HIV Exposure, Disease Acquisition and Progression Among Children: Role of Maternal Immunogenetics, Viral Genetic Diversity, HAART Exposure, Co-morbidities and Psycho-Social Status: (UZ-CHS Birth Cohort)
Conditions: HIV Infections, Immune Activation, Comorbidities
Sponsor: University of Zimbabwe

 
 
Version 2, 3 December 2015 
 
 
 
 
 
 
PROTOCOL 
Protocol ID: 
 
MRCZ/A/1968 
 
Brief Title: 
University of Zimbabwe College of 
Health Sciences Birth Cohort Study  
Acronym: 
 
UZ-CHS-BC 
 
Official Title:  
  HIV Exposure, Disease Acquisition and  
Progression among Children: Role of 
Maternal 
Immunogenetics, 
Viral 
Genetic Diversity, HAART Exposure, 
Co-morbidities 
and 
Psycho-Social 
Status: UZ-CHS Birth Cohort.  
 
Principal Investigator:  
Duri Kerina (PhD) 
 
Sponsor:  
 
 
University of Zimbabwe  
College of Health Sciences (UZ-CHS) 
      Department of Immunology 
 

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Co-investigators 
 
1. Professor E. Gomo 
Immunogeneticist 
Department of Laboratory Sciences, UZ-CHS. 
 
2. Professor FZ Gumbo 
Paediatrician 
Department of Paediatrics and Child care, UZ-CHS. 
 
 
3. Professor S. Rusakaniko 
Biostatistician 
Department of Community Medicine,  
University of Zimbabwe College of Health Sciences 
(UZ-CHS) 
 
 
 
Co-ordinating Centre (Sponsor) 
University of Zimbabwe College of Health Sciences Research 
Support Centre (UZCHS RSC) 
Parirenyatwa Hospital Grounds, Harare 
Telephone: +263 4 708020 
Email: rscsacoreoffice@gmail.com 
 
 

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Mentors/Advisory Committee 
 
1. Professor M.Z. Chirenje 
Obstetrician 
Department Obstetrics and Gynaecology, UZ-CHS 
Email: chirenje@uz-ucsf.co.zw 
 
 
2. Professor I. Chitsike 
Paediatrician 
Department of Paediatrics, UZ-CHS 
Email: chitsikei@gmail.com 
 
 
3. Professor L.S. Zijenah 
Immunologist 
Department of Immunology, UZ-CHS 
Email:lzijenah@gmail.com 
 
 
4. Professor B. Stray-Pedersen 
University of Oslo, Institute of Clinical Medicine 
Department of Women and Children 
Oslo University Hospital, Rikshospitalet 
0027 Oslo 
E-mail: babill.stray-pedersen@medisin.uio.no 
 

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Confidentially Statement 
This protocol is confidential and may not be given, without permission of the investigators’ to any 
other persons other than the prospective co-investigators, research site staff and relevant ethics 
committees and regulatory authorities. 
 
Statement of Compliance 
This research study will be conducted in compliance with the International Conference on 
Harmonisation Tripartite Guidelines on Good Clinical Practice protocol, local good Clinical and 
Laboratory Practice Guidelines in line with the ethical principles of the Declaration of Helsinki and 
requirements and guidelines of the Human Research Ethics Committee (HREC) that is; Joint 
Parirenyatwa Hospital and College of Health Sciences Research Ethics Committee and Medical 
Research Council of Zimbabwe. 

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Protocol Signature Page 
Sponsor Representative 
 ________________________  _____________________________  
 ____________________  
Name 
Signature 
Date 
 
Investigator(s) 
I agree to conduct this clinical study in accordance with the design and specific provisions of this 
protocol and will only make changes in the protocol after notifying the sponsor. 
I understand that I may terminate or suspend enrolment of the study at any time if it becomes 
necessary to protect the best interests of the study participants. 
I agree to personally conduct and supervise this investigation to ensure that all associates, colleagues, 
and employees assisting in the running of this study are informed about their obligations in meeting 
these commitments. 
I will conduct the study in accordance with Good Clinical Practice, the Declaration of Helsinki, and 
the moral, ethical and scientific principles that justify medical research. The study will be conducted 
in accordance with all relevant laws and regulations relating to clinical studies and the protection of 
study participants. 
I will ensure that the requirements relating to HREC review and approval are met.  
I agree to maintain adequate and accurate records and to make those records available for audit and 
inspection in accordance with relevant regulatory requirements. 
I agree to promptly report to the HREC any changes in the research activities and all unanticipated 
problems involving risks to human subjects or others. Additionally, I will not make any changes in the 
research without HREC approval, except where necessary to ensure the safety of study participants. 
Principal Investigator 
 ________________________  _____________________________  
 ____________________  
Name 
Signature 
Date 
Co-Investigator 
 ________________________  _____________________________  
 ____________________  
Name 
Signature 
Date 
Co-Investigator 
 ________________________  _____________________________  
 ____________________  
Name 
Signature 
Date 

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Protocol Summary 
Full Title 
HIV Exposure, Disease Acquisition and Progression Among Children: Role of Maternal Immunogenetics, 
Viral Genetic Diversity, HAART Exposure, Co-morbidities and Psycho-Social Status: UZ-CHS Birth 
Cohort 
Short Title 
UZ-CHS Birth Cohort 
Study 
Design 
Prospective cohort study  
Study 
Population 
Pregnant women from 28 weeks gestation and their infants 
Sample size 
1200 (600 HIV positive (+) and 600 HIV negative (-) 
Duration of 
participation 
A total of 27 months that is, plus or minus 3 months in pregnancy and a follow up period of 24 months 
after delivery. 
Study 
 intervention 
An observational study on how antenatal co-morbidities impact on pregnancy outcome, infant growth and 
development in this era of option B+ of prevention of mother to child transmission of HIV (PMTCT). 
Background 
Whilst the numbers of human immune deficiency virus (HIV) exposed and infected (HEI) infants are 
dwindling by the day, courtesy of the current effective PMTCT programs, the converse is true with (HIV) 
exposed but uninfected (HEU) infants. Despite being HIV uninfected HEU infants, have been shown to 
have higher morbidity and mortality mainly from infectious diseases just like their HEI counterparts. 
Understanding why and how this ever growing population of HEU infants is at high risk of acquiring 
infections is critical as their problems will be of major public health importance in the near future. However, 
before understanding HEU infants themselves, it is necessary to first understand the impact of 
theintrauterineenvironment which is key in shaping their immune development and their lifelong health in 
general. To achieve this, a new research paradigm involving an integrated approach is critical. There is need 
to clearly discern, in a holistic and synergistic approach involving different specialties, the role of antenatal 
comorbidities including maternal psychosocial well-being not mention the impact of highly active 
antiretroviral therapy (HAART) prophylaxis regimen on pregnancy outcome, infant growth, immune and/or 
neuro-development. More so, how similar or different these factors are among HIV+ and HIV- pregnant 
women remains poorly described. Evaluation of combinations of soluble biomarkers of different 
comorbidities may prove more powerful tool in predicting disease outcome. Undertaking research in such a 
manner within the college not only provides the mostly sought after evidence based clinical care for mothers 
and their children but also build research capacity within the College. In addition investigators also attain 
higher degrees for self-actualization not to mention institutional and national professional advancements.  
Primary 
Objectives 
1. To determine the prevalence and incidence of HIV and co-infections (hepatitis B virus (HBV) hepatitis 
C virus (HCV), cytomegalovirus(CMV)intestinal parasites) and determine how these impact on 
pregnancy outcome, infant growth and immune/neuro-development. 
2. To determine of maternal nutritional status and the prevalence and incidence of non-communicable 
diseases (diabetes, hypertensive disorders, cardiovascular diseases) and how these impact on pregnancy 
outcome and child growth 
3. To determine immunological and soluble biomarkers of HIV transmission, diseases progression for 
different comorbidities in isolation or combinations among mothers and infants. 
4. To correlate maternal plasma viral load, soluble biomarkers immune activation profiles and diseases 
progression and duration of HAART treatment among HIV+ mothers 
5. To determine any association of different HAART regimens and pregnancy outcome, infant growth and 
immune/neuro-development among HIV+ mothers 
Primary 
end points 
1. Prevalence, trends and incidence of comorbidities among HIV+ and HIV- mothers 
2. Pregnancy outcomes for HIV-/+ mothers with different comorbidities with or without different HAART 
regimens  
3. HIV and co-infections vertical transmission rates in the era of option B+ among mothers with chronic 
and acute HIV infection 
4. Soluble biomarkers for different comorbidities, acute, chronic HIV infection and vertical transmission 

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Table of Contents 
Investigators and Sponsor ............................................................................................................................... i 
Mentors/Advisory Committee ....................................................................................................................... ii 
Confidentially Statement .............................................................................................................................. iii 
Statement of Compliance .............................................................................................................................. iii 
Protocol Signature Page ................................................................................................................................ iv 
Protocol Summary ......................................................................................................................................... v 
Table of Contents .......................................................................................................................................... vi 
Table of Figures ........................................................................................................................................... vii 
List of Abbreviation and Acronyms ............................................................................................................ vii 
Executive Summary ...................................................................................................................................... ix 
Background .................................................................................................................................................... 1 
1.0 
Introduction....................................................................................................................................... 2 
1.1. 
HEU Infants Increased Susceptibility to Infections .......................................................................... 3 
1.2. 
Immunological Abnormalities of HEU Infants ................................................................................. 3 
1.3 
Responses to Vaccines among HEU Infants ..................................................................................... 7 
1.4 
Viruses: Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) ..................................................... 8 
1.5 
Other STIs and Role of Cervico-vaginal /gut Microbiota ................................................................. 9 
1.6 
Maternal nutritional Status .............................................................................................................. 12 
1.7 
Vitamin D status, GDM and Pregnancy .......................................................................................... 13 
1.8 
Hypertensive Disorders ................................................................................................................... 14 
1.9 
Host genetics ................................................................................................................................... 15 
1.10 
HIV Genetic Diversity .................................................................................................................... 16 
1.11 
Maternal Psycho-Social Factors ..................................................................................................... 17 
1.12 
Justification ..................................................................................................................................... 18 
2.0 
Research Questions ......................................................................................................................... 19 
2.1 
Main Hypotheses ............................................................................................................................ 19 
3.0 
Project Goals: .................................................................................................................................. 19 
3.1 
Main Objective ............................................................................................................................... 19 
4.0 
Methodology ................................................................................................................................... 20 
4.1 
Study Design ................................................................................................................................... 20 
4.2 
Study Sites ...................................................................................................................................... 22 
4.3 
Study Participants ........................................................................................................................... 22 
4.4 
Inclusion Criteria ............................................................................................................................ 22 
4.5 
Exclusion Criteria ........................................................................................................................... 22 
4.6 
Selection of the Participants............................................................................................................ 22 
4.7 
Sampling Procedure ........................................................................................................................ 22 
4.8 
Mothers’ Procedures ....................................................................................................................... 22 
4.9 
Infants Procedures .......................................................................................................................... 24 
4.10 
Laboratory Methods ........................................................................................................................ 25 
5.0 
Reference List ................................................................................................................................. 35 

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Table of Tables 
Table 1: Types and Total Volumes of Maternal Specimen Required ................................................................... 23 
Table 2: Type, Volume, Intervals of Collection And Purpose Of Maternal Specimens Required ....................... 24 
Table 3: Types and Total Volumes of Infant Specimen Required ........................................................................ 24 
Table 4: Type, Volume, Intervals of Collection and Purpose of Infant Specimens Required .............................. 25 
Table 5: Pictorial Depiction of the Stratification .................................................................................................. 32 
Table 6: Tasks and Time Frames .......................................................................................................................... 34 
Table of Figures 
Figure 1: Study Flow Diagram ............................................................................................................. 21 
List of Abbreviation and Acronyms 
µl 
 
microliter 
AIDS   
Acquired immune deficiency syndrome 
APOBEC3G 
Apolipoprotein-B mRNA-editing enzyme catalytic polypeptide-like-3G  
BCG 
 
Bacille de Calmette et Guérin 
BMI 
 
Body mass index  
BST-2  
Bone stromal tumour protein-2  
BV 
 
Bacterial vaginosis  
CD 
 
Cluster of differentiation 
CMV  
Cytomegalovirus  
CNS 
 
Central nervous system  
DNA 
 
Deoxyribonucleic Acid 
DPIC  
Depo-Provera injectable contraception  
EBV   
Epstein Barr virus 
Env 
 
Envelope gene 
g/dl 
 
grams per decilitre  
GDM  
Gestational diabetes mellitus  
Gp 
 
Glycoprotein 
GUD   
Genital ulcer disease  
HbA1c  
Glycosylated haemoglobin  
HBV 
 
Hepatitis Bvirus  
HCV 
 
Hepatitis Cvirus  
HDL   
High density lipoprotein  
HELLP  
Haemolysis, elevated liver enzymes and low platelet count 
HIV 
 
Human immunodeficiency virus 
HPV 
 
Human Papilloma virus  
HSE 
 
Herpes simplex encephalitis  
HSV 
 
Herpes simplex virus  
IFN-γ  
Interferon-gamma 

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IGF-1  
Insulin growth factor -1  
IgG 
 
ImmunoglobulinG 
IL 
 
Interleukin 
KIR  
 
Killer cell immunoglobulin-like receptors  
LTBI  
Latent TB infection  
MCP-1  
Monocyte chemoattractant protein-1 
Nef 
 
Negative factor 
NK cells 
Natural killer cells 
OIs 
 
Opportunistic infection  
PI 
 
Protease inhibitor  
PKPD-VD 
Pharmacokinetics- pharmacodynamics-viral dynamics  
PMTCT 
Prevention of mother to child transmission 
PNGs  
Potential N-glycosylation sites  
RFs 
 
Restriction factors  
SAMHD-1 
Sterile alpha motif histidine-aspartic domain-1 
SSA 
 
Sub-Saharan Africa 
STIs 
 
Sexually transmitted infections  
TB 
 
Tuberculosis 
Th-2 
 
T-lymphocyte helper-2  
TIBC  
Total iron binding capacity  
TLRs  
Toll like receptors  
TRIM5α 
Tripartite motif 5-alpha  
TST 
 
Tuberculin skin test  
TV 
 
Trichomonas vaginalis  
UZ-CHS 
University of Zimbabwe College of Health Sciences 
V3 
 
Envelope gene variable region 3 
VDR 
 
Vitamin D receptor 
Vif 
 
Viral infectivity factor 
Vpr 
 
Viral protein R 
Vpu 
 
Viral protein U 
VZV 
 
Varicella zoster virus 

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Executive Summary 
Viral, host immunogenetics and environmental factors associated with human immunodeficiency 
virus (HIV) non-acquisition and/or slow HIV disease progression among paediatric patients remain 
poorly described. The little that is currently known comes from HIV-1 subtype B studies. The role of 
innate immunity in protection against HIV infection and disease progression may not be over-
emphasised among paediatric HIV infected individuals whose adaptive immunity may not have fully 
developed. Since HIV-infected children progress more rapidly than adults and have fewer background 
cofactors such as drug use and co-infections, the effects of host genetic factors on HIV-1 disease may 
be more clearly identified. While the emphasis has been on increasing availability and coverage of 
efficacious antiretroviral regimens and strengthening health systems within PMTCT initiatives, there 
is also a need to address other maternal-related factors which are equally essential. Undiagnosed 
maternal HIV infection and co-morbidities, nutritional status, pyscho-social factors, unplanned 
pregnancies, delays in accessing antenatal care, non-disclosure of the pregnant woman’s HIV status to 
her partner and social demography are some of the most significant maternal risk factors associated 
with vertical transmission of HIV and/or infant morbidity including mortality. Equally important in 
determining infants ‘susceptibility to infections are maternal behavioural, biological and 
immunogenetic factors. How all these factors in isolation or combination modify the infants’, growth, 
neuro- and immune development remains poorly described. In view of varied population specific 
differences in the frequencies of protective or susceptibility alleles, it is important to strengthen 
research efforts towards defining unknown genetic propensity in our own population. Mathematical 
modelling of these potentially interactive genetic variables alongside with their respective 
immunological profiles may help to identify potential genetic or/and immunological markers with 
better diagnostic and/or prognostic values. Thus, there is need for a cohort study to clearly discern the 
causes and consequences of viral immune profiles and genetics in addition to social dynamics of 
disease acquisition which are impossible to determine with cross sectional studies. A holistic and 
integrative research approach involving different disciplines to predict diseases outcomes of the ever 
growing population of HEU may not be over-emphasised. This is only possible through development 
of sub-studies nested within this cohort, which may have different study designs, addressing other 
pertinent comorbidities research questions from different perspectives.Such a research approach is not 
only cost effective and efficient as it quickly yields incredible maternal and infant scientific 
observations. HEU and non-progressing HEI infants offer hope that natural and effective HIV control 
is possible at the same time providing an important insight into the development of the next 
generation of HIV/AIDS vaccines and immune-based therapies with minimal side effects. 
 

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Background 
The underlying reason behind the high HIV-1 prevalence in sub-Saharan Africa (SSA) remains 
elusive. Unlike in Europe and the United States of America(USA) where HIV is confined to 
intravenous drug users and homosexual men 1, the African epidemic is more widely distributed across 
the general population with heterosexual penile-vaginal as the main transmission mechanism 2. 
However, this observation is inconsistent with the low probability of heterosexual HIV transmission 
per coital act, as differences in sexual behaviour do not always translate to differences in HIV 
prevalence or incidence 3;4 pointing to the possibility of other precipitating co-factors that may 
increase per contact transmission rates in the region. Moreover, this heterosexual mode of 
transmission does not satisfactorily explain the relatively high proportion of HIV discordant couples 
common in the region who in some instances continue to bear children5. Thus, contact with an 
infected person represents only a necessary but not sufficient condition for HIV transmission as 
exposure to the virus without being infected is common, pointing to gaps in our current knowledge on 
HIV transmission. 
 
Contemporary scientific literature demonstrates that human immunogenetics coupled with ecological 
factors are important determinants of vulnerability to HIV infection and progression to AIDS 6. 
Synergistic relationships between HIV disease burden and viral genotypes, malnutrition including co-
infections have been observed, implicating them as possible HIV disease acquisition precipitating 
factors 7;8. On average 74% Africans are exposed to two or more parasitic, bacterial or viral infections 
whilst a good 26% grapple with six or more diseases including non-communicable diseases, some of 
which are side effect(s) attributed to intake of HAART 9-11. Relative to single pathogen infections, co-
infections may alter HIV transmission dynamics 12. Single and/or dual pathogen infection studies have 
quite common. Such studies are essential but they could have reported misleading conclusions taking 
cognisance that in real local life situation such solitary or double infections are rare. Thus, there is a 
paucity of information on the prevalence of co-morbidities and how these in isolation or/and 
combination modify or modulate HIV disease acquisition, immune responses progression, pregnancy 
outcome, neuro-cognitive development and response to therapy more so in children. The foregoing 
including other potential social-cultural factors may be central in fuelling the HIV-1 epidemic in SSA. 
Unless a holistic multi-disciplinary research approach is adopted that will at the same time generate 
critical mass of local professionals with relevant expertise complimented by cross fertilization of 
regional and international institutional collaborations, the problem of HIV will always remain. 

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1.0 
Introduction 
Use of effective prophylactic HAART in pregnancy has reduced vertical transmission rates of HIV 
down to about 1% from a high of more than 20% 13;14. Thus the immediate benefit of HAART in 
pregnancy remains unquestionable, implying that most infants can now escape HIV infection. 
Consequently, the number of HEU infants is increasing, representing about 30% of all children born 
in most parts of SSA15. Despite these positive and impressive developments, HEU children have been 
shown to have substantially increased morbidity and mortality predominantly from infectious causes, 
particularly between 2 and 6 months of age, compared with their HIV unexposed uninfected (HUU) 
counterparts16-21. This observation is of great concern taking cognisance that over and above their ever 
increasing numbers, HEU alongside the HIV exposed and infected (HEI) infants, are now maturing 
into adults22, entailing that any problems specifically associated with these groups of children will be 
of major public health importance. So far it is not yet clear whether in utero exposure to HIV-1 and/or 
other co-morbidities including exposure to prophylactic HAART during pregnancy and/or infancy 
may predispose these infants to such infections. 
 
Preterm deliveries have been the commonest adverse pregnancy outcome among Chinese HIV-
infected women, with such babies generally suffering from respiratory distress syndrome, dysplasia, 
intracerebral hemorrhage and necrotizing enterocolitis 23. Studies have also shown maternal HIV 
infection to be associated with being underweight and low height for age z scores among HEU infants 
24-26. Contrary to the above findings, a recent Botswana study has found no significant mortality 
difference by HIV exposure status and non-exposure in a neonatal intensive care setting but has 
identified low Apgar scores and extreme prematurity as important risk factors for mortality, pointing 
to the importance of other related factors 27.Maternal plasma and cervical HIV-1 RNA load are 
important risk factors for vertical transmission of HIV28. Little is known about trends of HIV RNA 
levels in HIV-infected women conceiving on HAART with or without viral load suppression and how 
this impacts on pregnancy outcome including infant development. Recent findings suggest that 
exposure to high maternal HIV-1 viremia in utero, even in the absence of perinatal transmission, 
affects the infant's immune system development resulting in health complications later in life29. The 
role of the mother-infant pair host genetics and HIV diversity in susceptibility to infections is equally 
critical. Thus, a better understanding of the role of all the foregoing factors, not to mention the 
obvious shortcomings associated with being born in an HIV affected household is essential for better 
clinical management of this ever growing HEU population with the long-term aim of addressing most 
of their unmet health needs. 
 

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1.1. HEU Infants Increased Susceptibility to Infections 
In spite of being HIV uninfected, HEU infants, like their HEI counterparts, have been shown to 
harbour enteropathogenic Escherichia. Coli (E. coli) and Cryptosporidium infections in their stool 
specimens, an observation that may explain the observed increased mortality 30. A low full blood 
count (FBC) at birth has been significantly associated with this increased infant morbidity and 
mortality 31. Nasopharyngeal bacterial colonisation with Staphylococcus aureus, Streptococcus 
pneumoniae, Moraxella catarrhalis including Haemophilus influenza have been shown to be 
prevalent among Tanzanian HEU infants at about 6 weeks of age 32. Acute bronchiolitis has been 
quite common and more severe among Brazilian HEU infants of less than 6 months of 
age33.Interestingly, pulmonary and extra-pulmonary tuberculosis (TB) have also been shown to be 
prevalent among HEU infants34. Malawian and South African studies on hospitalized HEU infants 
showed serious infectious morbidity due to Pneumocystis jiroveci pneumonia, Cytomegalovirus 
(CMV) colitis with perforation, Pseudomonas sepsis, haemorrhagic varicella, Group a Streptococcal 
meningitis, endocarditis and gastroenteritis 35-37. Early breastfeeding cessation has been associated 
with increased risk of serious gastroenteritis among HEU infants when compared with later 
breastfeeding cessation 38;39.Maternal prolactin hormone levels “rise with infant suckling. Thus, the 
more feedings, the higher the level of serum prolactin although stress can also elevate levels. (Cox, 
Owens, & Hartmann, 1996; Tay 1996). Studies have found out that breastfeeding decreased 
pneumonia incidence among Kenyans HEU infants with morbidity rates picking up after 
weaning40;41;42. Besides issues surrounding early cessation of breastfeeding, studies have indicated that 
HEU infants have a wide range of phenotypical and functional immunological abnormalities that may 
contribute to the high morbidity and mortality from infections.43;44. Such factors surrounding the 
abnormal infant immune development are not well described. 
 
1.2. Immunological Abnormalities of HEU Infants 
1.2.1. Adaptive Immunity Anomalies 
Cell-mediated immunity and T-lymphocyte maturation have been shown to be altered in HEU new-
borns and these abnormalities persist over time 45;46 implying that in utero exposure to HIV and/or 
HAART may cause some of these immune abnormalities. The thymus being the organ responsible for 
the maturation and selection of T lymphocytes is thus pivotal in allowing the development of a 
functional immune system 47;48. The role played by thymus size and output in the shaping of foetal 
immune development in HEU infants remain poorly described. Absolute numbers of lymphocytes are 
decreased in HEU infants with feotal exposure to maternal HIV-1 priming the infant immune system 
leading to an enhanced immune activation and altered T lymphocyte homing 49;50. Immune activation 
in HEU infants is also believed to modify interaction between the immune system and HIV 
pathogenesis51.Findings have demonstrated a significant disturbance in cytokine network among HEU 

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neonates exposed to HAART during pregnancy 52;53. The observation that ARV induces not only 
quantitative but also qualitative alterations in the cytokine profile warrants attention. Cytokine profile, 
when properly designed could complement the data from viral load and CD4+ T lymphocyte count in 
the analysis of the disease status, thus aiding in the decision making among possible therapeutic 
interventions aimed at persistently decreasing immune activation and chronic inflammation 54;55.Thus, 
soluble markers in body fluids is a reflection of immune status and therefore may be important in 
assessing HIV transmission, progression even mortality. Decrease in IL-2, IFN-γ and concomitant 
increases of IL-4 and IL-10 have been associated with a decline in antigen-specific immune 
responses, resulting in opportunistic infections 56;57. Elevated inflammatory biomarkers such as CRP 
and IL-6, have been associated with HIV disease progression and mortality. Chemokines and 
defensins in breast milk and cervico-vaginal secretions have been associated with HIV-1 
susceptibility and transmission. Trends in plasma and breast milk biomarker levels of immune 
activation and inflammation as interferon-γ-inducible protein-10 (IP-10), monokine induced by 
interferon-γ (MIG) and soluble CD14 (sCD14) levels, intestinal fatty acid binding protein (I-FABP) 
can be compared to HIV-1 RNA level and evaluated for associations with comorbidities. Biomarkers 
of the acute HIV infection, such as IFNγ, serum amyloid A, IP-10, IL-12, , IL-7 and IL-15, may help 
predict viral set-point variation and disease progression whilst IL-6, soluble (s) CD14, sCD163, and 
CRP, D-dimer, fibrin and hyaluronic acid can be clinically useful biomarkers in predicting 
comorbidities in HIV-1-infected patients both on and without HAART. Evaluation of combinations of 
soluble biomarkers of different comorbidities may prove more powerful tool in predicting disease 
outcome. Persistent exposure to viremia alters HIV-specific CD8 response through a tenacious 
immune activation process leading to naive CD8 T-lymphocyte exhaustion and skewed maturation of 
memory subsets 58. Increasing differences in lymphocyte subsets become more apparent as early as 
six weeks of age with total and naive CD4+ T lymphocytes being significantly higher in American 
HEU than HEI infants whilst CD8+ lymphocyte positive for human leukocyte antigen DR locus 
positive (HLA-DR+), CD45 RA+ HLA-DR+ and CD28+ HLA-DR+ were elevated in HEI infants 59. 
Soluble CD14 (sCD14) is secreted into plasma and serves as a biomarker of monocyte activation. 
elevated levels of sCD14 have been associated with CD8+ T-cell activation and with mortality in the 
setting of HIV infection. Altered CD4/CD8 and the memory/naive ratios have been observed at least 
in Italian HEU adults60. Studies have also shown alterations in immunoglobulin levels and higher B-
lymphocyte apoptosis among HEU infants 61;62. The role of pro-inflammatory lipids high density 
lipoprotein (HDL), in systemic immune activation in HIV infection remains largely unknown. All in 
all these findings suggest that foetal exposure to a chronically activated maternal immune system may 
be the reason behind the observed altered adaptive immune responses which are not yet fully 
developed. The innate immunity is more important at this tender age of development. 
 

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1.2.2. Innate Immunity Anomalies 
In view of the under-developed adaptive immunity and the limited exposure to pathogens in utero 
infants rely on the innate immune system for protection against infections. Innate immune cellular 
population include macrophages, dendritic cells, monocyte and natural killer (NK) cells. Findings 
have shown the dysfunctionality of the innate immunity of HEU infants. Alterations in NK cell 
subsets, activation, and cytolytic potential including cytokine production may contribute to the 
reduced ability to fight infections, a phenomenon observed among these infants 63;64. Identification of 
invading pathogens is through pathogen associated molecular patterns that are sensed by patterns 
recognition receptors such as toll like receptors (TLRs) and C type lectins. TLRs recognise both extra 
and intracellular pathogens. TLR agonist receptor binding culminate in the downstream production of 
cytokines, chemokines and antimicrobials. Defensins are effector molecules of the innate immunity 
with a broad antimicrobial spectrum 65. Alpha-defensin concentrations in breast milk have been 
associated with a decreased risk of intra-partum and post-natal HIV transmission 66;67. The role of 
innate immunity in protection against HIV acquisition and disease progression need not be over-
emphasised among paediatric HIV infected infants whose adaptive immunity would not have fully 
developed. When it comes to immunity against retroviruses, mammalian cells have evolved powerful 
mechanisms to limit or restrict viral replication through the production of restriction factors (RFs), 
constituting the so called intrinsic immunity. 
 
1.2.3. Intrinsic Immunity: Role of RFs 
Host constitutively express cellular antiviral proteins called RFs. Examples of such proteins include 
apolipoprotein-B mRNA-editing enzyme catalytic polypeptide-like-3G (APOBEC3G), bone stromal 
tumour protein-2 (BST-2) or tetherin integral membrane protein, tripartite motif 5-alpha (TRIM5α) 
and sterile alpha motif histidine-aspartic domain-1 (SAMHD1). However, HIV has developed 
strategies to counter the antiviral activity byencoding accessory/regulatory proteins viral protein u 
(Vpu), viral protein r (Vpr), viral infectivity factor (Vif) and negative factor (Nef) to escape 
destruction from host RFs.Like the innate immune responses, the expression of RFs is also induced by 
interferons. Role of RFs in HIV infection for both mother and new-born pairs has so far received very 
little attention yet they constitute the very first line of immune defense, much before of both the innate 
and adaptive immune responses kick in. Few recently published articles suggest the key roles played 
by APOBEC3G,TRIM5α, tetherin and SAMHD1 in facilitating immediate response to viral 
infections68-70. The mechanisms by which RFs inhibit viral replication and their potential contribution 
to HIV pathogenesis remain poorly described. To improve our understanding of such underlying 
mechanisms, there is need to compare and contrast the innate and intrinsic immune development 
between HEI, HEU and HUU infants especially in resource poor settings where risks to infectious 
diseases are remarkably high during infancy. Greater understanding of intrinsic immunity may 
facilitate the development of safer and more effective pharmacological agents for the treatment of 

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viral infections. Also blocking viral replication pharmacologically is HAART that is offered to 
pregnant women to reduce maternal infectiousness including vertical transmission of HIV. 
 
1.2.4 HAART in Pregnancy 
HAART has had undoubted preventive benefits in reducing vertical transmission of HIV. Adherence 
to HAART in pregnancy is crucial to optimize efficacy and curtail vertical transmission. Despite these 
remarkable successes, HAART use in pregnancy has been associated with adverse birth outcomes 
such as preterm delivery, small for gestational age, stillbirths, anthropometric parameters and body 
composition anomalies including birth defects 71;72. There is also need to explore possible etiologic 
role of HAART in cleft palate. HAART pose metabolic complications in HIV patients including 
pregnant women. Protease inhibitor (PI) use early in pregnancy has been associated with increased 
risk of prematurity and gestational diabetes mellitus development 73;74. ARVs have also been 
demonstrated to damage the mitochondria. Studies have suggested that polymorphisms in the 
mitochondrial DNA (mtDNA) may predispose one to ART related metabolic complications. 
Mitochondrial toxicity due to ARVs may explain mt-related metabolic outcomes such as neuropathy, 
lipodystrophy, dyslipidaemia, hyperlactaemia and insulin resistance among ART experienced 
individuals. Pharmacogenomics studies have linked ART related metabolic complications and 
treatment side effects to genetic polymorphisms in genes encoding drug metabolising enzymes and 
drug transporters. Birth weights for gestational age norms for Botswana infants have been shown to 
be lower at term than norms for black American infants. Standard values for birth weight by 
gestational age are not generally available for SSA, where HIV infection, HAART and other in utero 
co-infections exposures may impact on birth outcomes, yet these parameters are necessary if 
incidence and risk factors for intrauterine growth retardation are to be assessed. HAART may cause 
adverse lipid profiles and increased risk for cardiovascular events due to complex interactions 
between traditional risk factors and HIV infection itself in terms of ongoing endothelial dysfunctional 
immune activation/inflammation and increased risk of thrombosis75;76. Both lipoatrophy and 
lipohypertrophy are associated with increased cardio-metabolic risk and in metabolic disorders that 
likely lead to premature aging reported in HIV infected patients 77. Whether these complications are 
also experienced by the child and to what extent presents a knowledge gap. The need to quantify in-
utero exposures to HAART may not be over-emphasized, hence the need to explore the impact of 
these chronically administered medications on pregnancy outcome and child development. 
 
1.2.5 
HEU Infants and exposure to HAART 
After birth all HIV exposed infants must undergo HIV virological testing at the earliest possible 
opportunity to facilitate uptake of ART without any delay for infants with positive test results, in the 
process preserving their immune function and consequently reducing morbidity and mortality 78. 
Marston and et al.; have demonstrated that failure to access treatment results in a 52% net survival at 

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one year among perinatally infected infants 79. Thus the benefits of early infant diagnosis may not be 
overemphasised not to mention the reduced maternal psychological trauma associated with extended 
waiting time for the infant HIV diagnosis test results. 
HAART exposure in utero or postpartum has been associated with significant anaemia and 
neutropenia, persistent decrease platelets and lymphocytes as well as an increased risk of transient 
lactic acidaemia, and mitochondrial deoxyribonucleic acid (DNA) depletion 80;81. Mitochondrial 
dysfunctions following perinatal exposure to nucleoside analogues have been observed 82. Studies 
have observed a greater neurodevelopmental delay among HEI and HEU compared with HUU 
infants/children in resource-poor settings 83. Long-term effects of in utero and neonatal ART exposure 
on cognitive and academic development in HIV-exposed, uninfected school-age children are 
unknown. Other studies have shown that a greater proportion of HEI children receiving PI containing 
HAART to have hyperlactataemia, hyperlipidemia, high triglyceride concentrations compared to 
those not receiving ART 84;85. Low-density lipoprotein cholesterol has been significantly higher in the 
cord blood of PI-exposed infants versus those not exposed to PIs in utero 86. HIV-infected children 
have higher levels of biomarkers of vascular dysfunction than do HEU children. Unfavourable lipid 
profiles have been positively associated with interleukin (IL)-6, monocyte chemoattractant protein-1 
(MCP-1), fibrinogen, and P- and E-selectin, whereas increased HIV viral load has been associated 
with markers of inflammation (MCP-1 and C-reactive protein, CRP) and endothelial dysfunction 
soluble intracellular cell adhesion molecule-1 and soluble vascular cell adhesion molecule-1 
87.HAART exposure in feotal and early neonatal life has also been linked to lower immunoglobulin G 
(IgG) titres 61. The current extent of infant HAART exposure from breast milk remains unknown. All 
these observations call for the need for long-term follow-up of HEU and HEI infants with the aim of 
understanding the impact of maternal HAART treatment on infant toxicity, development of non-
communicable diseases and HIV-1 resistance mutations. 
 
1.3 
Responses to Vaccines among HEU Infants 
Following 6 months of HAART exposure, studies have also shown that more than half of previously 
immunized children still lacked positive measles antibody titres 88. HEU infants have been shown to 
frequently exhibit a range of immunological abnormalities indicative of a deficiency in their ability to 
develop immunological memory following vaccinations 89;90. Studies have shown that exposure to 
HIV in utero has been associated with significant alterations to CD4 and CD8T-lymphocyte responses 
to vaccines in infants91. Some vaccines effectiveness has been found to be similar among HEU and 
HUU infants, for example, the rotavirus vaccine 92. However, some studies have shown impaired 
humoral immune responses to anti-tetanus and Bacille de Calmette et Guérin (BCG) vaccines in HEU 
infants93-96. Lower measles antibody quality and quantity following vaccination have raised challenges 
for ascertaining the long-term protection of these children 97. Some studies support the need for a 

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second dose of the vaccine and for a booster dose of the diphtheria and polio or measles vaccines to 
maintain the necessary antibody concentrations in HEU and HEI African children98;99.BCG-specific 
T-lymphocyte proliferation and interferon gamma (IFN-γ) concentration are reduced in HEU infants 
showing a delay in immune system maturation 95. BCG vaccine administered soon after birth, before 
in utero and peripartum HIV infection exclusion may result in severe disease100;101. Studies have 
demonstrated that the immunogenicity of BCG vaccination among South African HEU infants is not 
compromised when delayed until 8 weeks of age102. Interestingly, obesity-associated factors have also 
been shown to interfere with vaccine immunogenicity and efficacy103.Thus, the effectiveness of 
childhood standard vaccines in SSA with a high prevalence of HIV infection remain poorly described. 
 
Foetuses can also be exposed to maternal co-infections that can modify immune system development 
and influence the new-born’s immune response to other infections. High maternal exposure to 
coinfections may contribute to high rates of infections among HEU 104.There is paucity of data 
regarding the immunogenetics of HIV/tuberculosis (TB)/Human papilloma virus co-infections and 
how their co-existence may modify or modulate host immune response, HIV /TB acquisition, disease 
progression or responses to treatment. An integrated approach in dealing with the conditions is also 
warranted in view of the hepatotoxicity due to anti-tuberculosis drugs105 that limit treatment options 
for patients co-infected with HIV and TB. 
 
1.4 
Viruses: Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) 
Liver diseases due to chronic HBV and HCV infections are now emerging as the leading causes of 
morbidity and mortality among HIV infected persons in SSA 106;107. Prognosis of such patients co-
infected with HIV and HBV or HCV is not well known not to mention the immunological, virological 
and biochemical including elastographic responses.HIV positive pregnant women co-infected with 
HBV/HCV have been shown to have an increased risk of liver enzyme elevation 108. A Nigerian study 
found an HBV prevalence of 4% among HIV positive pregnant women that was associated with 
induced abortion, blood transfusion and elevated baseline transaminase109. HCV and/ or HBV can be 
transmitted from mother to child during pregnancy and delivery 110;111. A 5% HBV prevalence and a 
3% HBV/HIV co-infection prevalence has been reported among South African pregnant women 
which was associated with history of induced abortion 112.The overall rate of HCV MTCT among 
HIV-negative mothers has been estimated at around 5% 113. However, co-infection with HIV raises 
this figure to 19% 114. An Indian study showed HCV prevalence in pregnancy of 3% that was 
associated with surgical procedures and blood transfusion 115. In the same study pregnancy 
complications such as pregnancy-induced hypertension and antepartum hemorrhage were more 
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outcomes are currently not known. The foregoing points to the fact that HCV/HBV/MTCT remains 
poorly described. In addition the trends and patterns of CMV infections warrant further investigation. 
 
1.4.1 
Viruses: Cytomegalovirus (CMV) 
CMV infection is the leading infectious cause of congenital hearing loss and neurodevelopmental 
disability in developed countries. CMV together with other herpes viruses such as HSV, Varicella 
zoster virus (VZV) also cause of significant ocular morbidity. Maternal plasma CMV DNA has been 
shown to be a high risk factor that may result in death in 2 years following delivery. The possible 
effects of transplacental viral infections include feotal loss.HIV-1 and CMV are important pathogens 
which are both transmitted via breastfeeding. CMV infection increases T lymphocyte activation and 
apoptosis contributing to the rapid disease progression in co-infected infants 116.Maternal CMV DNA 
can be measured in cervical secretions, breast milk or dried blood spots at birth 117. Infection of the 
foetus may cause congenital defects, mental retardation including cardiac anomalies or cerebral palsy, 
growth faltering and cognitive impairment 118;119.The prevalence of congenital CMV infection among 
HEU new-borns is around 3% in the USA. Maternal CMV sero-prevalence ranged from 84% to 100% 
in developed countries with CMV birth prevalence varying from 1% to 6%120;121. Studies have shown 
lower socio-economic status to be associated with CMV sero-positivity122. When CMV infection 
occurs early during gestation, it disturbs the neurogenesis of the central nervous system (CNS) while 
late-onset CMV infection affects brain growth and the development of the white matter, leading to 
leukomalacia and cyst formation123. CMV is one of the most common infections known to humans 
and is characterized by a self-limiting infection in healthy individuals. However, the emergence of 
HIV/AIDS has made it the leading cause of birth defects and developmental delays. CMV and HIV 
co-infections have been shown to increase infant morbidity, mortality and AIDS progression. 
Zambian HEU infants have shown poor growth and increased morbidity 124. The birth of congenital 
CMV has been shown to be high despite prenatal ARV prophylaxis and is associated with advanced 
maternal immunosuppression and breast milk CMV levels. Studies have shown that CMV may 
facilitate in utero transmission of HIV-1 by activation of cord blood mononuclear cells 125. 
Determinants of vertical CMV transmission in our setting of high maternal HIV-1 infection remain 
poorly-defined. More so being a neglected but growing problem with the advent of AIDS, congenital 
CMV infection leads to a wide spectrum of symptoms. The challenge is that these symptoms are not 
always obvious at birth. 
1.5 
Other STIs and Role of Cervico-vaginal /gut Microbiota 
Healthy vaginal microbiota is an important biological barrier to pathogenic microorganisms playing a 
pivotal role in diseases acquisition. Changes in the vaginal microbiota have been associated with 
several adverse pregnancy outcomes including premature birth and acquisition of HIV infection 126. 
Buve etal have hypothesised that variations in per sex-act transmission probability of HIV may in part 
be attributed to differences in the composition and function of the vaginal microbiota between high 

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and low income countries127. Microbial composition is affected by hormonal changes, vaginal 
douching practices including use of sex enhancing herbs/botanicals. A healthy vaginal microbiota is 
one predominated by hydrogen peroxide-producing Lactobacillus spp., mainly Lactobacillus 
crispatus, Lactobacillus jensenii, and Lactobacillus iners. Vaginal microbiota has been defined as BV 
when it is predominated by Gardnerella vaginalis and Atopobium vaginae including other anaerobic 
bacterial species. BV is associated with a distinct vaginal discharge syndrome, poor pregnancy 
outcomes, pelvic inflammatory disease, post-operative wound infections and endometritis after 
elective abortions 128;129. Recent data have linked specific vaginal microbes and urogenital infection 
with preterm birth 130. Additionally, BV predisposes women to infection by HIV as well as other 
STIs131;132. The aetiology of BV is unclear, though it is believed to involve loss of vaginal hydrogen 
peroxide-producing Lactobacilli and acquisition of complex bacterial communities that include 
fastidious BV-associated bacteria 133. Cervico-vaginal microbiota dominated Lactobacillus crispatus 
and to a lesser extent L. iners has been associated with a lower prevalence of HIV/STIs and a lower 
likelihood of genital HIV-1 RNA shedding 134.Studies have shown that disturbances of the vaginal 
microbiome especially the presence of BV, increases the risk of acquisition of HIV infection more so, 
with use of Depo-Provera injectable contraception (DPIC)135. Interestingly DPIC use has also been 
associated with increased HSV-2 prevalence 136;137. Contrary to the foregoing observations, some 
studies’ findings suggest that hormonal contraception is associated with lower rates of BV 
138.Gardnerella vaginalis has been significantly enriched in cases of HIV antepartum transmission 
compared with non-transmission 139. A better understanding of the composition of the microbiota in 
healthy and diseased states is important in identifying the role of newly identified microbes. Vaginal 
microbiota may also play a role in mediating susceptibility to Trichomonas vaginalis (TV) 140. If this 
relationship is causal, interventions that improve the vaginal microbiota could contribute to reductions 
in TV incidence. Maternal chlamydial infection, gonorrhoea, genital warts and syphilis are also 
associated with MTCT 141;142. Chlamydia trachomatis, which is asymptomatic in most women, causes 
significant adverse effects for pregnant women and neonates. Studies have observed an unexplained 
high HIV-1 incidence among pregnant women who would have been sero-negative at the first 
antenatal visit but sero-convert later during antenatal and post-partum periods 143;144 and this may have 
serious consequences for both the mother and infant. Hence, screening and early treatment of 
maternal infections during pregnancy may reduce paediatric HIV infections. However, there is need to 
re-visit the current protocol of screening for HIV only once during pregnancy as this may miss 
women who sero-convert during pregnancy. The incidence of HIV and vertical transmission rates 
during the breastfeeding period remain poorly described in most resource poor settings. Different 
studies suggest that some bacteria present in the maternal gut could reach the mammary gland during 
late pregnancy and lactation through a mechanism involving gut monocytes 310. Mammary dysbiosis, 
microbial imbalances may lead to mastitis, a condition that represents the first medical cause for 

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undesired weaning 145;146. Thus, modulation of maternal gut microbiota during pregnancy could also 
have a direct effect on infant health. 
 
1.5.1 
Parasites 
Helminthic and HIV-1 co-infections are common in SSA 6. Thus, this convergent distribution of the 
HIV and helminths’ infections is suggestive of a possible biologically plausible observation that 
persistent infection with helminths may exacerbates the HIV epidemic in the region 147;148.Helminths 
induced chronic immune-activation, altered immune cell distribution, strong T-helper-2 (Th2) bias 
and enhanced HIV replication facilitate faster progression to AIDS 149. It is also important to note that 
activation of Th2 bias is critical for a successful pregnancy which predispose the pregnant women to 
infections. Schistosoma hematobium which affects almost 200 million people in SSA has been shown 
to act as a co-factor for HIV transmission where parasite lesions create open portals for HIV and 
inflammation in the genital area making transmission more efficient. This increases the risk of HIV 
acquisition three-fold 150;151. Helminthiasis is a significant burden in pregnancy and it is also 
associated with anaemia 152.In SSA hookworms, malarial parasites and HIV are important factors 
implicated in anaemia of pregnancy 153. Anaemia in pregnancy has been defined as haemoglobin<11 
g/dl 154. Infections and inflammation have been of great significance in the aetiology of anaemia 
among pregnant Ugandan women155. A Nigerian study has demonstrated that intestinal helminth 
infection has a negative correlation with haemoglobin and packed cell volume, contributing to 
anaemia 156. In Peru T. trichiura infection was found to be a significant risk factor for anaemia in 
pregnant women 157.Maternal infections with parasites might affect the development of feotal 
immunity and susceptibility to post-natal infections independent of in-utero transmission of the 
pathogens 158;159.There is evidence that this may impact on the long-term responses to helminth and 
non-helminth antigens and to allergens 160;161.Prevalence of intestinal parasitic infections such 
asTrichuris trichiura, Entamoeba histolytica/dispar/moshkovskii, Ascaris lumbricoides and Giardia 
duodenalis, Entamoeba spp., E. nana and Blastocystis hominisremain poorly described among 
pregnant women in our setting. Parasitic intestinal infection with helminths and/or protozoa can lead 
to significant morbidity and mortality if not recognized and treated appropriately. There is also 
paucity of data regarding parasite(s) co-infection(s) with HIV and how their co-existence modify or 
modulate, host immune responses, HIV acquisition, disease progression and/or response to treatment 
in the background of a SSA population infested with a myriad of parasites. In addition to infections, 
food accessibility also contributes to the high rate of anaemia during pregnancy in our populations. 
The most common causes of anaemia are poor nutrition, deficiencies of iron and other micronutrients, 
malaria, hookworm disease, and schistosomiasis; HIV infection and haemoglobinopathies are 
additional factors. A Tanzanian study showed parasitic infections, vitamin D deficiency, low CD4 T-
cell count and high erythrocyte sedimentation rate as the main predictors of iron deficiency in 
pregnancy and the postpartum period 162. Severe anaemia is associated with increased incidence of 

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pre-term labour pre-eclampsia. Hence the need for nutritional education among pregnant women is 
critical. 
1.6 
Maternal nutritional Status 
Studies have shown that 30% of SSA population is malnourished 8. HIV infection further 
compromises the nutritional status of infected individuals. Malnutrition in turn worsens the effects of 
the disease by weakening the immune system consequently, hastening disease progression and early 
mortality 163;164. Pregnant women in SSA are at risk of poor nutritional status and adverse pregnancy 
outcomes as a result of poverty, food insecurity, sub-optimal healthcare facilities, frequent infections 
and pregnancies. Subsequently, obstetric complications, including hypertension, anaemia, neural tube 
defects, night-blindness, and low birth weight, maternal and perinatal mortality are common 165.  
Components leading to healthy pregnancy outcome include healthy pre-pregnancy weight, appropriate 
weight gain, physical activity during pregnancy, consumption of a wide variety of foods, appropriate 
vitamin and mineral supplementation, avoidance of alcohol, other harmful substances and safe food 
handling 166. Nutritional assessment needs to encompass changes in anthropometric, biochemical and 
clinical indicators throughout pregnancy 166. Due to some challenges associated with these 
measurements plasma insulin growth factor-1 (IGF-1) offers a potentially; simple but accurate single 
measurement of nutritional status alongside with total protein, creatinine, urea nitrogen, albumin, 
complement components C3/C4 and α-1 anti-trypsin167. Maternal anthropometry, haemoglobin 
concentration, serum iron, total iron binding capacity (TIBC) and serum albumin were assessed and 
the incidence of preterm labour was significantly higher in mothers with severe anaemia168. Anaemia 
is to be defined as Hb below 11.0 g dl-1 and severe anaemia as Hb below 7.0g dl-1 (WHO, 1991).Poor 
maternal bodyweight and low body mass index (BMI) have been associated with low birth weight in 
HIV-infected women 169;170. Birth weight is largely determined by maternal factors other than 
hyperglycaemia, with the most significant influences being gestational age at delivery, maternal pre-
pregnancy body mass index (BMI), maternal height, and pregnancy weight gain, the presence of 
hypertension, cigarette smoking and alcohol use. Anthropometric assessments of mid-upper arm 
circumference (MUAC) have also been shown to be an efficient, cost-effective screening tool for low 
birth weight in both HIV-infected and uninfected women 171. Low MUAC has been associated with 
poor maternal health outcomes, including anaemia. 172. Taking multivitamins during pregnancy may 
be an inexpensive and effective strategy to improve the health of the mother and baby. MUAC 
measurements of <11.0 cm among children has been associated significantly elevated risk of 
mortality173. An improvement in prenatal mean haemoglobin concentration linearly increased birth 
weight 174. On the other hand maternal obesity is associated with increased risk of gestational 
hypertension, preeclampsia, gestational diabetes and feotal macrosomia, high birth weight (>4,000 
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epidemiological studies have shown that women who develop gestational diabetes mellitus (GDM) 
are more likely to be vitamin D deficient 175. 
1.7 
Vitamin D status, GDM and Pregnancy 
Vitamin D insufficiency has now reached epidemic proportions, even in healthy individuals living in 
the tropics 176. Use of HAART has been a possible cause of hypovitaminosis D 176. Recent evidence 
links not only low maternal vitamin D status with increased risk of severe preeclampsia or small-for-
gestational age but also schizophrenia in the offspring. Other Foetal risks include intrauterine growth 
retardation, neonatal hypocalcemic seizures, impaired postnatal growth, rickets in infancy and 
cardiomyopathy with increased future risks of asthma and type 1 diabetes. Polymorphisms in the 
vitamin D receptor (VDR) gene may contribute to vitamin D-related disparities in feotal growth. 
Evidence from recent studies suggests an early prenatal influence of maternal vitamin D status on 
feotal skeletal development. 177. Vitamin D status has also been shown to have a significant inverse 
association with TB incidence 178;179. Vitamin D exerts its action through VDR and the gene 
polymorphisms associated with susceptibility or resistance to TB 180. Vitamin D, selenium and zinc 
play fundamental roles in determining susceptibility to infections including accelerated HIV disease 
progression 181;182. Currently little is known regarding the effects of HIV/AIDS and its treatment on 
the metabolism of vitamin D and pregnant income and susceptibility to infections. Being a potent 
immunomodulator, vitamin D’s impact on morbidity and mortality among infants remains poorly 
described. There is a particular need for maternal education on healthy diet and for interventions 
which aim to limit over consumption of excess calories or insufficient nutrition during feotal 
vulnerable developmental periods appears to result in a lifelong predisposition to obesity and adult 
disease, such as cardiac diseases and type 2 diabetes. Maternal vitamin D levels exhibit a significant 
negative relationship with glycosylated haemoglobin (HbA1c) levels, supporting a potential role for 
this vitamin in maintaining glycaemic control 183. Pregnancy is also a risk factor for impaired glucose 
metabolism. GDM is defined as glucose intolerance of variable degree with onset or first recognition 
during pregnancy. Carrying a male fetus has been associated with poorer maternal beta cell function 
and an increased risk of GDM 184. Studies have given conflicting results regarding the contribution of 
PIs to impaired glucose tolerance and GDM in pregnant HIV-infected women. Betatrophin, also 
known as TD26/RIFL/lipasin/ANGPTL8/C19/f80, is a novel protein predominantly expressed in 
human liver. Betatrophin plays a significant role in the regulation of lipid metabolism and glucose 
homeostasis, positively correlating with weight gain during pregnancy, systolic blood pressure, very 
low-density lipoprotein levels , fasting insulin level and homeostatic model assessment insulin 
resistance 185;186. 
 
GDM is associated with many adverse neonatal and maternal outcomes miscarriage, infants being 
large for gestational age (LGA) requiring a Caesarean delivery. A growing body of literature supports 

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a relationship between intrauterine exposure to maternal diabetes and risk of a metabolic syndrome 
childhood obesity, hypertension, dyslipidaemia, and glucose intolerance later in life. Glycosylated 
haemoglobin values (HbA1C) levels are associated with increased risk for malformation. HbA1C is a 
reliable measure of chronic glycemic control without the need for a fasting or timed sample. Infants 
born to mothers with diabetes have been shown to exhibit higher levels of cardiovascular risk 
biomarkers for endothelial damage and inflammation, higher leptin levels, BMI, waist circumference 
and systolic blood pressure but decreased adiponectin levels. Maternal GDM, low socioeconomic 
status and preterm infants have been associated with an increased risk of neurocognitive development 
especially the attention-deficit/hyperactivity disorder (ADHD) and compromised neurobehavioral 
functioning 187. GDM birth anomalies also involve cardiovascular diseases (CVD). CVD complicates 
pregnancies with congenital heart disease being the most common pre-existing condition and 
hypertension as the most common acquired condition. 
 
1.8 
Hypertensive Disorders 
Hypertensive disorders during pregnancy represent the major cause of maternal morbidity with 
chronic hypertension occurring in up to 5% of pregnant womenworldwide188. Mild to moderate 
hypertension in pregnancy is defined as systolic blood pressure of 140-159 mmHg or diastolic blood 
pressure of 90-109 mmHg. Hypertension during pregnancy is classified into three main categories: 
chronic hypertension, gestational hypertension, and preeclampsia (high blood pressure and 
proteinuria) with or without pre-existing hypertension. The occurrence of postpartum depression and 
anxiety is higher in pre-eclamptic women 188. Chronic hypertension is defined as blood pressure 
>140/90 mm Hg that either precedes pregnancy or develops before 20 weeks gestation usually also 
persists beyond 42 days postpartum. Chronic hypertension is significantly higher among obese 
women 189.Hypertension during pregnancy is more likely in women with high baseline systolic blood 
pressure and those with higher MUAC 190. Diet has been suggested to play a role in pre-
eclampsia.Maternal serum concentration of the soluble receptor-1 of tumor necrosis factor-alpha 
(TNF-R1) a predictor of development of pre-eclampsia. Oxidative stress and endothelial cell 
dysfunction of the placenta have been implicated in the development of hypertension during 
pregnancy and vitamin intake can reduce oxidative stress and improve endothelial function 
191;192.Endothelial dysfunction biomarkers include VonWillebrand factor (vWF), platelet derived 
micro particles (PMPs), and endothelin-1 that are easily analysed by ELISA. Other serum/plasma 
biomarkers associated with increased risk of hypertensive disorders of pregnancy that correlate with 
adverse pregnancy outcomes include soluble corin, vascular endothelial growth factor, matrix 
metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 (TIMP-1). Haemolysis, elevated liver 
enzymes and low platelet count (HELLP) syndrome is a risk factor for preeclampsia. HELLP is a life-
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the presence of hypertension disorder plus microangiopathic haemolysis, elevated liver enzymes and 
low platelet count, less than 100000. Serum aspartate aminotransferase (AST) and lactic 
dehydrogenase lactic dehydrogenase levels of greater than 70 U/L, and 600 U/L, respectively. HELLP 
syndrome not only increases maternal morbidity and mortality but also that of the fetus. 
Thrombocytopenia is defined as a platelet count of less than 150 x 103 µl. It is commonly diagnosed 
and has attracted interest from the researchers in pregnant women especially among hypertensive 
pregnant women. Gestational thrombocytopenia defined as a platelet count of less than 150 x 10 3 µl 
is recognized as a major cause of thrombocytopenia particularly in hypertensive pregnant women 
during the third trimester. Hypertension during pregnancy increases feotal growth retardation, preterm 
deliveries, still births and perinatal deaths, yet its causes remain unclear 190. Hypertension and 
pregnancy outcomes remain poorly described in our setting more so comparing and contrasting 
between HIV positive and negative pregnant women. HIV-infected women using HAART before 
conception have been shown to be at increased risk of developing preeclampsia/eclampsia during 
pregnancy among Latin American women193. Contradicting evidence exists concerning the 
relationship between HIV infection, HAART use and hypertensive disorders in pregnancy. In addition 
there are indications of hypertensive disorders of pregnancy being associated with high infant blood 
pressure. Moreover, the trends blood pressure changes across childhood remain poorly described.A 
history of pre-eclampsia or pregnancy induced hypertension is an important prognostic factor for 
micro- and macro-vascular complications later in life. CVD factors such as obesity, diabetes, and 
hypertension hyperlipidemia, and thrombophilia which are associated with increased risk of 
spontaneous abortion, preterm labour or premature rupture of membranes, and acute arterial or venous 
thromboses during pregnancy. Of course just like in any condition, host genetic factors also play a 
significant role. 
 
1.9 
Host genetics 
A myriad of host and viral factors act in tandem to cause vertical transmission of HIV. Understanding 
the mechanisms, host and viral factors associated with vertical transmission of HIV will help to 
identify appropriate interventions and suitable antiretroviral chemoprophylaxis regimens to reduce or 
eliminate it. Increasing data support host genetic factors as important determinants of HIV-1 
susceptibility, vertical transmission and disease progression with the CC chemokine CCL3 and HLA 
taking the centre stage 194-198. Relative resistance to HIV infection has been associated with genetic 
polymorphisms. The killer cell immunoglobulin-like receptors (KIR) play a fundamental role in the 
innate immune system. KIR genes are incredibly polymorphic both in the number of genes an 
individual carries and in the number of alleles identified 199. However, their interactions with HLA 
molecules, leading to the modulation of activity in NK cells, mainly related to killing pathogen-
infected cells remain poorly described. Typing of HLA loci B, Cw, and killer cell KIR molecules has 

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shown that the reciprocal equilibrium between inhibitory and activatory NK receptors and their 
ligands favour NK activation among HEU adults 199. A trend toward increased early HIV-1 
acquisition among Kenyan infants presenting in HLA A*29 and increased late HIV-1 acquisition via 
breast milk for both Cw*07 and Cw*08. HLA B*18 may protect breast-feeding infants against both 
early and late HIV-1 acquisition, a finding that could have implications for the design and monitoring 
of HIV-1 vaccines targeting cellular immune responses against HIV-1200. Placental HLA-G1 
expression has been shown to be up-regulated 4 times more in placentas of HIV-1 infected mothers 
with infected babies compared to uninfected babies 201. Single nucleotide polymorphisms and copy 
number variations have been documented for defensins chemokines and RFs which are natural 
inhibitors of HIV infection 202-205. These results demonstrate a significant relationship between genetic 
variants of beta-defensin-1 gene, RFs genes, viral load, and vertical transmission of HIV-1, thus 
supporting a critical role of innate immunity in paediatric HIV-1 infection. However, such studies are 
rare in the region. 
 
1.10 
HIV Genetic Diversity 
HIV-1 genetic diversity in the HEI may also affect viral transmissibility, pathogenicity and responses 
to antiretroviral therapy. Variable region 3 (V3) of the envelope (env) gene for CCR5 and CXCR4 
determination is a key determinant of vertical transmission 206;207. HIV-1 env gp (glycoprotein)120 
variable regions exhibit an unusual pattern with encoded amino acids (asparagine, serine and 
threonine) that leads to the creation of new N-linked glycosylation sites, which helps the virus to 
escape from the immune pressure 208;209. Potential N glycosylation sites (PNGs) glycans and amino 
acid length polymorphism have been shown to play essential roles in disease progression with 
changes potentially affecting the capacity of the virus to replicate 210;211. Studies have shown that at 
birth all HIV infected infants’ env amino acid sequences are initially of the same length as the 
maternal ones with amino acid sequence length polymorphism observed from six months postpartum 
212. However, other studies have observed no clear trends in changes in neither the env PNGs nor 
median amino acid lengths with disease progression 213. Genetic analyses revealed that rapid 
paediatric progressors receive and maintain a genetically homogeneous viral population throughout 
the disease course whilst slow progressors’ exhibit low levels of variation 212 initially, but attain 
higher levels of diversity over time a phenomenon important in HIV transmission linkages. 
Phylogenetic and phylodynamic studies have identified clusters of new infections occurring along 
geographic routes and in different groups214-216. Late diagnosis of HIV remains prevalent and 
represents missed opportunities for early treatment. The combination of phylogenetic analyses of HIV 
sequences with patients' demographic data allows greater understanding of local HIV transmission 
and necessary knowledge for designing prevention strategies. 217. The host immune response to HIV 
in early life is complex. Understanding the relationships between acquired immune responses to HIV, 
innate immune responses that include chemokines and their receptors, and host genetic influences that 

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affect these immune processes in HIV-exposed infants will help identify critical components of 
protective immunity. This helps in the identification of infants at risk of acquiring HIV-1 infection 
and/or at risk of more rapid disease progression. Results from such studies will vastly improve our 
knowledge of HIV-host interactions. Further elucidation of these processes will contribute to the 
rational design of vaccines and the development of novel preventative and therapeutic anti-HIV 
strategies for children exposed to HIV. Having discussed all the foregoing factors shortcomings 
associated with maternal psycho-social issues and being born in an HIV affected household may also 
play a pivotal role. 
1.11 
Maternal Psycho-Social Factors 
Undiagnosed maternal HIV infection prior to conception, unplanned pregnancies, delays in accessing 
antenatal care, non-disclosure of the pregnant women’s HIV status to her partner and low levels of 
education are some of the most significant maternal risk factors associated with vertical transmission 
and or infant death 218-220. Directly disclosing a positive HIV sero-status to family members can have 
psychological and physiological health benefits 221. Hence disclosure of HIV sero-status by women to 
their sexual partners is critical for the success of the PMTCT programme as an integrated service in 
antenatal care. Pregnant HIV-negative women and their unborn babies remained at risk of HIV 
infection owing to the resistance of their partners to go for HIV testing 222. Poverty, lack of support, 
HIV, witchcraft and child illness have been identified as causes of worry in the perinatal period 223;224. 
Adverse maternal health and feotal outcomes have been shown to be more prevalent in socio-
economically-disadvantaged women 225. Depression and its impact on maternal and child health has 
important implications with depressed mood being associated with alcohol use, less breastfeeding and 
low birth weight 226;227. Smoking increases the risk of morbidity and mortality and is particularly 
harmful to pregnant mothers. Chronic maternal marijuana use has been associated with early sex in 
offspring 228.Studies have shown that alcohol use and HIV sero-status of partner predict high-risk 
sexual behaviour among patients receiving antiretroviral therapy 229;230.Alcohol-exposed pregnancy is 
a significant public health problem as alcohol consumption is associated with feotal abnormalities and 
long-term cognitive problems depending on the amount consumed, drinking pattern, and time of 
gestation. Feotal alcohol exposure is a leading cause of developmental disabilities and mental 
retardation 231. Prenatal cocaine exposure has been linked to child behaviour problems228. Screening 
for alcohol use disorders and substance uses are important to reduce alcohol consumption during 
pregnancy and associated problems in infants. Intimate partner violence defined as actual or 
threatened physical, sexual, psychological and emotional abuse by current or former partners is a 
global public health concern 232;233.Hence there is need to manage health related stress. There is 
significant current interest in the degree to which prenatal exposures to stress influence infant 
outcomes. Such maternal psycho-socio and demographic factors also play a role in infant mortality 
and necessary interventions for common perinatal mental disorders may be required.  

18 
 
Version 2, 3 December 2015 
 
 
 
There is paucity of information regarding the impact of environmental factors related to being born in 
an HIV-affected household especially of low social economic status and how this impacts on child 
care practices. Limited data exists which explores the possible associations between the mothers’ 
psychosocial and economic wellbeing during the prenatal and postnatal period and increased 
morbidity and mortality among HEU infants in the first year of life. It is against this background that 
in addition to investigating the abnormal immunity of HEU and the associated maternal genetic and 
environmental factors. For the holistic and integrative research, numerous other related sub-studies 
addressing other coinfections, NCD and maternal psycho social aspects will be developed for a full 
assessment of the common comorbidities. Each of these comorbidities in isolation and/or combination 
may be assigned characteristic immune biomarkers assayed from one blood plasma sample and such a 
development may lead to powerful tool to optimize and individualize treatment and care. Other sub 
studies linked to this study also seeking to determine the maternal psychological wellbeing such as 
stress and depression including other important co-infections such as Tb, herpes simplex virus (HSV), 
human papilloma virus (HPV), bacterial vaginosis, (BV) infections gut and vaginal microbiomes 
associated with high morbidity and mortality among HEU infants will be developed. In general, 
research questions will fall into seven thematic areas namely, co-infections, non-communicable 
diseases, nutrition, immunogenetics, pharmacokinetics/dynamics/genomics, psychosocial and child 
growth and development. See Appendix 6.1.  
1.12 
Justification 
Viral, host immunogenetics and environmental factors associated with HIV non-acquisition and/or 
slow HIV disease progression among paediatric patients remain poorly described. The little that is 
currently known comes from HIV-1 subtype B studies. The role of innate immunity in protection 
against HIV infection and disease progression may not be over-emphasised among paediatric HIV 
infected individuals whose adaptive immunity may not have fully developed. Since HIV-infected 
children progress more rapidly than adults and have fewer background cofactors such as drug use and 
co-infections and the effects of host genetic factors on HIV-1 disease may be more clearly identified. 
Most current researches are done in “silos” (focused on single, isolated aspects of immune defense) 
downplaying systemic and interconnected nature of the arms of the immune system. Yet in real life 
situation disease acquisition, progression and even response to therapy are multi factorial with 
environmental and immunogenetic factors playing critical roles. There is need for a cohort to clearly 
discern the causes or/ and consequences of viral immune profiles which is impossible to determine 
with cross sectional studies. HEU and non-progressing HEI children offer hope that natural and 
effective HIV control is possible and can provide important insight to the development of the next 
generation of HIV/AIDS vaccines and immune-based therapies. There is need for a comprehensive, 

19 
 
Version 2, 3 December 2015 
 
integrative and a holistic approach to predict diseases outcomes.HIV infection and co-infections, 
immune profiles, nutritional status that may influence transmission, disease progression and immune 
development and these will be assessed from baseline.  
2.0 
Research Questions 
1. Is the increased morbidity and mortality of HEU infants associated with increased exposure 
to maternal HIV load, other maternal co-infections (HBV, HCV, CMV, intestinal parasites) 
and how do these affect pregnant outcome and child growth and development? 
 
2. Is the incidence of comorbidities, anaemia, diabetes, malnutrition and hypertensive 
disorders similar between HIV positive and HIV negative pregnant women and how do 
these affect pregnant outcome and child growth and development? 
 
3. Is there a difference in biomarkers of inflammation and immune activation among mothers 
(HIV+/HIV-) and their (HUU/HEU/HEI) infants with different comorbidities? 
2.1 
Main Hypotheses 
1. There is no difference in the prevalence and incidence of co-morbidities (anaemia, 
malnutrition, hypertension, diabetes, HCV, HBV, CMV) and immune profile of HIV+ and 
HIV- pregnant women and the impact of these comorbidities on pregnancy outcome and 
infant growth and development within 24 months post-delivery is similar. 
2. There is no difference in biomarkers of inflammation and immune activation profiles 
among HIV+ and HIV- mothers with different comorbidities and their HEI, HUU HEU 
infants. 
3.0 Project Goals: 
1. The main goal of this study is to characterise co-morbidities, immunogenetics of pregnant 
women and their infants in Harare and ascertain their roles in disease acquisition and 
transmission 
2. Contribute to the knowledge base on the role of HIV and human immunogenetics on 
acquisition and disease progression and co-infections in children. 
3.1 
Main Objective 
This study aims to establish a birth cohort to investigate the role of HIV and comorbidities on 
vertical HIV transmission, immune development and disease progression in children 0-2 years, 
HEU, HEI and HUU as the control group. 
 
Objectives 
A. All Mothers Regardless of HIV Status 

20 
 
Version 2, 3 December 2015 
 
1. To determine the prevalence and incidence of coinfections (intestinal parasitic infections, 
HBV, HCV, CMV) malnutrition and non-communicable diseases (NCDs) (gestational 
diabetes mellitus, hypertensive disorders and CVD) risk factors over 24 months and relate all 
these comorbidities to: 
a. 
Pregnancy outcome, infant growth, development, morbidity and mortality 
b. 
Biomarkers of inflammation and immune activations 
Infants 
2. To determine vertically transmitted infections and incidence non-communicable diseases 
among infants. 
3. To determine clinical and immunological profiles of HEI, HEU and HUU infants. 
B. 
HIV+ Mothers: Chronic HIV infections 
1. To determine vertical transmission rates of mothers with acute and chronic HIV infection on 
option B+ 
2. Determine any association(s) between different option B+ drug regimens, adverse pregnancy 
outcome, viral load suppression and CD4/CD8 counts on mothers and their infants 
C. HIV+ Mothers; Acute HIV infections,  
1. To determine the incidence of HIV in the postpartum period over the 2 years 
2. To determine the viral set points, HIV suppression trends and biomarkers of immune 
activation 
3. To determine the HIV and host genetic diversity and possible transmission linkages 
Infants 
To characterise natural killer cell phenotype and function longitudinally from cord blood at birth 
up to 2 years and relate to infant HIV acquisition , morbidity and mortality 
4.0 Methodology 
 
4.1 Study Design 
A prospective cohort study of 1200 pregnant women (600 HIV positive and 600 HIV negative). 
Figure 1 below is the study flow diagram. 

21 
 
Version 2, 3 December 2015 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Coinfections; HCV, HBV, CMV, intestinal parasitic infections 
 
Viral load re-testing with be done for all Unsuppressed HIV load every 3 months 
Figure 1: Study Flow Diagram 
Key 
*Pregnancy outcome include spontaneous abortions, birth defects, multiple births, low birth weights preterm infants, birth 
with prolonged hospitalization, birth requiring neonatal intensive care, maternal, perinatal and infant deaths. 
**Coinfections include HBV, HCV, CMV and intestinal parasites 
***NCD, non-communicable diseases (hypertensive disorders, malnutrition, diabetes) 
**** For acute HIV infections ONLY 
*****Adverse feotal outcomes include preterm birth (<37 weeks of gestation) low Apgar score<7, low birth weight, Fetal 
macrosomia, birth weight of more than 4,000 grams, birth defects, stillbirth (death of a fetus weighing ≥500g or ≥22 weeks 
gestation if weight is unavailable), early neonatal death (death between 0-6 days of life), neonatal death (death within the 
first 28 days of life), neonatal jaundice, infant death, intensive care unit admission, hospital admissions including 
documented local clinic treatments 
 
 
1200 eligible pregnant women 
Demographics, physical examinations, history of STIs, assessment of nutritional status, co-
infections**, NCDs***. 
600 HIV+ chronic HIV infection with or without 
HAART, duration type of, stigma assessment, Viral 
load, CD4/CD8 biomarkers of immune activation. 
Prevalence and incidence of coinfections** and 
NCD***
600 HIV- Immune activation profiles, HIV 
incidence, Prevalence and incidence of 
coinfections and NCD 
Pregnancy outcome*, Vertical transmission of co-
infections  
Pregnancy outcome*, vertical transmission of co-
infections 
± 28 HEI infants 
± 60 Acute HIV infections 
Incidence of acute HIV sero 
conversion in breastfeeding vertical 
transmission rate and linkages, 
diversity****
±540HUU 
infants 
± 572 HEU infants, 
NCDs acquisition, 
Viral load, CD4%, NCDs 
acquisition, immune activation, 
neurodevelopment assessment 
NCDs acquisition, 
immune activation, 
neurodevelopment 
assessment 
NCDs acquisition, immune 
activation, neurodevelopment 
assessment 
 
HEI, ± 12 
NK cells profiling, 
host/viral genetic, NK 
cell profiling, gut/ 
vaginal microbiome 
characterisation plus 
12 HEU and 12 HUU 
matched controls
HEU, 
± 48 
NK cells 
profiling, immune 
profiles
Incidence vertical transmission of co-infections & NCDs, Infant and Maternal morbidity and mortality,  
Child (neuro) development, adverse feotal outcomes***** 

22 
 
Version 2, 3 December 2015 
 
4.2 
Study Sites 
City of Harare poly-clinics, that offer maternal and child health services. 
4.3 
Study Participants 
ANC mothers registering at the ante-natal clinicsat 28-36 weeks gestation will be enrolled. Upon 
delivery, babies will be automatically recruited into the study and be followed up as mother-baby 
pairs for two years. 
4.4 
Inclusion Criteria 
 
Pregnant woman 18 years of age and above at 28-36 weeks gestation. The date of the last 
menstrual period (LMP) will be used to estimate gestational age. 
 
Planning to deliver at the study sites 
 
Able to give informed consent 
o Willing to be followed together with her baby from delivery up to two years 
o Willing to provide the required specimens 
4.5 
Exclusion Criteria 
Women with severe obstetric complications and serious psychiatric disorders will be excluded. 
4.6 
Selection of the Participants 
Potential participants will be identified during routine ante-natal care (ANC) visits at the City of 
Harare Polyclinics. The potential participants will be briefed about the study and those who verbally 
agree to participate in the study will be given the participant information sheet to allow voluntary 
participation and informed decision. All mothers with a positive HIV status will be encouraged to 
enrol in the study. HIV negative mothers will be recruited systematically.  
4.7 
Sampling Procedure 
The whole population of HIV+ mothers and systematic sampling of HIV- mothers will done. For very 
HIV positive pregnant mother, the 10th HIV negative mother will be recruited, taking cognisance of 
the 12% HIV prevalence in this population. 
4.8 
Mothers’ Procedures 
 
A questionnaire will be administered at enrolment from which information regarding their socio-
demographics, sexual behaviour and reproductive health issues including obstetrical records will 
be obtained. Antiretroviral use and regimen will be recorded for all the HIV+ mothers. 
Assessment of nutritional status/BMI will be done using standard anthropometric indices. A 
physical examination will be done at every visit by the study clinician. Mothers will be enrolled at 
28-36 weeks gestation and followed up at delivery, 10 days and 6, 10 , 14, 24, 36, 48, 72 and 96 
weeks postpartum thus totaling 11 visits during the study period. Data can still be collected in 
cases of missed visits provided the participant turns up within the seven day window period of the 
scheduled visits. The participant may be asked to discontinue if she consecutively fails to turn up 
for at least two successive scheduled study visits without any reason(s).  
 
Biological specimens blood, cord blood, amniotic fluid, breast milk urine and stool will be collected 
at different visit with the respective quantities and purposes as shown in the Table 1 below.  

23 
 
Version 2, 3 December 2015 
 
 
Table 1: Types and total Volumes of Maternal Specimen Required 
 
Total Volume of Specimen Required 
Visit 
HIV 
Status 
Blood* 
(mL) 
Stool** 
(grams) 
Breast 
Milk* 
(mL) 
Urine *** 
(mL) 
Amniotic 
Fluid (mL) 
Cord Blood 
(mL) 
1 
In Pregnancy 
HIV+ 
30 
20 
20 
- 
100 
100 
50 
50 
 
HIV- 
25 
2 
Delivery 
HIV+ 
10 
20 
20 
 
100 
100 
50 
50 
50 
50 
HIV- 
5 
3 
7-10 days pp 
HIV+ 
- 
If delivery 
visit is 
missed 
some of the 
samples are 
collected.  
10 
10 
100 
100 
 
 
 
HIV- 
- 
4 
6 Weeks/1½Months pp 
HIV+ 
20 
20 
20 
10 
10 
100 
100 
 
50 
 
HIV- 
10 
5 
10 Weeks /2½ Months 
pp 
HIV+ 
20 
20 
20 
10 
10 
100 
100 
 
 
HIV- 
10 
6 
14 Weeks, 3½Months 
pp 
HIV+ 
20 
20 
20 
10 
10 
100 
100 
 
 
HIV- 
10 
7 
24 Weeks/6 Months pp 
HIV+ 
20 
20 
20 
10 
10 
100 
100 
 
 
HIV- 
10 
8 
36 Weeks/9 Months pp 
HIV+ 
20 
20 
20 
10 
10 
100 
100 
 
 
HIV- 
10 
9 
48 Weeks/1 year 
pp 
HIV+ 
20 
20 
20 
10 
10 
100 
100 
 
 
HIV- 
10 
10 
72Weeks /11/2 years 
HIV+ 
20 
20 
20 
10 
10 
100 
100 
 
 
HIV- 
10 
11 
96 Weeks/2 years 
HIV+ 
20 
20 
20 
10 
10 
100 
100 
 
 
HIV- 
10 
*Generally blood samples, amniotic fluid cord blood and breast milk will be tested for trend in immune profiles and coinfections. 
**Stool samples will be tested for intestinal parasites 
*** 
Urine 
for 
urinalysis

24 
 
Version 2, 3 December 2015 
 
 
Table 2: Type, Volume, intervals of collection and Purpose of Maternal Specimens Required 
Procedure 
/Test 
Specimen  
Required 
(volume) 
1 
Volume/gra
m 
2 
labour 
3 
7-10 Day 
pp 
4 
6 wks  
pp 
5 
10 wks  
pp 
6 
14 wks 
 pp 
7 
24 wks 
 pp 
8 
36 wks 
 pp 
9 
48 wks  
pp 
10 
72 wks 
 pp 
11 
96 wks  
pp 
CMV / 
HBV/ HCV 
serum(1 mL) 
 ( 
 
 
 
 
 
 
 
 
 
 
 
 
HIV testing/all 
negatives  
Plasma (1 mL) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
STI serology 
 
 
 
 
 
 
 
 
 
 
 
 
Parasites 
At least 3 specimens 
Stool(10 g) 
Anal swab 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
FBC/Differentials/pla
telets  
Plasma (5mL) 
 
 
 
 
 
 
 
 
 
 
 
Markers of immune 
activation, cytokines 
levels and trends 
Blood (10mL) 
 
 
 
 
 
 
 
 
 
 
 
Cord 
blood 
(10mL) 
 
 
 
 
 
 
 
 
 
 
 
Amniotic fluid 
(10mL) 
 
 
 
 
 
 
 
 
 
 
 
Breast milk(10 
mL) 
 
 
 
 
 
 
 
 
 
 
 
Urine  
urinalysis 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Biochemistry 
Panel 
Plasma (5 mL) 
 
 
 
 
 
 
 
 
 
 
 
Sugar (3 mL) 
 
 
 
 
 
 
 
 
 
 
 
Restriction factors 
Cord blood 
 
 
 
 
 
 
 
 
 
 
 
Breast milk 
 
 
 
 
 
 
 
 
 
 
 
FOR HIV POSITIVES ONLY 
T cell profile 
PBMCs 
 
 
 
 
 
 
 
 
 
 
 
HIV RNA load* 
Whole blood 
* 
 
 
 
 
 
 
 
 
 
 
HLA/KIR host 
genetics 
 
 
 
 
 
 
 
 
 
 
 
 
HIV genetic diversity 
for 
Transmission 
linkages** 
Whole blood 
 
 
 
 
 
 
 
 
 
 
√ 
*Re-testing with be done for all Unsuppressed HIV load every 3 months 
** for all acute HIV infections 
 
4.9 
Infants Procedures 
From delivery infections, immunological profiles, co-infections patterns and trends, mortality  will be 
determined and , feeding practices development , responses to standard vaccines, general health will 
be assessed. Types of specimen required and the test to be assayed are shown in Tables 3 and 4. 
 
Table 3: Types and total Volumes of Infant Specimen Required 
 
 
Specimen Required 
Visits 
Baby Status 
Blood* 
Stool** 
Urine*** 
PBMCs**** 
 
1  
 
HIV+ 
 
 
 
 
 
HIV- 
 
2 
HIV+ 
 
 
 
 
 
HIV- 
 
3 
HIV+ 
 
 
 
 
 
HIV- 
 
4 
HIV+ 
 
 
 
 
 
HIV- 
 
5 
HIV+ 
 
 
 
 
 
HIV- 
 
6 
HIV+ 
 
 
 
 
 
HIV- 
 
7 
HIV+ 
 
 
 
 
 
HIV- 
 
8 
HIV+ 
 
 
 
 
 
HIV- 
 
9 
HIV+ 
 
 
 
 
 
HIV- 
 
10 
HIV+ 
 
 
 
 
 
HIV- 
 
11 
HIV+ 
 
 
 
 
 
HIV- 
 
*Generally blood samples will be tested for trend in immune profiles and coinfections. 
**Stool samples will be tested for intestinal parasites 
*** Urine for urinalysis 
****PBMC of infants born from mothers with acute HIV infection for NK cell phenotyping 

25 
 
Version 2, 3 December 2015 
 
 
Table 4: Type, Volume, intervals of collection and Purpose of Infant Specimens Required 
Procedure 
/Test 
Specimen  
Required 
(volume) 
1 
Delivery 
2 
7-10 Day 
pp 
3 
6 wks  
pp 
4 
10 wks  
pp 
5 
14 wks 
 pp 
6 
24 wks 
 pp 
7 
36 wks 
 pp 
8 
48 wks  
pp 
9 
72 wks 
 pp 
10 
96 wks  
pp 
CMV / 
HBV/ HCV 
serum(1 mL) 
 ( 
 
 
 
 
 
 
 
 
 
 
 
 
Serum (1 m)L 
- 
 
 
 
 
 
 
 
 
 
 
 
 
HIV testing/all 
exposed infants 
Plasma (2 mL) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Parasites 
Stool 
(5 
grams) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
FBC/Differentials 
 
 
 
 
 
 
 
 
 
 
 
Markers of immune 
activation, cytokines 
levels and trends 
Blood,  
 
 
 
 
 
 
 
 
 
 
 
Urine dip stick  
Urine (5 ml) 
 
 
 
 
 
 
 
 
 
 
 
 
 
Biochemistry 
ALT, 
AST, 
albumin,  
 
 
 
 
 
 
 
 
 
 
 
Sugar 
 
 
 
 
 
 
 
 
 
 
PMBC for infants born from Mothers with acute HIV infections 
HEI Infants only on First HIV positive sample, thereafter every 3 months 
T cell profile 
Whole blood 
 
 
 
 
 
 
 
 
 
 
 
 
 
HIV RNA load* 
Whole blood 
 
 
 
 
 
 
 
 
 
 
HIV genetic diversity 
for 
Transmission 
linkages** 
Whole blood 
 
 
 
 
 
 
 
 
 
√ 
All the tests will be done locally except for transmission linkages assay and NK cell phenotyping that will be done in South Africa. 
 
4.10 
Laboratory Methods 
4.10.1 
Sample Preparation and Storage 
On the day of collection, clotted samples will be centrifuged for 5 minutes at 3000rpm. The 
supernatant will then be removed and placed in appropriately labelled serum cryo-tubes. All samples 
will be stored in the Department of Medical Laboratory Science at -70°C to -80°C for subsequent 
analysis.Full blood count, renal function, thyroid function, lipid profile, glucose levels, bone profile, 
and liver profile will be done at baseline, 6 weeks and thereafter every 6 months 
 
4.10.2 
Determination of Full Blood Count (FBC) 
FBC report of white blood counts (WBC) and differentialswill give an idea about the presence of 
anaemia, infection, inflammation or blood disorders. Peripheral blood for FBC analysis will be sent to 
the laboratory in an EDTA tube and analysed using a Mindray Haematology Analyser, BC3600within 
six hours of collection. Results will be grouped into white and red cell parameters including platelets 
 
4.10.2.1 White Blood Cells Parameters 
The FBC provides a total white cell count (WCC x 109)/white blood cell count (WBC) and an 
automated differential WCC which typically includes information the absolute and % of each type of 
white cell about neutrophils, lymphocytes, monocytes, eosinophils and basophils. 
4.10.2.2 Red Blood Cells Parameters 
Iron deficiency anaemia will determined according to WHO guidelines where haemoglobin (Hb) level 
below 11gm/dl in pregnant women constitutes anaemia with haemoglobin below 7gm/dl indicating 
severe anaemia. Iron deficiency anaemia is suspected if the red cells show a microcytic hypochromic 
classification. An mean corpuscular volume (MCV) between 80 and 97 fL is normocytic, above 97 fl 

26 
 
Version 2, 3 December 2015 
 
is macrocytic and below 80 fl is microcytic. An mean corpuscular haemoglobin concentration 
(MCHC) between 31 and 34% is normochromic, above 36% is hyperchromic and below 31%, it is 
hypochromic. A diagnostic postpartum Hb level 10.0 g/dLwill be used. 
4.10.2.3 Platelet Counts 
Gestational thrombocytopenia will be defined as a platelet count of less than 150 x 10 3 µL in 
hypertensive pregnant women during the third trimester. Platelet count of <100 000 µL will be an 
indicator ofincreased chance of pre-eclampsia in hypertensive pregnant women during the third 
trimester. 
4.10.3 
Biochemistry Determination 
4.10.3.1 Blood Glucose Determination 
Blood will be collected in grey topped tubes (potassium oxalate and sodium fluoride) for HbA1C 
analysis and random blood glucose levels. A cutoff value of 6.5% will identify undiagnosed diabetes 
whilst individuals with A1C ≥5.6% will be assumed to have an increased risk for future diabetes 
according to the Glycohemoglobin standardization guidelines. The glucose levels will be analysed on 
Mindray BS200 Chemistry Analyser. All the blood tests will be conducted at Immuno-Path 
Diagnostic Laboratory Services. 
4.10.3.2 Kidney Function Profiles 
Urea and electrolytes, essentially sodium and potassium including lactate dehydrogenase, will be done 
on mothers’ and infants’ serum or plasma samples baseline, 10 days, six weeks and every 6 months 
postpartum. 
 
4.10.3.3 Liver function Profiles 
Albumin, bilirubin, hepatic enzymes; alanine transaminase (ALT), specific to the liver and aspartate 
transaminase more widely distributed are markers of damage to cells. Alkaline phosphatase (ALP) 
and gamma glutamyl transpeptidase (GGT) are markers of cholestasis. 
 
4.10.3.4 Lipid profiles  
Risk factors of CVDs of borderline and high risk ranges for cholesterol, triglycerides, high-density 
lipoprotein cholesterol (HDLc), low-density lipoprotein cholesterol (LDLc) cholesterol and 
cholesterol/HDL ratio at baseline will be determined using Mindray BS120 automated analyserat 
baseline, 10 days, six weeks and every 6 months postpartum. 
 
4.10.3.5 Bone Profiles  
Longitudinal changes in ionised calcium, phosphorus, albumin and alkaline phosphatase levels in 
pregnancy (baseline), lactation (10 days pp, six weeks, 6 months) and weaning (earliest visit then after 
6 months).Serum parathyroid hormone (PTH) including serum 1,25 (OH)2 Vitamin D level will be 

27 
 
Version 2, 3 December 2015 
 
determined at baseline, 6 weeks, 6, 12 and 24 months postpartum. <25 nmol/L will be the cut off 
value for 25-OH Vitamin D deficient. 
 
4.10.4 
Determination of Serum prolactin Levels 
Serum prolactin levels in pregnancy, 7-10 day pp, 6 weeks and thereafter 6 monthly will be 
determined using Sandwich (quantitative) AbcamR. Prolactin Human ELISA Kit (ab108655) 
according to the manufacturer’s instruction. The serum concentration of prolactin will be given in 
ng/mL which will be correlated with the reported breastfeeding patterns and frequencies. 
 
4.10.5 
Determination of Coinfections 
HBs Ag/anti-HB/anti-HCV/anti-CMV will be screened at enrolment and at 24 month postpartum for 
the mothers using qualitative rapid immunochromatographic assays. These will coinfections will be 
tested in the peripheral blood, cord blood including amniotic fluid. Stool specimen will be examined 
for intestinal parasitic infections at the age of 7-10 days and six weeks old for the infants. 
4.10.6 
Determination of Intestinal Parasites 
10 grams stool sample will be collected at enrolment, delivery six weeks and every six months for 
helminths and protozoa diagnosis. Sample will be processed with 24 hours into smears, stained 
appropriately and then examined under the light microscopy. 
 
4.10.6.1 
Helminths  
Cestodes (tapeworms) Taenia, Diphyllobothrium, Hymenolepis, Dipylidium, Echinococcus, and 
Spirometra be diagnosed by collecting at least three stool samples and checking for ova and 
parasites.Enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR) assay 
may help confirm a diagnosis; depending on the species involved. Enterobius vermicularis associated 
anal pruritus will be made through microscopic identification of ova and female pinworms from 
perianal swabs. Round worm; Ascaris lumbricoides diagnosis will be made by examination of stool 
ova and parasite under the microscopy. Hookworms, Ancylostoma duodenale and Necator americanus 
diagnosis will be made by means of stool ova and parasite examination. Intestinal trematodes/Flukes, 
Fasciolopsis buski, Heterophyes heterophyes, Metagonimus yokogawai, and Echinostoma diagnosis is 
will be made by means of stool ova and parasite examination. Microsporidia ofEncephalitozoon 
hellem and E intestinalis species diagnosis will be made via stool microscopy. Schistosoma ova will 
also be examined including the determination of the serum circulating antigens 
 
4.10.6.2 
 Protozoa 
Balantidium coli, Dientamoeba fragilis, Giardia lamblia, Entamoeba histolytica, Cryptosporidium 
including Cystoisospora belli diagnosis will be made on at least 3 different smears of stool specimens 

28 
 
Version 2, 3 December 2015 
 
and will be examined for trophozoites, cysts or oocytes on a microscopy and complemented by 
antigen detection with ELISA and or PCR. 
4.10.6.3 Infants stool 
Infant stool collected from delivery will be diagnosed for E. coli and Cryptosporidium infections. 
 
4.10.7 
Determination of soluble biomarkers of inflammation and immune activation  
Several biomarker profiles  association with different comorbidities may become a power tool for 
individualised treatment and care. 
4.10.7.1 Markers of inflammation 
Hs CRP including inflammatory cytokinesIL-6, IL-10 and IL-17 will be determined. 
 
4.10.7.2 Markers of immune activation 
Plasma INFγ and interferon gamma inducing protein 10 (IP 10) (CXCL10) chemokine whilst in 
breastmilk RANTES (CCL5) and MIP-1β (CCL4) chemokine will be assayed. For mothers with acute 
HIV infection HβD-2 ,IL-10, IL-12, IL-15, IL-17, IL12p70 determination from cervico-vaginal lavage 
and plasma IL-7. 
 
4.10.7.3 Markers of Microbial translocation in coinfections 
Lipopolysaccharide, soluble (s)CD14, endogenous endotoxin core antibody (EndoCAb) and bacterial 
16S DNA complemented by marker of enterocyte damage, intestinal fatty acid binding protein (I-
FABP) will be determined. 
 
4.10.7.4 Markers of Monocyte and macrophage activation 
SCD14, sCD163, sCD40L and IL6 will be determined. Markers of tissue fibrosis and coagulation  
associated with CVD related comorbidities as fibrinogen, D-dimer, a fibrin degradation product 
associated with endothelial dysfunction and hyaluronic acid biomarker of tissue fibrosis will be 
determined. 
 
4.10.7.5 Cytokine level Determination, Flow Cytometry Method 
IL-2,4,6 10, TNF IFN and IL17A will be determined in a single sample (50 µL) using the Becton, 
Dickinson (B cytometric Bead Array (CBA) human Th1/Th2/Th17 cytokine kit, catalogue number 
560484 in either EDTA plasma or serum according to the manufacturer’s instructions. 
4.10.8 
HIV Diagnosis 
Qualitative rapid immunochromatographic assays, SD Bioline HIV-1/2 3.0 (Standard Diagnostics 
Inc., Kyonggi-do, South Korea) and Abbott’sDetermine®HIV-1/2will be used to detect HIV-1/-2 

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Version 2, 3 December 2015 
 
antibodies in mothers venipuncture whole blood specimens. Western blot test will be used as a tie 
breaker for any indeterminate test results. 
4.10.9 
HIV-1 RNA Load Determination 
Maternal and infant plasma samples were quantified for HIV-1 RNA load using an automated 
TaqMan Roche Amplicor 1.5 Monitor Test (Cobas AmpliPrep/Cobas TaqMan, Roche Diagnostics, 
Branchburg NJ), according to the manufacturer’s instructions. The detection level is 40 copies/mL. 
4.10.10 
Infants’ Qualitative HIV-1 DNA PCR Test 
Detection of infants’ HIV-1 infection will be determined using a qualitative 1.5 Roche Amplicor HIV-
1 DNA PCR kit (Roche Diagnostics Incorporation, Branchburg, New Jersey).This qualitative HIV-1 
proviral DNA PCR tests will be done on all HIV exposed infants ’plasma samples/from birth up to 15 
months past partum. 
 
4.10.11 
CD4 cell counts enumeration 
Absolute and percentage CD4+ T lymphocytes will be enumerated for all HIV positive mothers and 
infants using a Partec Cyflow counter (Cyflow, Partec, Munster, Germany) within 6 hours of blood 
collection.. 
4.10.12 
Viral load Determination 
Maternal and infant plasma viral load will be determined using automated Roche AmpliPrep COBAS 
AMPLICOR HIV-1 Monitor (Version 1.5) Ultrasensitive Assay according to the manufacture’s 
instruction. All unsuppressed viral load based on a threshold of ≥ 1000 copies/mL will be re-run after 
3 months. 
4.10.13 Markers of immune activation and senescence at enrolment and exit whole blood sample 
Determine maternal activation markers, profiling by flow cytometry.CD38 on both CD4+ and CD8+ T 
cells. Senescence marker: CD28-CD57+ will be determined. 
4.10.14 
NK cell phenotypic and functional analysis 
Phenotype (CD56+ dim and bright) and function on PBMCs. 
4.10.15 
Maternal MUAC Measurements 
MUAC will be used as an indicator or predictor of nutritional outcomes. The subject’s left arm should 
be bent at the elbow at a 90 degree angle, with the upper arm held parallel to the side of the body. 
Measure the mid-point distance between the bony protrusion on the shoulder and the point of the 
elbow will be measured. Maternal MUAC cutoffs of <22 cm to ≤24 cm relative to the median of the 
sample will be used with the following assumption that; 
1. If MUAC is <23.5 cm, BMI is likely to be <20 kg/m2 
2. If MUAC is >32.0 cm, BMI is likely to be >30 kg/ m2 
 

30 
 
Version 2, 3 December 2015 
 
4.10.15.1 Infant Measurements 
Among children 6-60 months old assessment for acute malnutrition will be done using the 2009, 
WHO and UNICEF recommendation of a MUAC cut off value of <11.5 cm  
 
4.10.16 
Determination of Hypertensive Disorders 
Any one of the following will be enough to make a diagnosis of hypertension in pregnancy; 
4.10.16.1 Chronic hypertension 
 
Preexisting hypertension: A woman diagnosed of hypertension either before she fell pregnant 
who may be already on treatment or first diagnosed after 20 weeks gestation and persisting after 
12 weeks postpartum 
 
A woman with a blood pressure greater than or equal to 140/90mmHg on at least 2 separate 
occasions 4hours apart, anytime during the pregnancy. An Obstetrician will diagnose the 
following complications: 
4.10.16.2 Increased chance of pre-eclampsia 
BP ≥ 160/110mmHg, Proteinuria ≥ 2g/24 hrs, ≥ 2+ dipstick and platelets < 100 000 µL, urine protein 
(mg/dL)/creatinine (mg/dL) ratio ≥0.3, -elevated serum lactate dehydrogenase (LDH) level and 
activity, elevated serum transaminase levels-AST, ALT; persistence epigastric pain/headache/visual 
disturbances 
4.10.16.3 Eclampsia Seizures 
New onset of grand mal seizure activity or unexplained coma during pregnancy or postpartum period 
in a woman with hypertension in pregnancy or postpartum period. 
4.10.16.4 
HELLP syndrome 
1. Hemolysis is associated with the following: 
 
Total bilirubin >1.2 mg/dL  
 
LDH >600 U/L  
2. Elevated liver enzymes: 
 
Aspartase aminotransferase (AST) >70 U/L  
 
Alanine aminotransferase (ALT 
 
LDH >600 U/L  
3. A platelet count < 100,000/μL  
Sub-classification of HELLP based on the severity of thrombocytopenia: 
 
Class 1 - Platelet count >50,000/μL  
 
Class 2 - Platelet count 50,000-100,000/μL  
 
Class 3 - Platelet count 100,000-150,000/μL  
 
4.10.17 
Transmission linkages 
Mothers who seroconvert after enrolments will have host/viral genetics factors determined 
transmission linkages assessment. Peripheral blood mononuclear cells will be collected for these 
HUU, HEI and HEU infants for NK cell phenotype and functional analysis.  
 

31 
 
Version 2, 3 December 2015 
 
4.10.17.1 
Nucleic acid extraction and DNA Amplification 
Total RNA was extracted from plasma using the NucliSENS isolation kit, based on the Boom et al., 
method Briefly samples are lysed in a lysis buffer containing a chaotropic agent, guanidine 
thiocyanate. Cells, bacteria and viruses in the sample are lysed whilst proteins such as nucleases are 
denatured and inactivated. DNA and RNA bind to silica particles and everything else is washed 
following several washing steps with the wash buffer. Finally the nucleic acids are eluted from the 
silica particles using the elution buffer. Purified DNA will be stored at -80 for genotyping.Taq 
polymerase is a thermostable DNA polymerase named after the hot spring Thermusaquaticus 
bacterium. The primary PCR amplified an approximately 800-base pair (bp) fragment spanning the 
V3 and V4 region of the envelope (positions 6948–7537) on the HIV-HXB2 genome using HIV 
primers ENV 2 and NY3. Secondary or nested PCR amplified a 535-bp env gene fragment using a 
PCR Thermal cycler. Detection and quantification of secondary PCR amplicons were done using a 
1% agarose gel electrophoresed together with a standard mass ladder and then stained with SYBR 
safe stain.  
 
4.10.18 
Sample Size Determination 
Pocock’s formula was used to determine sample size (n) required: 
 
 
 
Where    
 and 
are proportion of success in population 1 and 2 
 
 and 
 are values of standard normal distribution 
 
α= Significance level 
 
= Power 
3% incidence rate has been assumed to factor in for the a high risk of vertical transmission in the 
breastfeeding period for the few anticipated acute HIV infection234.With the advent of cell phones the 
% loss to follow up may not be that bad and cannot be similar for the first and second year. Factored 
in the worst case scenario of a 15% and 20% loss to follow-up during the first and second year 
respectively, a minimum sample size required computed using STATA is 1200 (600 HIV+ and 600 
HIV-) mothers. Assuming a 15% and 20% loss to follow up in the first and second year of the study 
respectively, should leave us with at least the 417 infants. The sample size has power to predict 
diseases outcomes of HEU children which must culminate in their improved management. The 
sample size has not factored in sample sizes of sub-studies linked to it in the Appendices’ Section 
6.2. Should there be sub-studies, they should fit within this parent protocol. These aim to address all 
the research gaps highlight throughout the literature review. 
 
 
 

32 
 
Version 2, 3 December 2015 
 
Table 5: Pictorial Depiction of the Stratification 
 
a) 
HIV Chronic Infection 
b) HIV Acute 
Infection 
c) HIV Negative 
Outcomes 
Comorbidities 
Treatment 
experienced 
Treatment 
naive 
 
 
Prevalence and incidence of 
comorbidities, comorbidities trends, 
relating comorbidities to 
immunological profiles, pregnancy 
outcomes, vertical transmission of 
infections, patterns and 
transmission linkages, infant/child 
growth and neurodevelopment  
Immune Profiles for different comorbidities combinations 
HBV 
 
 
 
 
HCV 
 
 
 
 
CMV 
 
 
 
 
Anaemia 
 
 
 
 
Diabetes 
 
 
 
 
Nutritional Status 
 
 
 
 
Hypertensive 
disorders 
 
 
 
 
 
4.10.19 
Ethics Statement 
Ethical approval will be sought from the necessary institutional review boards, JREC and MRCZ. All 
study participants will give written informed consent. Permission for storage and future use of 
biological specimens, external shipping of specimens including genetic testing of blood samples will 
also be sought. This observational study involves minimal risks to both the mother and the baby hence 
one parental consent is sufficient. However, in case of death of the mother re-consenting will be done 
from either the father or any family member who shall have legal responsibility for the care and 
custody of the baby. In case of a child death, the mother will cease to continue participating in the 
study. 
Permission has already been obtained from Harare City Health.The sensitivity of HIV infection and 
sexuality means that the mother’s emotional response is a potential risk. Anxiety associated with 
sample testing and waiting for the results for HIV exposed children may also be experienced. 
Nevertheless the study will minimize such risks by providing the necessary education and counselling 
through the research nurses and where necessary, referring them to the appropriate clinics. Health 
education and counselling on pregnancy, delivery and childcare will be given to mothers on a 
continuous basis at every visit. The study will offer consultation and basic treatment to both the 
mother and child. Whenever necessary referrals will made through the clinic.  
Each sub-study will apply for their respective ethical approvals to all the relevant ethical boards 
before they can access the data and/or specimens. However, some sub-studies may require separate 
consent forms should they entail collection of additional data from study participants.  Those that rely 
on specimens and data collected through main study will need no additional informed consent.  
4.10.20 
Adverse Event Monitoring and Reporting 
All Severe Adverse Events (SAEs): prolonged hospitalisation, life threatening, congenital birth 
defects and significant disabilities including deaths will be reported to MRCZ and sponsor within 24 
hours of first becoming aware of it. Regular monitoring visits to the investigative sites will be 
conducted by UZ-CHS Research Support Centre (RSC) monitors in accordance with a site monitoring 

33 
 
Version 2, 3 December 2015 
 
plan which will be finalised by RSC before screening begins. The frequency and duration of visits 
will be sufficient to allow monitoring of: 
1. Compliance with the informed consent process and documentation 
2. Adequacy of maintenance of participant files 
3. Accuracy of transfer of data from participant trial files into the CRFs 
4. Compliance with the current version of the protocol 
5. Ongoing assumption by the PI of primary responsibility for conduct of the study 
6. Storage and handling of biological specimens. 
7. Compliance with all regulatory requirements 
 
4.10.21 
Data Management Plan 
Study data will be collected and managed using Research Electronic Data Capture(REDCap) tool 
hosted at the UZ RSC. Quality assurance on the accuracy of data entry will employed including 
independent double entries. Data analysis will be carried out using statistical packages such as; Stata 
version 13 and Statistical Package for the Social Sciences SPSS. Categorical data will be compared 
using chi squared test. Continuous variables will be compared between groups using analysis of 
variance (parametric) and sum rank (non-parametric) tests. Continuous variables will be correlated 
using regression models.  
 
4.10.22 
Data analysis 
The data were collected and analyzed using SPSS (version17.0, Chicago, IL). Viral load values were 
log10 transformed. The Student’s t-test was used to compare means. Regression analysis was used to 
investigate the associations Tests of statistical significance included the 95% confidence interval of 
unadjusted relative risks, two-sided p values based on Chi-square and Fisher’s exact tests. Sequences 
were assembled using the Vector NTI Advance 10 program. Alignment was attained using Gene Doc, 
BioEdit, and Clustal X2 sequence alignment programs including manual editing to ensure that 
deletions or insertions did not alter the reading frame.  
 
4.10.23 
Budget 
The baseline study will cost is 120 000 US dollars and enrolment is expected to start in July 2015 and 
the study will continue following children up to 24 months. The current budget should be sufficient 
for baseline surveys. However, additional funding for the study will be sourced as the study 
progresses. Please note that for sub-studies the respective MPhil or PhD students will bring or source 
own funding for to answer their specific research questions. Activities and task are shown in Table 6 
below. 
 

34 
 
Version 2, 3 December 2015 
 
Table 6: Tasks and Time Frames 
TASK 
WHEN 
WHO 
HOW LONG 
RESOURCES 
COST 
Get ethical approval 
Y1 m1-3 
PI 
3 months 
PI 
$700 
Communication 
Y1-2 
Coordinator/ driver 
24 months 
Mobile phone hand set x 2, air time, data 
bundles 
$1000 
Training 
Y1 M1 
Team 
2 weeks 
Teas/ lunches 
$1000 
Meetings 
Y1-2 
Team 
weekly 
Teas 
$ 1000 
Recruit patients 
Y1 m5-8 
Midwives,  
Coordinator 
3 months 
Stationery & copying 
$1000 
Participant 
transport 
reimbursement 
Y1-2 
Research Nurse 
24 months 
$5/visit x 12 visits 
$ 70 000 
Transport (fuel, vehicle 
hire/ services) 
Y1-2 
Driver 
24months 
 
$5000 
Sample 
preparation 
& 
storage 
Y1-2 
Team Members 
24 Months 
Tubes, pipettes, boxes, accessories, 
PMBCs , plasma for storage 
$5000 
Lab assays (local) 
Y1-2 
Team Members 
24 Months 
Kits, haematology, chemistry, urinalysis, lipid 
profile, T cell profiles, viral load 
20 000 
Data entry, 
Y1-2 
Biostatistician 
24 Months 
Laptops x 3, dongle, memory sticks. 
$ 2500 
Allowance 
Y 1 M 1-6 
PI 
6 months 
Research 
nurses, 
driver, 
secretary 
, 
coordinator and lab scientist, data clerks 
$15 000 
Conferences  
Y2 
PI/co-investigators 
 
Travel and subsistence, tickets 
$5 000 
Publications 
Y2 
Team Members 
 
Publication fees 
$ 2000 
Y: Year 
 
4.10.24 
Research Outcomes 
 
Characterisation of comorbidities of HIV negative and HIV positive mothers 
 
Immune activation profiles for different comorbidities 
 
HIV vertical transmission rate in the era of option B+ among mothers with chronic HIV infection 
 
HIV incidence and postpartum vertical transmission among mother with acute HIV infection 
 
Transmission linkages of acute HIV infection 
Research outcomes will entail improved knowledge of HEU infants immunological abnormalities and 
genetics resulting in their better clinical management. There will also be technology transfer in 

35 
 
Version 2, 3 December 2015 
 
molecular biology and genetics techniques through the establishment of research networks both 
locally and internationally. The project will generate a critical mass of local professions with diverse 
expertise as they attain higher degrees, MPhils and PhDs from sub-studies. At least ten (10) papers 
will be published from the parent study material. 
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