 9.5 year period, reported a similar incidence of 2.1 per 1000 live births (CDC., 1997; Schrag et

al., 2000; Towers et al., 1999). In contrast, another USA study presented a much lower LOGD rate of 0.4 per 1000

live births during 1990 and 1998 (Mullaney, 2001).

Although the incidence for LOGD is lower than the incidence for EOGND; the LOGD incidence had not been

regularly reported in other countries outside the USA. In the USA prior to 1998 Schuchat reported that the EOGND

incident rate had remained constant (Schuchat, 1998) and in 2004 it was reported that a reduction in the incidence had

not occurred (Gibbs et al., 2004). Other authors have also concurred stating the incidence of LOGD had not

decreased and was unaffected by current EOGND IAP treatment (Schrag et al., 2000; Zangwill et al., 1992).

Mortality & Morbidity of LOGD

It is difficult to evaluate whether neonatal LOGD morbidity and mortality rates are increasing or decreasing. What

can be established is that morbidity and mortality rates resulting from LOGD vary dramatically from country to

country as well as regionally within a country (CDC., 1997; Gotoff, 2000a; Mullaney, 2001). In 2004 an USA study


reported a LOGD mortality rate of 1.6% during a three year period from 1995 to 1998 and 1.5% for a earlier period

from 1985 to 1991 (Lukacs et al., 2004). These are the lowest reported morality rates throughout the USA. Other

USA studies found that the mortality rate associated with LOGD ranged from 2 to 10% (Dillon et al., 1987; Fletcher

& Gordon, 1990; Schrag et al., 2000).

In 2000, the UK reported a LOGD morality rate of 17% (Beardsall et al., 2000), the highest ever rate possibly

published. In the same UK study it was reported that 50% of all the infants who contracted LOGD would suffer from

morbidity (Beardsall et al., 2000). This UK study investigated women from public low socio economic hospital areas

which may account for the high morality and morbidity rates (Beardsall et al., 2000).

1.3 GBS Disease in the General Population

1.3.1 GBS Disease in Pregnant Women Importantly GBS had been reported as a pathogen in pregnant women causing: i) reproductive tract infections

including; postpartum endometritis (10 to 20%), puerperal sepsis and chorioamnionitis; and, ii) postpartum

complications including: UTI’s, bacteremia, wound infections associated with caesarean delivery, and infrequently

meningitis (Farley et al., 1993a; Faro, 1981; Fletcher & Gordon, 1990; Gibbs & Blanco, 1981; Persson et al., 1988).

1.3.2 GBS Disease in the Foetus Maternal asymptomatic LGT GBS colonisation may cause the foetus to abort or a neonate to be born stillborn or to be

delivered preterm (Katz & Bowes, 1988). To prevent these adverse pregnancy outcomes women and their partners

need to be screened prior to conception as well as early in the pregnancy for the presence of this bacterium. GBS

screening of the mother should continue throughout the pregnancy as GBS may recolonise the LGT of the mother

after the administration of antibiotic treatment (Jeffery & Royal, 2002; Knox et al., 1997). Perhaps a protocol for

early gestational detection of GBS needs to be designed and implemented which may result in the reduction in

adverse pregnancy outcome cases. This protocol may help to reduce the number of miscarriages that occur, especially

in cases where a woman has had a previous miscarriage.

Preterm labour may be caused by the presence of GBS in the amniotic fluid (Kenyon et al., 2001). Kenyon et al.

(2001) studied microbial colonisation of the cervicovaginal and amniotic fluid in women with preterm labour and they

found GBS was present (Kenyon et al., 2001). They concluded that preterm delivery can occur when GBS has


invaded the placental membranes decreasing the membranes’ tensile strength and elasticity causing it to rupture

(Schuchat, 1998; Stoll et al., 1996). It had also been suggested that GBS produces proteases that degrade the

placental tissue and similar mechanisms may promote membrane rupture causing miscarriage and preterm delivery

(Kenyon et al., 2001). Given that preterm delivered neonates are at greater risk of EOGND (Oddie & Embleton,

2002) and that GBS causes preterm delivery then early third trimester screening may decrease the incidence of

neonates that are born preterm and as a consequence decrease the associated GBS neonatal infections of those groups

of neonates.


1.3.3 GBS Diseases in Infants & Children Invasive GBS disease is rare in children past infancy (three months and older) (Young et al., 1996) since infants

develop antibodies to GBS by three months of age (Isaacs & Royle, 1999; Jeffery & Royal, 2002). A USA study of

20 million children in 1998 reported that 2% of these children will develop invasive GBS disease (Edwards & Baker,

2003) and 10% of these cases will develop meningitis (CDC, 1997). In the majority of these cases, these children

were also suffering from predisposing conditions such as human immunodeficiency virus (Edwards & Baker, 2003).

The mortality rates for children with GBS disease is about 9%, much higher than the mortality rates reported for GBS

infected neonates (Edwards & Baker, 2003). Individual reports of GBS disease in infants and children have included

manifestations such as necrotizing fasciitis, endocarditis (Lang et al., 2003) and epiglottitis (Young et al., 1996).

Epiglottitis is a life threatening disease in children reported usually between the ages of two and six (Young et al.,

1996). Prior to the introduction of the HIB vaccination Haemophilus influenzae type b (Hib) was the most common

cause of epiglottitis. Epiglottis caused by GBS was extremely rare and it was not reported prior to 1995 (Young et al.,

1996).

1.3.4 GBS Disease in Adults In the USA it has been reported that 50,000 women annually are affected by antepartum, intrapartum and postpartum

GBS related illness (James, 2001) and that two thirds of all invasive GBS disease affects the adult non pregnant

population (Katz & Bowes, 1988; Nizet, 2002). In 2001, it was reported that approximately 4.4 per 100,000 adult

males and non pregnant women over the age of 15 years of age would develop invasive GBS disease annually (Farley

et al., 1993b). GBS disease in the adult population generally does not cause death (James, 2001). However, the

elderly population, those over 60 years of age are at greater risk of GBS infection and the annual rate of disease for

this cohort is 18 per 100,000 (Farley et al., 1993b). Another USA study reported the disease rate in adults over the

age of 65 at 28.3 per 100,000 (Katz & Bowes, 1988; Nizet, 2002). Immunocompromised people are also at greater

risk of GBS disease, especially elderly people who have a predisposing condition such as diabetes mellitus,

malignancy and liver cirrhosis (Liu et al., 1997). They can develop illnesses such as bacteremia, pneumonia,

endometritis and necrotizing fasciitis (Bayer et al., 1976). GBS had also been reported as a cause of UTIs in both

men and women (Fletcher & Gordon, 1990; Munoz et al., 1992; Persson et al., 1986a; Persson et al., 1988). Munoz

et al., (1992), found that 2% of UTIs in non pregnant adults was due to GBS (Munoz et al., 1992).

There are a number of risk factors associated with GBS disease within an adult population. These include: (i) age,

individuals 60 years of age or above are at greatest risk; (ii) ethnicity, African American people are at greater risk than


other ethnic groups within the USA; (iii) ethnicity plus age, African American people over the age of 60 years; and,

(iv) people with chronic underlying illnesses, such as diabetes mellitus, and HIV infection and cancer (Colford et al.,

1995; Farley et al., 1993b; Isaacs, 1998; Schwartz et al., 1991).

1.3.5 Treatment of GBS Disease in the General Population Schrag et al., (2000) stated that there is an increasing burden of GBS disease in non pregnant adults and children

(Schrag et al., 2000). If GBS is identified as the causative agent of disease through routine bacteriological culture

then these infections can be treated by the administration of antibiotics such as penicillin G.

1.4 Prevention of GBS Neonatal Infections The GBS neonatal disease potential is related to the GBS prevalence in the adult population and GBS LGT

colonisation of the mother during pregnancy. A report published in 1999 by Garland and Kelly reviewed 57,000

mothers over an eight year period. They reported that 30,197 women were screened and treated for GBS and

subsequently no cases of EOGND were reported in neonates born to these women. In the same period, 26,915

mothers who were not screened for maternal GBS colonisation gave birth to 27 neonates who developed EOGND

(Garland & Kelly, 1995). Of these 27 infected neonates eight died (Garland & Kelly, 1995). IAP administered to

mothers in labour can prevent neonatal GBS colonisation at birth (Boyer et al., 1983; Dillon et al., 1987; Jeffery &

Royal, 2002). Reports have demonstrated that GBS neonatal infections can be prevented when late gestational

screening based protocols that incorporate the administration of IAP are implemented (Boyer et al., 1983; Dillon et

al., 1987; Isaacs & Royle, 1999; Jeffery & Royal, 2002).

1.4.1 Prevalence of GBS in Adult Populations Knowledge of GBS prevalence in adult populations would lead to a greater understanding of the EOGND and LOGD

potential. The prevalence of GBS in pregnant and non pregnant women has rarely been reported in Australia. A few

hospital studies have identified regional GBS colonisation rates for pregnant women ranging from 12 to 25.8%

(Connellan & Wallace, 2000; Garland & Kelly, 1995; Garland et al., 2000; Gilbert, 2002; Jeffery & Royal, 2002;

Knox et al., 1997). In 1993, a small study of 162 pregnant women attending a public antenatal clinic in Toowoomba,

Queensland, reported a GBS colonisation rate of 16.7% (Knox, 1997). Based on USA data, McLaughlin and

Crowther (2000) proposed that 10 to 30% of pregnant women in Australia could be colonised with GBS of the LGT.

Gilbert et al., (2002) reported an Australian carriage rate of 25.8% while in 1995, Garland and Kelly reported a


colonisation rate amongst pregnant women at 23% (Garland & Kelly, 1995). A lower rate of 12 to 15% was reported

in 2002 from the Royal Prince Alfred Hospital (RPA) in Sydney; however, only vaginal samples were obtained and

cultured using non selective media (Jeffery & Royal, 2002). These rates may be underreported as an increase in the

detection rate can be achieved by; i) 20 to 40% when both vaginal and rectal or vaginal and perianal samples are taken

in conjunction and tested (Jamie et al., 2004; Quinlan et al., 2000); ii) using selective media which can increase

detection by up to 50% in cases where non selective media were used (1999); and, iii) subculturing samples that have

been inoculated in an enrichment broth media (Daley & Garland, 2004; Rauen et al., 2005).

GBS colonisation rates vary within a country as well as regionally between countries (Table 3). Within the USA

women in Columbia had a GBS colonisation rate of 26.4%, compared to rates in Washington (17.5%), Oklahoma

(13.6%), Texas (9.2%), and Lousianna (20.8%) (Regan et al., 1991). Regional rates as high as 35% have been

recorded in the USA (Boyer & Gotoff, 1986; Mercer et al., 1995b; Molnar et al., 1997).


Table 3: Maternal & Neonatal GBS Prevalence Colonisation %

Year Reference

Country

Swab Type

Number Maternal Neonatal


It has been reported that an Indigenous Australian neonate has a much greater chance of contracting GBS sepsis

compared to an Non Indigenous Australian (Isaacs & Royle, 1999). This may suggest that the prevalence of adult

GBS colonisation amongst Indigenous women is appreciably higher than for Non Indigenous Australian women.

In the USA, regional prevalence data is dominated by ethnicity and age of that population group (Anthony et al.,

1979; Morales et al., 1986; Pass et al., 1979). African American women have higher GBS colonisation rates than

other ethnic groups throughout the USA except in New York where they have the second highest rate with Hispanics

from the Caribbean having the highest rate (Schuchat, 1995; Schuchat, 1998; Zangwill et al., 1992). African

American women aged younger than 20 years of age and from a low socio economic environment have higher GBS

colonisation rates than other American women ethnic groups. African American women also have a higher incidence

of preterm delivery and have a much greater risk of giving birth to a neonate who may develop EOGND (Regan et al.,

1991). Australian Indigenous women have an increased risk of delivering their neonates preterm (Isaacs & Royle,

1999); therefore, these neonates are at greater risk of GBS disease.

1.4.2 Prevention through Treatment of the Colonised Mother Early detection and administration of antibiotics to pregnant mothers can prevent GBS infection from occurring in the

foetus and/or neonate. Data from USA and Australian studies have stated that EOGND has been significantly reduced

because of the use of selective IAP which disrupts the vertical transfer of GBS from the mother to the neonate during

labour (Boyer et al., 1983; Chen et al., 2001; Isaacs & Royle, 1999; Janek et al., 2004; Jeffery & Royal, 2002;

Morales et al., 1986). A reduction rate in EOGND of 50 to 80% has been reported (Isaacs & Royle, 1999; Jeffery &

Moses Lahra, 1998; Lukacs et al., 2004; Schrag et al., 2000). Even though the rate of EOGND has been significantly

reduced, no prevention strategy has been implemented which could reduce the incidence of GBS related miscarriage,

stillbirths, preterm delivery or LOGD. Miscarriage could be prevented if GBS screening occurred during the first

trimester and asymptomatic maternal GBS bacteruria could be identified and the mother treated (Jeffery & Royal,

2002). Other gestational screening for GBS should be implemented to reduce the incidence of preterm delivery,

stillbirths and LOGD(Jeffery & Royal, 2002).

Daley 2004 suggested that GBS should be detected using the following optimal GBS detection method. Swabs should

be collected from both vaginal and rectal regions 35 to 37 weeks gestation, either by a health care worker or self

collected (after informed instruction and diagram) and samples should be cultured on selective media after enrichment

(Daley & Garland, 2004). Vaginal/perianal sampling is the preferred detection method compared to vaginal/rectal


sampling (Jamie et al., 2004). Results of the different tests are similar and collecting a rectal sample is invasive

(Jamie et al., 2004).

Pregnant women diagnosed with GBS colonisation can be treated at intrapartum with either IV infusions of penicillin

G (5 million units for the first dose and then 2.5 million units every four hours) or ampicillin (2g for the first dose then

1g every four hours) (Gotoff, 2000a). Alternatively a women allergic to penicillin can be administered clindamycin

and erythromycin (Gotoff, 2000a). Cefazolin can also be used if the woman is allergic to penicillin but who are at

low risk of anaphylaxis (Apgar et al., 2005) or if the isolate is known to be resistant to clindamycin or erythromycin.

It has been established that IAP administration for at least two hours is more effective at reducing EOGND than one

hour of antibiotic treatment. An EOGND rate of 46% was reported when less than one hour of antibiotics treatment

was administered to the mother. Whilst administration of antibiotics for one to two hours resulted in a 29% neonatal

infection rate, a further reduction in EOGND at 2.9% was reported when IAP was administered for two to four hours.

After four hours of maternal IAP treatment an EOGND rate of 1.2% was reported (de Cueto et al., 1998).

To reduce the incidence of GBS neonatal disease, IAP is best administered to the GBS positive mother during labour.

Whilst the results from the current gold standard (culture) method for GBS detection is not rapid and could not be

used to detect GBS during labour, a PCR diagnostic test may provide the GBS status of the intrapartum mother.

1.4.3 Prevention through Treatment of the Neonate Neonatal GBS infection is best prevented by disrupting the transfer of GBS from mother to neonate prior to delivery.

Isaac 1998, reported the study by Siegel and colleagues where intramuscular penicillin was administered to neonates

after the birth for the prevention of EOGND (Isaacs, 1998). This practice was reviewed by Woodgate et al. (2004)

and their conclusions did not support this practice (Woodgate et al., 2004).

1.4.4 Prevention through Treatment of Sexual Partners GBS is a sexually transmitted disease as up to 65% of partners shared identical GBS serovars, which is consistent

with sexual transmission (Bliss et al., 2002; CDC, 1996; Fletcher & Gordon, 1990; James, 2001; Persson et al., 1988).

In 2004, in the USA the GBS colonisation rate of participants who had experienced sex doubled when compared to

participants that were sexually inexperienced (Manning et al., 2004). Still, no current literature on the concurrent


treatment of both GBS colonised partners and the effect that his treatment may have on the incidence of GBS foetal

and neonatal disease. Treatment of both the mother and their partner may lead to a decreased incidence of GBS

colonisation and subsequently a decreased incidence of miscarriage, preterm delivery, EOGND and LOGD.

1.4.5 EOGND Prevention Protocols EOGND prevention protocols that included guidelines for the screening and the administration of selective IAP have

reduced the incidence of neonatal GBS disease (Isaacs & Royle, 1999). The neonate can become infected in utero or

by vertical transmission from the female LGT during birth (American et al., 1997; Centre, 1996). Women with GBS

LGT colonisation have a one in 10 chance of giving birth to a neonate with immediate symptoms of GBS infection

(James, 2001). The chance of neonatal infection can be further reduced to a 1:200 chance if IAP penicillin is

administered to the mother during labour followed by post delivery monitoring (James, 2001).


Current Prevention Protocol (CDC & ACOG)

In 2004, the Centre for Disease Control (CDC) and American College of Obstetricians and Gynecologists (ACOG)

recommended that all pregnant women be screened for GBS during late gestation (36+ weeks), by culture method and

administration of IAP to all women with a GBS positive colonisation status (termed Protocol 4 in Table 4) (CDC,

2004b; Schrag et al., 2002). Garland and Kelly 1995 and other studies have concurred that a culture based screening

protocol during late gestation is considerably more effective when compared to all previous prevention approaches

(Table 4) (CDC, 2004b; Garland & Kelly, 1995). This current screening protocol can prevent up to 78% of all

EOGND (Table 4) (CDC, 2004b; Schrag et al., 2002). The CDC also recommended that antimicrobial susceptibility

testing be conducted when women colonised with GBS are at high risk of penicillin anaphylaxis (CDC, 2004b).

Evaluation of Previous EOGND Prevention Protocols

For the past decade four different GBS neonatal prevention protocols have been implemented and all of these

protocols to varying degrees were successful in reducing the incidence of EOGND (Table 4). The first

recommendations in 1992 were responsible for three protocols based on either a risk based (non screening)

management protocol or a screening based protocol (Benitz et al., 1999a; Gotoff, 2000a).

The risk based (non screening) management protocol (Protocol 1) evaluates the mother during labour for signs and

symptoms of GBS colonisation by monitoring for obstetric risk factors (refer to 1.4.6 page 28). When obstetric risk

factors are present during labour clinicians will then administer IAP (Table 4). During labour approximately 40% of

pregnant women may express obstetric risk factors associated with GBS colonisation (Garland & Kelly, 1995);

therefore, this approach does not significantly reduce the incidence of EOGND (Table 4).

Alternatively screening based protocols (Protocols 2 and 3) were implemented and included screening of all pregnant

women at approximately 28 weeks gestation to determine their GBS status (Table 4). If the mother tested positive

for GBS, then clinicians would either administer IAP to all women who were positive for GBS (Protocol 2) or

alternatively use the screening test as a diagnostic tool and only administer antibiotics to GBS positive women if other

risk factors presented themselves (Protocol 3) (Table 4) (Benitz et al., 1999a; Gotoff, 2000a).

In comparison to no protocols being implemented, all Protocols 1 to 4 decreased the incidence of EOGND (Table 4).

A risk based management or Protocol 1, although cheaper, only prevents up to 40% of all EOGND compared to

screening protocol 2 which would prevent significantly more at 65 to 68% of all EOGND (Table 4) (Daley &


Garland, 2004). Protocol 3, which uses the screening result as a diagnostic tool would prevent only 60% of all

EOGND cases because up to 40% of women colonised with GBS are asymptomatic during labour (Benitz et al.,

1999a; Gotoff, 2000a). Screening and antibiotic administration protocols will not prevent all EOGND cases because

of the transient nature of GBS.

Table 4: Protocols used to prevent GBS neonatal disease

Protocol No.

Description

Prevents EOGND

1 Risk based Management. No maternal GBS screening but administration of IAP based on maternal obstetric risk factors observed during labour (Benitz et al., 1999a; Gotoff, 2000a).

41%

2 Screening 28 Weeks and Intrapartum Antibiotics. LGT screening of women at 28 weeks gestation and administration of IAP to all women with a positive GBS status during labour (Benitz et al., 1999a; Gotoff, 2000a).

65-68%

3 Screening and Intrapartum Antibiotics only if Obstetric Risk Factors present themselves. Routine antenatal screening for GBS at 28 weeks gestation and treatment only when additional obstetric risk factors for GBS sepsis arise.

60%

4 Current recommended protocol 36+ Week Screening Protocol & Intrapartum Antibiotics. Screening at 36+ weeks gestation and administration of IAP to all women with a positive GBS LGT sample. This the current recommended prevention protocol outlined by the CDC (CDC, 2004b; Schrag et al., 2002).

78%

Prevention of EOGND can be best achieved if GBS can be detected during labour. A rapid PCR assay would be the

most accurate indicator of maternal GBS colonisation during labour and this practice would eliminate the majority of

all EOGND and prevent resistant strains from developing. Connellan and Wallace in 2000, reported that only 6% of

Victorian hospitals studies were able to identify risk factors associated with maternal GBS colonisation of the mother

appropriately (Connellan & Wallace, 2000).

Current Culture Based Method for GBS Detection

Currently GBS colonisation of the mother is detected using microbiological culture, the ‘gold standard’, with test

results available 48 hours later. Vaginal and perianal or vaginal and rectal clinical specimens are collected, placed in

transport medium and transported to a microbiology laboratory. In the laboratory, specimens are directly plated onto

a selective blood agar plate then inoculated into a selective (type) enrichment broth that is incubated overnight then

streaked onto a selective blood agar plate. Plates are incubated for 12 hours. Verification of the suspected GBS

colonies can be carried out using CAMP or latex agglutination tests.

Prevention Protocols Utilised within Australia


An Australia wide protocol has not be designed or implemented for the prevention of EOGND. In Australia,

universal screening occurs in NSW and Victoria with IAP administered based on those screening results. This

method was effective as The Prince Alfred Hospital in Sydney had a very low incidence of GBS neonatal disease at

0.2 per 1000 live births (Isaacs & Royle, 1999; Jeffery & Royal, 2002). In Sydney in 1988, the King George V

Hospital introduced maternal screening at 28 weeks and administration of IAP to all women who tested positive for

GBS resulting in a decreased incidence of EOGND (Isaacs & Royle, 1999; Jeffery & Royal, 2002). Connellan and

Wallace 2000 reported that GBS prevention strategies varied widely amongst Victorian hospitals although screening

and administration of IAP was widely practised (Connellan & Wallace, 2000). The Royal Women’s Hospital in

Melbourne screened women for GBS at 28 weeks gestation and instigated treatment only when additional obstetric

risk factors for GBS sepsis arose (Garland & Fliegner, 1991).

In Australia many medical providers have put in place detailed protocols for the management of EOGND and

protocols of some Australian medical providers and the type of prevention protocol (refer to table 4) they utilise are

outlined in table 5. These screening protocols used in Australia can be compared to protocols used in the USA and

UK (Table 5).


Table 5: Comparisons of Screening Protocols within Australia, USA & UK Location Year Protocol

No. Prevent EOGND

Reference

AUSTRALIA Queensland non Screening Based Policy Except Redland Hospital Melbourne Recommend Screening RWH (Melbourne, Vic) New South Wales Recommend Screening PAH (Sydney, NSW)

2003

2002


Queensland’s policy is largely a result of a EOGND incidence which was considered low when approximately 13

EOGND cases within an eight month period from January to August in 2002 had occurred (Jenkins-Manning, 2004).

Queensland’s protocol does not take into account the following considerations that also suggest that the current annual

EOGND incidence may be understated. Firstly, identifying GBS as a possible cause of early onset neonatal disease is

difficult as detection of GBS infection in neonates is very low if detection is from a single small volume of blood

(Gotoff & Boyer, 1977; Negre et al., 2004). GBS can be site specific and therefore, not able to be detected from that

particular blood sample (Gotoff & Boyer, 1977; Negre et al., 2004). Low GBS detection may also occur because of

the type of culturing technique used. The levels of GBS that can be detected will be affected by the type of sample(s)

taken from the participant, the type of transport medium used or whether a growth medium was selected. Secondly,

the maternal GBS colonisation rate of Brisbane and Australian women has not been established. The neonatal GBS

disease potential of Brisbane or Australia can only be calculated once regional and national prevalence has been

established. Thirdly, the current Queensland policy does not take the mother’s ethnicity into consideration and it

maybe possible that the GBS prevalence of Indigenous women compared to the GBS prevalence of Non Indigenous

women in Brisbane or Australia may be higher. A higher prevalence amongst Indigenous women may coincide with

the fact that Indigenous neonates are at greater risk of EOGND and in some towns where the population of Indigenous

people is higher then that town may have a greater EOGND potential. In the past due to documentation and

procedures, it was impossible to identify all Indigenous people let alone the number of Indigenous neonates who

contracted EOGND. Historically, the recorded incidence of GBS disease amongst Indigenous neonates is not

accurate because the person’s ethnicity was not a requirement of the form or formally documented until the early

2000s. Today collection of ethnic data would only be accurate if every person attending medical agencies was asked

his/her ethnicity.

Although Queensland’s protocol offers a maternal EOGND prevention protocol that includes the administration of

IAP to mothers who express obstetric risk factors during labour, this procedure will not identify up to 60% of mothers

who will give birth to an EOGND neonate (Benitz et al., 1999b; Gotoff, 2000b).

1.4.6 Signs & Symptoms of GBS Colonisation Gestational & Intrapartum GBS Colonisation

A mother may express signs and symptoms of GBS colonisation during gestation as well as in labour. During

gestation UGT GBS infection may results in miscarriage, preterm rupture of membranes (PROM) (before the onset of

labour) (Jeffery & Royal, 2002) and recurrent UTIs (Fletcher & Gordon, 1990; Persson et al., 1986a; Persson et al.,


1986b; Schuchat, 1995). During labour in some cases, the women may show signs or symptoms of GBS colonisation.

The obstetric risk factors that can be monitored during labour include: PROM for 18 hours or greater (OR 2.39, 95%

CI, 1.38-4.14) (Adair et al., 2003); an intrapartum fever of 38oC or greater (OR 4.65, 95% CI, 2.48-8.69) (Adair et al.,

2003); preterm birth (< 37 weeks) (OR 10.4, 95% CI, 3.9-27.6) (Oddie & Embleton, 2002) and/or low birth weight

(≤2500gm); and, preterm labour (Jeffery & Royal, 2002). Postpartum a mother previously colonised with UGT GBS

may develop clinical chorioamnionitis (Benitz et al., 1999a; Oddie & Embleton, 2002; Schuchat, 1995; Weisman et

al., 1992).

The mother can be asymptomatically colonised with GBS during labour and the neonate can still be at risk of GBS

colonisation and potential EOGND disease. The incidence of neonatal EOGND is low; however, the following seven

maternal complications have been associated with EOGND infections and adverse pregnancy outcomes. A neonate is

at a greater risk of EOGND when the mother is heavily colonised with GBS during delivery (Jeffery & Royal, 2002)

or when the baby is delivered preterm (Garland & Kelly, 1995; Jeffery & Royal, 2002). A preterm baby has an OR

10.4 (95% CI, 3.9 to 27.6) greater chance of GBS infection compared to a term delivered neonate (Schuchat, 1995).

A neonate born to a mother with symptoms of PROM for more than 18 hours (Garland & Kelly, 1995) has an OR of

25.8 (95% CI, 10.2 to 64.8) (Oddie & Embleton, 2002) chance of contracting EOGND. In contrast, neonates born to

mothers with a ruptured membrane before the onset of labour (Benitz et al., 1999a) have an OR of 11.1 (95% CI, 4.8

to 25.9) chance of developing EOGND (Oddie & Embleton, 2002). The neonate is also at risk of EOGND if the

mother expressed signs of fever during labour. These neonates have an OR 10.0 (2.4 to 40.8) of developing a GBS

infection (Oddie & Embleton, 2002). Again the risk of EOGND would be increased when their mothers were

diagnosed with maternal GBS sepsis (Garland & Kelly, 1995).

Demographic Factors Associated with GBS Colonisation

A number of studies have investigated the demographic factors and past medical history of mothers who have given

birth to EOGND infected neonates. In Australia, risk factors associated with EOGND may be similar to factors

reported elsewhere. These risk factors include the age of the mother (younger than 20) (CDC, 2004a; Jeffery &

Royal, 2002), her ethnicity (Towers et al., 1999), the number of her previous pregnancies (multiparous), (Towers et

al., 19

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