Epidemiology of Congenital Cytomegalovirus in a High HIV Prevalence Setting, South Africa
Conditions: Cytomegalovirus Congenital, Hearing Loss, Neurodevelopmental Disorders, Immunogenicity, Neonatal Death, Stillbirth
Sponsor: University of Witwatersrand, South Africa

CONGENITAL CMV IN A HIGH HIV PREVALENT 
SETTING, SOUTH AFRICA 
 
PhD Protocol 
Version 3, 22nd March 2017 
Jayani Pathirana 
 
Supervisors: Professor. Shabir Madhi 
                 Dr. Michelle Groome 
 
 
 
 
 
 
 
 
 
 

CANDIDATE’S 
SURNAME: 
[Please print] 
PATHIRANA 
FIRST 
NAME/S: 
JAYANI 
CHITRAMALI 
STUDENT 
NUMBER: 
1368789 
CURRENT  
QUALIFICATIONS: MBBS, MSc 
TEL:    
CELL:+27711406911 
E-MAIL: 
pathiranaj@rmpru.co.za 
FAX: 
DEGREE FOR WHICH PROTOCOL IS BEING SUBMITTED: PhD 
PART-TIME OR FULL-TIME: FULL TIME 
FIRST REGISTERED FOR THIS 
DEGREE: 
TERM :    13TH MAY 
YEAR:2015 
DEPARTMENT: SCHOOL OF PUBLIC HEALTH 
TITLE OF PROPOSED RESEARCH: CONGENITAL CYTOMEGALOVIRUS INFECTION IN A HIGH HIV 
PREVALENT SETTING, SOUTH AFRICA 
CANDIDATE’S SIGNATURE: 
DATE:26/02/2016 
SUPERVISOR 1 (NAME & SURNAME): SHABIR MADHI 
% Supervision : 50% 
SUPERVISOR’S QUALIFICATIONS: MBBCh, FCP, PhD 
SUPERVISOR’S DEPARTMENT: NATIONAL INSTITUTE FOR COMMUNICABLE DISEASES: DIVISION OF 
NHLS, PROFESSOR OF VACCINOLOGY, UNIVERSITY OF WITWATERSRAND, MRC RESPIRATORY AND 
MENINGEAL PATHOGENS RESEARCH UNIT 
SUPERVISOR’S ADDRESS / TEL / E-MAIL: NATIONAL INSTITUTE FOR COMMUNICABLE DISEASES: 
DIVISION OF NHLS, 1 MODDERFONTEIN ROAD, NHLS, SANDRINGHAM, 2131/+27113866137 
/ShabirM@nicd.ac.za 
SUPERVISOR 2 (NAME & SURNAME): MICHELLE GROOME 
 
% Supervision : 50% 
SUPERVISOR’S QUALIFICATIONS: MBBCh(Wits), DCH(SA), MSc(Med) Epi & Biostats (Wits) 
SUPERVISOR’S ADDRESS / TEL / E-MAIL: RESPIRATORY AND MENINGEAL PATHOGENS RESEARCH 
UNIT, CHRIS HANI BARAGWANATH ACADEMIC HOSPITAL, SOWETO/ +2711 983 4283/ 
groomem@rmpru.co.za 
 
SUPERVISOR 3 (NAME & SURNAME): 
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SYNOPSIS OF RESEARCH: 
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SYNOPSIS OF RESEARCH CONTINUED 
Introduction 
Cytomegalovirus (CMV) infection is the most common congenital infection worldwide and the leading 
cause of non-genetic sensorineural hearing loss (SNHL) in childhood1. Prevalence of congenital CMV 
infection  ranges from 0.6 to 5% dependent on the population studied1,2.  Up to 17% of all infected 
newborns are at risk of permanent neurologic sequelae, including SNHL; motor, cognitive and visual 
impairment; microcephaly; seizures and cerebral palsy3,4. Morbidity data on congenital CMV infections 
has mostly been from high income countries (HIC)2. It is necessary to establish whether congenital CMV 
infection in South African children presents the same public health impact as in high resource countries 
and determine the effect of HIV on CMV infection.  
 
Aim 
The aim of this project is to determine the epidemiology of congenital CMV infection and incidence of 
neurological sequelae until 12 months age in a high HIV prevalent setting in Soweto, Johannesburg.  
 
Methods  
This study will be nested within a larger cohort study of 35000 mother infant dyads to investigate a sero-
correlate of protection against invasive GBS disease (HREC 140203) currently underway at Chris Hani 
Baragwanath Academic Hospital (CHBAH) in Soweto. A cross sectional study design screening 2200 
mother-newborn pairs enrolled in V98_28OBTP that consent to this study will be undertaken identifying 
cases of congenital CMV by saliva PCR. A longitudinal cohort study following up the cases of congenital 
CMV matched to a CMV negative cohort of newborns will measure the incidence of and risk factors for 
neurological sequelae until infants are 12 months of age. To measure the association between congenital 
CMV infection and neonatal death and stillbirths, a longitudinal study on causes of death in under 5 
children by Minimally Invasive Tissue Sampling (MITS), which is currently underway (HREC 150215), 
will include tissue processing for CMV. Maternal CMV seroprevalence will be determined by serological 
methods as well as the association between vaginal CMV shedding and congenital CMV infection.  
 
These studies will identify CMV seroprevalence in pregnant women which is thought to be high (>70%) 
and will determine the prevalence of congenital CMV infection in Soweto through universal newborn 
screening which has only previously been done in symptomatic newborns at CHBAH. This study will also 
provide the first estimates on the incidence of SNHL and other neurological impairment in CMV infected 
newborns, adding to the current discussions on increasing resource allocation to universal newborn 
hearing screening. The measure of the effect of HIV exposure on the prevalence of congenital CMV and 
the burden of subsequent neurological disability will add to the growing global knowledge on congenital 
CMV in various settings impacting the prevention, diagnosis and management of infected infants.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Contents 
1 
Introduction ..............................................................................................................................................1 
 
Biology and Immunology of CMV ..................................................................................................1 
 
Epidemiology of Congenital CMV Infection ...................................................................................1 
 
Problem statement ............................................................................................................................5 
 
Justification for the study .................................................................................................................5 
2 
Aim and Objectives ..................................................................................................................................6 
 
Primary Objectives: .........................................................................................................................6 
 
Exploratory objective .......................................................................................................................6 
3 
Research Methods ....................................................................................................................................7 
 
Study Site .........................................................................................................................................7 
 
Overview of study ............................................................................................................................8 
 
Objective 1, 2 and 5 (exploratory) methods .....................................................................................8 
 
Objective 3, 6 and 7 Methods ........................................................................................................13 
 
Objective 4 Methods ......................................................................................................................17 
4 
My role and responsibilities ...................................................................................................................19 
5 
Ethics .....................................................................................................................................................19 
 
Informed consent and confidentiality ............................................................................................19 
 
Results Dissemination ....................................................................................................................20 
6 
Funding ..................................................................................................................................................20 
7 
Timeline .................................................................................................................................................21 
8 
References ..............................................................................................................................................22 
9 
Appendices .............................................................................................................................................27 
 

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*Refer to Appendix 1 for list of abbreviations 
1 Introduction 
Cytomegalovirus (CMV) infection is the most common congenital infection worldwide and the 
leading cause of non-genetic sensorineural hearing loss (SNHL)* in childhood 1. Global 
prevalence of congenital CMV infection  is estimated at 0.64%, varying across countries (0.2 – 
6%) with higher prevalence’s in lower and lower- middle income countries (LMIC) 1,2.  
Seventeen percent of all infected infants are at risk of permanent neurological disability3,4. The 
impact of congenital CMV infection in neonates is not well recognised as it is mainly 
asymptomatic at birth with the inability to attribute neurological impairment in children 
retrospectively to in-utero CMV infection. 
 Biology and Immunology of CMV 
Human CMV is a DNA virus of the Herpesviridae family5 and viral transmission occurs through  
body fluids (blood, saliva, breast milk, vaginal fluid, semen and urine) spreading to multiple 
organs (kidney, liver, lung, brain)6. CMV demonstrates strain diversity and mixed infection with 
multiple strains in an individual is common, whilst  immunity to CMV is strain specific7,8. 
Following primary infection, the virus establishes lifelong persistence9 and is usually 
asymptomatic in the immunocompetent. Secondary infection occurs with reactivation of a latent 
virus or infection with a new strain in a previously CMV seropositive person9. Strain specific 
antibodies (IgM and IgG) and a broad T cell response is activated, limiting viral replication and 
preventing disease, although unable to completely eliminate the virus10,11.  
 Epidemiology of Congenital CMV Infection 
Maternal infection: Low socioeconomic status, black race, crowded living conditions and 
younger age are associated with higher CMV seroprevalence1,2. Consequently, maternal 
seroprevalence may be over 80% in LMIC 12. A maternal CMV seroprevalence of 86.6% was 
found in a South African study13  but is expected to vary with socioeconomic disparity.  Primary 
infection in HIC commonly occurs in adulthood and in women of reproductive age, during 

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pregnancy1,14In lower resource settings CMV infection occurs at young age when exposed to 
infected maternal saliva, breast milk and because of over-crowded conditions1,14.   
Congenital infection: Viral transmission to the foetus can occur through the maternal-foetal 
placental interface increasing with gestational age.  Foetal effects of infection are more severe if 
infected in the first 16 weeks of gestation when organs are developing and may lead to abortion 
and stillbirths11,15 Transmission rates are higher during primary maternal infection (30 -35%) 
compared to women with secondary infection (1.4%)2. Counterintuitively, there is greater 
prevalence of congenital CMV in high CMV seroprevalent populations compared to low 
seroprevalent populations who are at higher risk of primary CMV infection.  This is explained by 
there being more pregnant women at risk of CMV reactivation and reinfection in high 
seroprevalent populations coupled with other risk factors2. A recent systematic review of studies 
from LMIC, showed population based congenital CMV prevalence ranging from  0.6 – 6.1% 
(e.g. Panama 0.6%, Gambia 5.4%, China, 6.1% )12 compared to 0.2-2.0% in HIC (Chile 0.2%, 
USA 1.2%, Finland 2.0%) 2,4.  
Diagnosis of congenital CMV infection: CMV isolation by culture or positive polymerase 
chain reaction (PCR) test in urine, saliva, blood or tissue taken at biopsy within the first 2 weeks 
of life is diagnostic of congenital CMV16. Acquired CMV during delivery can be detected after 2 
weeks incubation and CMV isolation beyond 2 weeks age cannot reliably be concluded as in 
utero infection16. CMV PCR in urine and/or saliva is favoured as the standard of diagnosing 
congenital infection17. 
Clinical significance and long term outcome of congenital CMV: Most newborns with 
congenital CMV are asymptomatic at birth (85-90%)18. Of the 10-15% of symptomatic newborns 
clinical manifestations range from mild, non-specific findings to multi-organ system 
involvement18. The most frequent signs are petechiae, jaundice and hepatosplenomegaly. Central 
nervous system (CNS) signs include microcephaly, hearing impairment, chorioretinitis and/or 

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optic atrophy and seizures 1,5,18.  Of the symptomatic neonates, about 50% are small for 
gestational age, 33% are born prematurely18 and 5% die in the neonatal period18. 
Permanent neurological sequelae occurs in 40-58% of symptomatic and 14% of asymptomatic 
infants at birth4. Children with symptomatic birth infection may have multiple neurological 
sequelae including SNHL (30-60%), cerebral palsy and mental retardation (50-80%) visual 
impairment (20-35%) and neurodevelopmental behaviour disorders (30-60%)4,19. The most 
common sequela in infants asymptomatic at birth are SNHL (10%) with other complications 
occurring at a lower frequency: motor deficits (5%) and chorioretinitis (2%)18.  
Effect of HIV: An estimated 3% of congenital CMV infection occurs in HIV exposed infants 1,20 
compared to 1% in HIV unexposed infants. HIV infected infants have an even higher prevalence 
of congenital CMV (10.3%) as found in a French perinatal cohort21. In this study, HIV and CMV 
co-infected newborns had a 3-fold greater risk of symptomatic congenital CMV infection at birth 
(23%) than HIV exposed but uninfected  infants (6.7%)21. CMV co-infection may also contribute 
to HIV disease progression with increased risk of mortality and faster progression of CNS 
disease in survivors 1,22. However, the incidence of vertical CMV transmission in HIV infected 
women decreased following  introduction of antiretroviral therapy (ART) during pregnancy 21.  
Data on congenital CMV infection among HIV exposed infants in sub-Saharan Africa are 
limited. A Kenyan study of infants born to mothers on perinatal Zidovudine, identified 
congenital CMV rates of 29% (4/14) in HIV-infected infants and 2.7% (1/37) in HIV exposed 
but uninfected infants23. Zambian newborns admitted to a referral neonatal unit had a congenital 
CMV prevalence of  3.8% (15/395) whilst prevalence in HIV exposed newborns was 11.4% 
(9/79)24. Although HIV prevalence in South African pregnant women is one of the highest in the 
world (29.9%)25, a single study has evaluated the relationship between congenital CMV and HIV 
exposure. In Cape Town, Manicklal et al (2013) found a congenital CMV prevalence of 2.9% 
(22) in 748 HIV exposed babies26. Maternal CD4+ count <200 cells/μL was independently 

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associated with congenital CMV26. Clinical outcomes in these infants and prevalence of 
congenital CMV among HIV unexposed newborns were not investigated. 
Perinatal and Postnatal CMV infection:  Perinatal CMV infection is defined as CMV infection 
after 22 completed weeks (154 days) of gestation and seven completed days after birth. Postnatal 
CMV acquisition is CMV infection acquired any time after birth through consumption of breast 
milk from CMV seropositive, lactating mothers is common.Most term infants with perinatal 
CMV infection are asymptomatic, as maternally transferred CMV IgG antibody provides 
adequate protection against disease 27. However, studies have identified a significant risk for 
low-birth-weight (LBW), premature infants for the development of CMV disease such as 
pneumonia, hepatitis or sepsis-like illness27. Data on severity and incidence of disease and long 
term impact in these infants is currently lacking requiring further study.  
Current therapeutic options: Treatment for maternal and newborn CMV infection is limited. 
Anti-CMV hyperimmunoglobulin is efficacious in preventing transmission from primary 
maternal infections but its value in secondary infection is uncertain2. Some European countries 
offer universal maternal CMV screening or foetal anomaly ultrasonography to identify 
ventriculomegaly, intracerebral calcification and microcephaly28. Foetal infection is confirmed 
by amniocentesis, not commonly available in LMIC, and women may be offered termination of 
pregnancy. Targeted CMV screening in symptomatic newborns and/or fail universal hearing 
screening occurs in the USA1. Screening programmes have not been widely implemented due to 
lack of awareness, high costs and lack of rapid diagnostic tests29.  
Ganciclovir (GCV) or Valganciclovir is administered to symptomatic CMV infected neonates, 
associated with improved audiologic outcomes1. However GCV’s effectiveness in asymptomatic 
cases has not been fully assessed1. Both symptomatic and asymptomatic infants should be 
monitored regularly for neurological disability.  

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Economic Impact: The effect of congenital CMV disease on quality of life and cost to the 
health system led the Institute of Medicine (IOM) in the USA (1999) to declare development of a 
CMV vaccine as a highest priority3. The IOM estimated that approximately $1.9 billion per year 
is spent toward medical and educational costs for congenitally CMV infected children in the 
USA1 compared to costs of developing a CMV vaccine of $360 million29. The health economic 
implications of congenital CMV in low income countries is under-recognised or unknown30.  
Impairment of immune response  The efficacy of infant vaccines in Sub-Saharan Africa has 
consistently been lower than in high income countries for a number of vaccines (e.g. measles 
vaccine, rotavirus vaccine) 31. It has been postulated that CMV infection, due to its large 
recruitment of T-cells may have a role to play in modulating immune responses to infant 
vaccinations but only a few groups thus far have investigated this in children31–33.  
Problem statement Morbidity data on congenital CMV infections has largely been from HIC 
as a result of maternal primary infection, whereas data from low-income, high secondary 
infection and high HIV prevalent settings are lacking. In the South African public health sector, 
only symptomatic newborns have CMV testing whilst newborn hearing screening is conducted 
in high risk babies born premature, symptomatic or admitted to ICU. Asymptomatic, CMV 
infected infants at birth are an at risk group of developing disabilities which only become 
apparent when children are older and interventions less effective. Studies have demonstrated up 
to 15% of stillbirths have lab confirmed CMV infection34. Any association between neonatal 
deaths and congenital CMV infection is yet to be described.  
  Justification for the study  
There is no robust data on the immunology, virology, mortality, disease and disability burden 
from congenital CMV infection in South Africa. The effect of HIV on congenital CMV and 
subsequent neurological sequelae is also largely unknown in black Africans. Early detection of 
SNHL and neurodevelopmental disorders could enable timely intervention to improve quality of 

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life. If sequelae are significantly higher in HIV exposed infants, this study may contribute to 
discussions towards a targeted screening programme for CMV in HIV exposed infants. With the 
development of a CMV vaccine in progress35, it is necessary to establish baseline disease 
epidemiology determining whether congenital CMV infection in South African children presents 
the same public health impact as in high resource countries and whether screening programmes 
or a vaccine is necessary or cost effective in our setting. Most available data suggest that 
antibody positivity, is not associated with protection from vertical transmission to a foetus or 
infant or with protection from re-infection. However, some studies suggest that antibody avidity 
and/or neutralisation activity may be associated with protection from vertical transmission. We 
wish to explore this further and also assess if either measure is associated with maternal HIV 
infection.  
2 Aim and Objectives 
The theme of this project is to determine the epidemiology of congenital CMV infection and 
incidence of sequelae until 13 months age in a high HIV prevalent setting in Soweto, 
Johannesburg.  
 Primary Objectives:  
 
2. 1. Determine the CMV seroprevalence, antibody avidity and (if possible) anti-CMV 
neutralising antibody activity in blood plasma of both HIV infected and uninfected  pregnant 
women at the time of delivery and explore associations between these measures and risk of 
congenital and postnatally acquired CMV infection in the first year of life.To compare the 
prevalence of congenital CMV infection and incidence of symptomatic infection in HIV-
exposed and HIV-unexposed neonates  

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3. To determine the incidence and risk factors for neurological sequelae in congenitally CMV 
infected neonates from birth up to 12 months of age compared to a control group of neonates 
without congenital CMV 
4. To determine by minimally invasive tissue sampling (MITS) the association between 
congenital CMV infection and:   1) Neonatal deaths less than 3 weeks of age and 2) 
Stillbirths  
 Exploratory objectives 
5. To explore the association between maternal CMV shedding in vaginal fluid and breast milk 
and the risk of congenital CMV infection assessed by quantitative CMV PCR.  
6.  Determine the incidence of postnatal CMV acquisition, in infants not CMV infected at birth, 
and explore possible risk factors, including maternal HIV status and the magnitude of 
maternal CMV shedding in vaginal fluid and breast milk. 
7. Explore differences in immune responses to the primary and booster series of vaccinations 
(DTaP-Hib/HepB, PCV rotavirus and measles vaccines) between congenital CMV cases, 
postnatally acquired CMV cases and non-CMV infected controls at 6, 12 and 13 months of 
age. 
3 Research Methods  
 Study Site 
This study will be conducted at Chris Hani Baragwanath Academic Hospital (CHBAH) in 
Soweto, a secondary-tertiary level facility serving a population of 1.2 million. The annual birth 
cohort in Soweto is approximately 28000, with 75% occurring at CHBAH and the rest at 
midwife operated units or a nearby district level hospital. 

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 Overview of study 
This will be an epidemiologic cross-sectional study, with a prospective cohort component (refer 
to Appendix 2 for study schema). Objectives 1, 2 and 5 will be nested within a larger cohort 
study enrolling mother-infant dyads at birth or antenatally to investigate a sero-correlate of 
protection against invasive Group B Streptococcal disease in newborns ( protocol number 
V98_28OBTP, HREC no. 140203, Appendix 4). In V98_28OBTP, research staff at 
labour/delivery wards identifies women enrolled antenatally or consents potentially interested 
women in labour for participation. Maternal and infant demographic and clinical data, maternal 
blood, vaginal swab and infant cord blood are collected for laboratory testing. Monitoring for 
subsequent hospitalization of the infant at CHBAH in the first year of life is undertaken daily in 
all general paediatric and neonatal wards in an established surveillance known as Babies of 
Soweto Study (BoSS). As of November 2015, 20 000 mother-newborn dyads are enrolled, and is 
anticipated to continue until October 2016.  
Objective 4 will be nested within a prospective study of cause of death in under-5 children by 
Minimally Invasive Tissue Sampling (MITS) currently underway at CHBAH (Appendix 13, 
HREC no. 150215). Stillborn babies are also enrolled into this study.  
A summary of objectives, methods and data analysis for this PhD is given in Appendix 3. 
 
Objective 1, 2and 5 (exploratory) methods 
Study design: Cross-sectional study design with consecutive sampling 
Study participants: Mothers meeting the inclusion criteria in table 1 and their newborns.  
Table 1: Maternal inclusion and exclusion criteria 
Inclusion criteria  
Exclusion criteria  
- Enrolled in V98_28OB 
- Age ≥18y 
- Residing in Soweto and would be available for study follow-up 
- Consents to enrol self and baby in study 
- Baby born alive 
- <18 years age 
- Visiting Soweto 
- Declines consent 
 

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Enrolment will be performed by study staff stationed in labour and delivery around-the-clock. 
The informed consent form (ICF) used in V98_28OBTP (Appendix 4) also allows collection of 
data and samples from mothers and newborn to test for CMV. 
 
Data collection: Data will be collected by maternal interview and examination of hospital 
notes of the mother and newborn, recorded onto the V98_28OBTP CRF (Appendix 5) and BoSS  
CRF (Appendix 6) if symptomatic. Table 2 describes the maternal and newborn data of interest 
and figure 1 illustrates the data collection process. 
Table 2: Maternal and newborn data of interest 
Maternal data 
Newborn data 
Demographic: age, race,  and contact details 
Demographic: date of birth, gender, race  
Clinical data: antenatal care details, past 
obstetric history, parity, gestation at delivery, 
delivery details, co-morbidities, HIV status 
and CD4 count, ART use 
Clinical: gestational age at birth, birthweight, 
head circumference, APGAR scores, HIV 
exposure, symptoms at birth, discharge or 
admission details 
 
Newborn saliva samples will be collected by placing an Orocal swab in the mouth for at least 1 
minute. All symptomatic neonates for congenital CMV infection (Appendix 11) will also be 
screened for other congenital infections which include syphilis, toxoplasmosis, rubella and 
herpes as is the standard of care at CHBAH. 
 
 
 
 
 

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Case Mothers 
Control 
Mothers 
Obj 1: Maternal CMV 
seroprevalence, antibody 
avidity and neutralisation  
Blood – IgG and IgM 
                    n=200 
Figure 1: Data collection process for Objectives 1, 2 and 5 
 
 
 
 
 
 
 
 
All children with any clinical finding are referred to the neonatology department at CHBAH for 
clinical management as per standard of care. If a newborn PCR indicates CMV infection, the 
mother will be contacted telephonically to return to the site within two weeks of birth. At this 
visit the implications of the positive CMV result will be explained and additional consent for the 
continued follow up of the child (refer to Appendix 7 for ICF and Appendix 8 for schedule of 
study visits). Should consent be confirmed, a second saliva swab and a urine sample will be 
collected for PCR to confirm CMV infection. Babies that test CMV negative on both the second 
saliva swab and urine will not be enrolled as a case as the saliva positivity may be a result of 
CMV infected breast milk contamination. However, they will be referred for hearing screening 
as standard of care. Cases testing positive on both saliva and urine or urine alone will continue as 
cases for objective 3.  
Mothers of CMV cases will have a vaginal swab and breast milk collected for quantitative PCR 
by a research nurse or the investigator. A subset of mothers that tested seropositive for CMV but 
gave birth to CMV uninfected neonates will also be recalled as controls for the case mothers in a 
Consent and enrol V98_28OB mothers in CMV study 
N=2160 
Obj 2: Screen 2160 newborn saliva for CMV by 
qPCR (1077 HIV Exposed, 1077 HIV 
Unexposed) 
HIV Exposed 
CMV+ 
n=31 
HIV Unexposed 
CMV+ 
n=11 
HIV Exposed 
CMV─ 
N=1046 
HIV Unexposed 
CMV─ 
n=1066 
Confirm CMV+ in cases Saliva 
and urine qPCR 
n=42  
Visit 2: ≤2 weeks post 
delivery  
Visit 1: At delivery  
Obj 5:  CMV vaginal and breast 
milk shedding in case and control 
mothers (1:1) 
Vaginal fluid and breast milk 
qPCR.  n=84  

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1:1 ratio, matched by HIV infection, for vaginal fluid and breast milk collection within 2 weeks 
of delivery.  
Sample size: Objective 1: Assuming the estimated CMV prevalence is 86% in pregnant 
women13 200 mothers will be needed to estimate the true value with a desired precision of 5% 
and confidence interval of 95%.  
Objective 2: Prevalence of congenital CMV in HIV exposed neonates is 2.9%26 and in 
unexposed  neonates is 1.0%1. Thus, a total sample of 1662 neonates with 831 HIV exposed and 
831 unexposed (1:1 ratio) for 80% power, are required to detect a 2.9 fold difference in CMV 
prevalence between the 2 groups. This will be increased to 2160 neonates, to accommodate 
objective 3, to determine clinical sequelae in CMV infected infants (section 3.3.1, Sample size). 
After adjusting for 10% being non-evaluable, 1077 HIV exposed (providing 31 CMV infected) 
and 1077 HIV unexposed infants (providing ~11 CMV cases) are required. Ranges of  
sample size estimates for varying CMV prevalences by HIV exposure are given in Appendix 9.   
Objective 5: The number of case mothers tested will be determined by the total number of CMV 
infected neonates that are identified during screening estimated at 42.  HIV infection and age 
matched control mothers will be allocated in a 1: 1 ratio giving a total sample size of 84. 
Laboratory testing: Maternal blood and vaginal swabs, newborn saliva and urine collected will 
be stored at -70 °C until processed for CMV serology (IgG and IgM in blood) and DNA by PCR 
assays (vaginal fluid, saliva, urine) at the RMPRU Laboratory or other collaborating laboratory 
in South Africa. The turnover time for PCR testing is expected to be within 5 working days of 
sample collection.  Refer to Appendix 10 for laboratory methods of sample processing. 
Data Analysis: Table 3 describes the planned data analyses, per objective. All analyses will be 
done on STATA/IC version 13 for Windows, Texas, USA, in consultation with a biostatistician.  

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Table 3: Data analysis plan 
Objective 
Data Analysis 
1. Determine the 
seroprevalence of 
Cytomegalovirus 
infection (CMV) in HIV 
infected and uninfected 
pregnant women 
attending CHBAH 
Table of maternal demographic and clinical characteristics as 
proportions  
Maternal CMV seroprevalence expressed as a percentage 
stratified by HIV status (95% confidence interval) 
Chi square test  to compare reactivations/recurrent infections 
determined by IgG avidity and IgM presence compared to 
latent infection stratified by HIV status  
2. Compare the prevalence 
of and risk factors for 
congenital CMV infection 
in HIV exposed and 
unexposed newborns 
Prevalence of congenital CMV infection in HIV exposed and 
unexposed infants expressed as a percentage with 95% 
confidence interval 
Multiple regression analysis to adjust for potential 
confounders: gender, gestational age at birth, birth weight, 
birth order, symptoms at birth 
In HIV exposed infants, chi square tests for association 
between: 
- 
Congenital CMV infection and maternal ART use 
- 
Congenital CMV infection and severity of maternal 
immunosuppression (CD4+ count  levels) 
To explore the association 
between maternal CMV 
shedding in vaginal fluid 
and breast milk  and the 
risk of congenital CMV 
infection assessed by 
quantitative CMV PCR. 
Chi square test for association between vaginal CMV  
shedding and congenital CMV infection 
- 
Stratify by varying ranges of viral shedding (high, 
moderate, low – quantification to be determined) 
- 
Stratify by HIV infection though will have low power 
for this  
 
Potential challenges:  Objective 1, 2 and 5: Mothers may be reluctant to be enrolled in a study 
for an infection which is unknown to them. To mitigate this, a patient information leaflet will be 
provided to improve understanding and includes the potential benefit to the infant should he/she 

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be diagnosed to have congenital CMV (Appendix 7). Training of researchers in effective 
communication will be conducted. 
Objective 5: CMV viral shedding is cyclical, highest during primary infection; reactivation or 
reinfection which when occurs during pregnancy may be transmitted to the foetus. This study 
may be limited by the collection of samples after delivery at which point, viral shedding may 
have ceased since pregnancy.  
 Objective 3, 6 (exploratory) and 7 (exploratory) Methods 
Study design: Prospective longitudinal cohort study of CMV infected and uninfected neonates 
Study participants: The cohort will consist of newborn infants identified as laboratory confirmed 
CMV infected from objective 2 forming the cases and a subset of neonates identified as CMV 
uninfected at birth chosen as controls. Each case will be matched to 2 controls by age, 
gestational age at birth (±2 weeks) and HIV exposure. Within 2 weeks of age, cases and controls 
will be recalled to the site to consent for follow up and initiation of investigations as illustrated in 
figure 2.  
Data collection: Postnatal CMV acquisition: A saliva and urine sample from each control infant 
will be collected at the 2 weeks visit. At subsequent follow up visits, infant saliva and urine will 
be collected from those that tested CMV negative at the preceding visit.   
Incidence of neurological sequelae: neurological sequelae is one or more of: unilateral or  
bilateral SNHL, developmental deficit, unilateral or bilateral ocular abnormality, abnormal 
cerebral imaging, microcephaly, or seizures not related to an acute systemic infection or CNS 
insult (refer to Appendix 11 for case definition of each sequela).  
 
 

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Figure 2: Schedule of study visits and follow up investigations  
 
 
 
 
 
 
 
 
 
 
 
 
Mothers will receive a reminder phone call 1 week prior to each visit. Cranial ultrasonography 
will be conducted at the neonatology unit and fundoscopic examination at St. John’s eye 
hospital. Hearing screening will be conducted at the audiology department as per standard of 
care by automated auditory brainstem evoked response audiometry (AABR). Developmental 
assessment will be performed by myself at the research clinic through a developmental 
assessment tool (Ages and stages questionnaire or Bayley III scales of infant and toddler 
development or equivalent). Refer to Appendix 12 for list of follow up data. Standard operating 
procedures to ensure standardisation of assessments between assessors will be developed. 
Consent CMV+ cases n=42  
Visit 2: ≤2 weeks 
Consent CMV– controls n=84  
Cases with 
neuro sequelae  
Cases without 
neuro sequelae  
Controls with 
neuro sequelae 
Controls without 
neuro sequelae  
Visit 3: 2 months  
Control infants: Saliva and urine  
All: Anthropometry, Cranial ultrasonography, hearing screening fundoscopy 
Control infants: Saliva and urine from infants negative for CMV at visit 2  
All: Anthropometry, hearing screening, developmental testing  
Visit 4: 6 months  
Control infants: Saliva and urine from infants negative for CMV at visit 3  
All: Anthropometry, hearing screening, developmental testing, blood draw 
Visit 5: 12 months  
Control infants: Saliva and urine from infants negative for CMV at visit 4 
All: Anthropometry, hearing screening, developmental testing, blood draw 
Visit 6: 13 months  
All: Blood draw  

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Blinding of the assessors to the status of CMV infection will be maintained at each assessment. 
If impairments requiring clinical management are identified, the assessor will be provided with 
the laboratory data. Any impaired infant will be referred to specialist CHBAH paediatric clinics 
for clinical management. HIV-exposed infants are tested for HIV-infection by PCR at birth, 6 
weeks of age and again at least 3 months post breast-feeding cessation, as per standard of care in 
South Africa. Should a child test HIV-infected, they will be referred to the HIV clinic at 
CHBAH. 
Infant specific vaccine induced immune responses: Cases and controls identified will be 
vaccinated as per the immunisation schedule in South Africa with hexavalent DTaPHibHepBIPV 
at 6, 10 and 14 weeks and PCV and rotavirus vaccine at 6 and 14 weeks of age. Mothers will be 
encouraged to access vaccines at the vaccination clinic at RMPRU and counselled on ensuring 
their infants are vaccinated at the scheduled time. At the 3rd  study visit when infants are aged 6 
months a venous blood sample will be collected by appropriately trained study staff. The volume 
of blood collected will be not more than 5ml following the recommendations by HREC 
(medical) on blood volumes from paediatric research participants. 
Laboratory Venous blood will be centrifuged and aliquots of serum will be stored at -70oC. 
Antibody to diphtheria toxoid, tetanus-toxoid, pertussis toxoid, filamentous hemagglutinin 
(FHA) and hepatitis B surface antigen (HBsAg) will be measured by an in-house Luminex 
multiplex immunoassay. Immune responses to the primary series of PCV will be measured by a 
standardized enzyme immunoassay (EIA) to test for vaccine serotype–specific capsular 
immunoglobulin (Ig) G antibody concentrations and opsonophagocytic assay. Titres of rotavirus-
specific IgA will be determined in enzyme-linked immunosorbent assays36. Refer to appendix 10 
for the serological correlates of protection 37. 

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Sample size: The anticipated prevalence of neurological sequelae in CMV uninfected controls  
with uneventful foetal or perinatal periods is estimated at 1%38. Prevalence of neurological 
sequelae in CMV infected infants is expected to be higher at approximately 17%4. For 80% 
power, the difference that could feasibly be detected, is 17-fold i.e. 1% vs 17%. Allowing for a 
loss to follow up of 10%, 42 CMV infected infants and 84 controls (n=126) with an allocation 
ratio of 1:2 are required (Appendix 9).  
Data Analysis: Table 3 describes the data analysis plan to determine the incidence and risk 
factors for neurological sequelae in congenital CMV infected infants  
Table 3: Data analysis plan  
Objective 3 
Incidence calculation of one or more neurological sequelae  for cases and controls at 2 weeks, 2 
months, 6 months and 12 months of age dependent on investigations done at each visit 
Comparison of proportions with permanent sequelae (hearing impairment and developmental 
deficit) at each visit between cases and controls using a Z test to compare ratios, including 95% 
confidence intervals. 
Adjust for potential confounders (IUGR, symptoms at birth, gender and co-morbidities) by 
multiple logistic regression analysis to establish factors contributing to neurological sequelae.  
Objective 6 
Incidence of postnatal acquisition of CMV in newborns will be calculated at 2 months, 6 
months and 1 year of chronological age. Incidence of neurological sequelae in postnatally 
infected infants will be calculated 
Objective 7 

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 The magnitude of specific antibody response between cases and controls will be compared 
using the unpaired t test when data are normally distributed. In the scenario that the distribution 
is non-normal the Mann-Whitney test will be used instead. Normality will be tested using the 
Shapiro-Wilk test, where the assumption of non-normal data will be concluded based on a 
significant result. Frequency plots and P-P plots will also be used to confirm the normality 
assumption, or not.  
Simple correlations will be assessed using Pearson or Spearman correlation in the case of 
normal or non-normal distribution, respectively. The resulting correlation coefficient will 
indicate the strength of association, whereas the significance test will provide the evidence of a 
linear correlation or not in the population.  
 
Potential challenges: To account for loss to follow up and timely completion of investigations, a  
window period of 30 days is allowed from the date of each scheduled visit in which to perform 
the investigations. Transport costs will be reimbursed with R150 per scheduled visit and all 
investigations will be provided at no cost. Neurological sequelae in congenital CMV infected 
children may manifest beyond 12 months of age and long term follow up to 6 years is 
recommended4. Hence, it is possible that this study may not detect all sequelae by 12 months of 
age and no differences may be observed between the 2 groups. 
Parents may be reluctant to consent to a blood draw at 6 months of age and will be thoroughly 
counselled on the purpose and processing of the blood samples. The informed consent form has 
been amended to include the purpose of the blood draw and each mother will be given a new 
consent form to sign with the option of refusing to participate in the blood draw but continue 
with the rest of the study procedures. 
 Objective 4 Methods  
Study design: This study will be nested within a broader study conducting Minimally Invasive 
Tissue Sampling (MITS) on death of children and stillbirths currently underway in CHBAH, 

18 
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ongoing until end 2017 (HREC no. 150215), and includes testing for CMV. I perform MITS in 
this study and will be involved in data analysis as it relates to CMV and neonatal deaths or 
stillbirths. 
Study participants: Stillbirths weighing ≥1000g and neonatal deaths weighing ≥750g, less than 3  
weeks of age from non-traumatic causes at CHBAH. Parents/guardians provide written consent  
after death, following grief counselling and provision of an information leaflet.   
Data collection: Ante-mortem clinical data will be extracted from patient records in the BoSS 
CRF (Appendix 6). Core biopsy specimens of lung, liver, kidney, brain tissue, placenta 
(stillbirths) and fluid specimens of cerebrospinal fluid (CSF) and blood will be sampled from 
each enrolled case within 72 hours of birth.  
Sample size: This will be a descriptive study, and sample size is based on the number of 
deaths/stillbirths which occur over a18-24 month period. From current experience, we anticipate 
enrolling approximately 200 stillbirths and 100 neonatal deaths, providing 80% precision to 
determine that at least 5% of these stillbirths and neonatal deaths are associated with CMV.  
Laboratory: Testing and examination for CMV infection by histopathological methods will take  
place at the National Health Laboratory Services (NHLS). Molecular testing (PCR) will occur at 
the RMPRU laboratory by in house assays. HIV PCR and antibody ELISA will be done to 
determine infection and exposure in utero as has been approved in the parent protocol (HREC 
no. 150215). Refer to Appendix 13 for the full tissue sampling protocol of the study. 
Data analysis:  Descriptive statistics presenting the number of deaths with CMV infection by  
birthweight, gestational age at birth and age at death in neonates. Multiple regression analysis 
will adjust for confounding factors such as HIV infection and exposure, prematurity, co-
morbidity and co-infection with other pathogens. The odds of CMV disease will be compared to 

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the prevalence of congenital CMV in the general population, stratified by HIV-exposure status, 
as measured in Objective 2. 
Study challenges: Although the majority (75%) of parents approached thus far, consent to the  
procedure, enrolment of neonatal deaths has been limited due to cultural practices of burying 
neonates immediately after death before study staff are able to approach the parents for consent. 
In contrast, we are enrolling approximately 5 stillbirths per week. This challenge will be 
mitigated by engaging with attending paediatricians to inform the study team more timeously if 
deaths occur. MITS in aborted foetuses is not being done in the current study but is an important 
demographic in describing the outcomes of congenital CMV infection.  
4 My role and responsibilities 
I am responsible for protocol development and running of procedures specific to this study. I will 
coordinate the follow-up of infants over 12 months and conduct the developmental assessments. 
I currently participate in quality control of data in the paediatric (BoSS) surveillance and project 
administration.  For the MITS study, I perform tissue sampling, participate in project 
administration and will contribute to data analysis as it relates to CMV. 
5 Ethics 
 Informed consent and confidentiality 
The V98_28OBTP (HREC 140203) and MITS (HREC 150215) protocols are approved and 
currently in progress at CHBAH. Approval for integrating this proposal into these studies and the 
additional follow up of infants until 12 months of age is sought. Written informed consent from 
mothers allowing participation will be obtained. Participant contact details and identifiers will be 
stored independently of source documents, only available to myself and research assistants 
responsible for interviewing mothers. Transport and incidental costs will be reimbursed at R150 

20 
Version 3: 22 March 2017 
per visit. Infants will benefit from screening and follow up as early intervention will be possible 
in cases where clinical sequelae are identified within 12 months. Following the conclusion of this 
study, infants with CMV infection will have hearing monitoring until the age of 6 years by the 
audiology department. 
 Results Dissemination 
This PhD will be achieved by publication in peer-reviewed journals, where they will also be  
available to the public (refer to Appendix 14 for proposed manuscripts). Summary results will be 
reported to the HREC on completion of the study. Results may be presented at local and 
international scientific meetings.  
6 Funding  
Funding for the 28OB study is provided by Novartis. Funding specific to this CMV study will be 
provided by the RMPRU. Refer to Appendix 15 for budget details.  

21 
 
7 Timeline 
2015 
2016 
2017 
2018 
AugSepOct Nov Dec Jan FebMarAprMay Jun JulAugSepOct Nov Dec Jan FebMarApr May Jun Jul AugSepOct Nov DecJan FebMarAprMayJun Jul Aug Sep
Literature review 
Postgrad proposal 
preparation 
Ethics assessment (for 
follow-up) 
Postgrad proposal 
submission 
28OBTP sample 
collection for CMV 
Stillborn and newborn 
MIA sample collection 
Sample processing for 
CMV 
Follow up cohort 
enrolment 
Cohort follow up 
Data collection 
Data cleaning and 
analysis 
Write up  
Submission 

22 
 
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Appendices