African Prospective Study for the Early Detection and Identification of Cardiovascular Disease and Hypertension (African-PREDICT Study)
Conditions: Cardiovascular Diseases, Hypertension, Health Risk Behaviors, Arterial Stiffness, Young, Renin Hypertension
Sponsor: North-West University, South Africa

 
i 
 
 
 
 
The African-PREDICT Study 
Study Protocol 
 
The African Prospective study on the Early Detection and 
Identification of Cardiovascular Disease and Hypertension 
 
 
 
 
 
 
 
Principal Investigator: Prof. AE Schutte 
Co-Principal Investigator: Prof CMC Mels 
Original Version: 13 August 2018 
Revised 31 May 2022 
 
 
 
 
 
 
 

 
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Table of Content 
 
1. 
Study Title ....................................................................................................................................... 1 
2. 
Executive Summary ......................................................................................................................... 1 
3. 
Problem Statement and Motivation ............................................................................................... 2 
4. 
Research Aims, Objectives and Hypotheses ................................................................................... 3 
4.1 
Aims......................................................................................................................................... 3 
4.2 
Objectives................................................................................................................................ 3 
4.3 
Hypotheses ............................................................................................................................. 4 
5. 
Research Design .............................................................................................................................. 4 
6. 
Target Population and Endpoints ................................................................................................... 5 
7. 
Sample Size and Power Calculations ............................................................................................... 6 
8. 
Human Research Participants ......................................................................................................... 8 
8.1 
Approval Health Research Ethics Committee (HREC) ............................................................. 8 
8.2 
Recruitment and Eligibility Criteria ....................................................................................... 10 
8.3 
Screening for Eligibility to Participate ................................................................................... 12 
8.4 
Informed Consent ................................................................................................................. 14 
9. 
Detailed Research Methodology................................................................................................... 15 
9.1 
Data Collection for the African-PREDICT study ..................................................................... 15 
9.2 
Questionnaire Data ............................................................................................................... 16 
9.3 
Cardiovascular Measurements ............................................................................................. 17 
9.4 
Body Composition and Physical Activity Assessments ......................................................... 19 
9.5 
Assessment of Target Organ Damage (TOD) ........................................................................ 19 
9.6 
Biological Sampling and Biochemical Measurements .......................................................... 22 
9.7 
Rationale/Justification for the Measurements ..................................................................... 25 
10. 
Statistical Analyses Plan ............................................................................................................ 27 
11. 
Data Handling and Management .............................................................................................. 29 
12. 
Timeframe ................................................................................................................................. 30 
13. 
Budget ....................................................................................................................................... 31 
14. 
Remuneration ........................................................................................................................... 32 
15. 
Data Dissemination Plan ........................................................................................................... 32 
16. 
Expected Outputs, Outcomes and Impact ................................................................................ 34 
16.1 
Expected Outcomes .............................................................................................................. 34 
16.2 
Expected Outputs .................................................................................................................. 35 

 
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16.3 
Impact: Improvement in health outcomes ........................................................................... 35 
17. 
Reference List ............................................................................................................................ 35 
 

 
1 
 
1. Study Title 
African PRospective study on the Early Detection and Identification of Cardiovascular Disease and 
HyperTension (African-PREDICT) 
2. Executive Summary 
Background and Problem Statement: Recent global analyses have indicated that the highest blood 
pressures worldwide are recorded in black populations. The vulnerable cardiovascular profile of 
Africans is believed to result from a combination of factors such as rapid urbanisation, abnormal 
sodium handling, elevated vascular resistance and arterial stiffness. The frequent underdiagnoses 
and ineffective treatment of hypertension in general but especially in Africans, result in severe 
complications, such as stroke, heart and kidney disease. Since diagnosis and treatment are generally 
unsuccessful in black populations – especially in low and middle-income countries – prevention is 
key to curb the rapidly increasing incidence of death and disability from cardiovascular disease. 
Cardiovascular risk prediction in black populations worldwide is inadequate since we do not have a 
clear understanding of the complex mechanisms underlying the development of cardiovascular 
disease. Main Aim: In the “African PRospective study on the Early Detection and Identification of 
Cardiovascular disease and Hypertension” (African-PREDICT) we aim to identify early markers or 
predictors for the development of cardiovascular disease in black South Africans. Only by 
understanding the early pathophysiology of disease development, and by identifying markers as 
potential screening indicators, predictors or targets for intervention, will we be able to implement 
successful prevention programmes in Africans at younger ages. We therefore aim to track and 
monitor change in young, normotensive black and white individuals (aged 20-30 years) over 10-20 
years. To achieve this we will perform detailed cardiovascular and novel biomarker measurements, 
as well as behavioural and biopsychosocial assessments every 4-5 years in order to identify and 
understand early changes in cardiovascular function, and specific predictors contributing to the 
development of hypertension and target organ damage. Methodology and Measurements: From 
2013-2017 we recruited, screened and assessed of 1202 apparently healthy participants (black 
N=606 and white N=596) with equal sex distribution. Five-year follow-up take place from 2018-2022. 
A wide range of basic and advanced measurements are taken within a Hypertension Clinic to get a 
highly detailed profile of the participants at each visit. We obtain: (1) relevant questionnaire data 
including medical history, lifestyle, social status, traditional risk factors (age, gender, smoking, 
alcohol intake) and validated questionnaires on dietary intake, personality and psychosocial profile; 
(2) Biological samples for biomarker analyses (serum, plasma, spot urine and 24-hr urine) are taken 
and preserved for the short and long-term at -80°C. We will assess a wide range of traditional and 
novel biomarkers related to hypertension and cardiovascular disease (including amongst others, lipid 
profile, glucose, glycated hemoglobin, C-reactive protein, interleukin-6, vitamin D, full blood count, 
sodium, potassium, creatinine, renin, aldosterone, angiotensin II, markers of oxidative stress and 
nitric oxide bio-availability, cortisol, sex hormones, insulin, C-peptide, leptin and other adipokines, 
angiogenic markers, the insulin-like growth factor-axis, soluble urokinase plasminogen activator 
receptor, Nt-proBNP, fibulin-1 and novel markers not yet identified). These samples will also be 
analysed in an attempt to identify bio-signatures in terms of the –omics sciences (genomic, 
metabolomic and proteomic profiles) as predictors of cardiovascular deterioration; (3) 
anthropometric measurements, bio-electrical impedance measurement of body fat and lean mass, 

 
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and 7-day physical activity monitoring; (4) A range of cardiovascular assessments: 24-hour blood 
pressure, central arterial pressure, and cardiovascular stress reactivity tests with continuous finger 
blood pressure; and (5) Assessments of early target organ damage including urinary albumin-to-
creatinine ratio, carotid intima-media thickness, ECG, echocardiography, pulse wave velocity, and 
retinal microvascular calibre and dilation during provocation with a light flicker test. Timetable: The 
project was approved and is endorsed by the National and Provincial Department of Health, and was 
approved by the Ethics Committee of the North-West University in 2012 (NWU-00001-12-A1). 
Screening commenced in November 2012, and research participants started entering the study from 
6 February 2013. The Health Research Ethics Committee approved continuation of the study on an 
annual basis.. Anticipated Outcomes: This project will increase our understanding of the complex 
mechanisms involved in the aetiology of early cardiovascular changes in relatively young individuals 
from African and European ancestry, which will (1) improve our ability to identify individuals at risk 
before the development of cardiovascular diseases (CVD), and (2) predict the development of future 
hypertension and related CVD. Both outcomes will make it possible to develop better individualised 
and population-based prevention of CVD contributing to better quality of life. These results will not 
only be applicable to Sub-Saharan Africa but are expected to have a broader impact with regards to 
white and black populations globally. Results are expected to have significant scientific impact by 
means of publications in high-impact journals; and also to translate directly into novel preventive 
healthcare policy and practices – translating into preventive measures regarding the development of 
CVDs in clinics throughout South Africa. 
3. Problem Statement and Motivation 
A recent systematic analysis undertaken in 5.4 million participants indicated that blood pressure (BP) 
has on average decreased worldwide since 1980. However, region-specific inspection of this data 
shows that in Africa, the mean SBP actually increased. In addition, the highest mean BPs recorded 
worldwide were in African countries.1 The WHO’s Study on Global Ageing and Adult Health including 
adults (aged > 50 yrs) in low and middle income countries, confirmed this by reporting that South 
Africa (SA) presented with the highest prevalence of hypertension ever reported in a nationally 
representative survey, with nearly 4 in 5 participants presenting with hypertension.2 
Unfortunately, current practices to treat hypertension in Africa are overwhelmingly ineffective, 
evidenced by appallingly low control rates: 7.8% in SA,2 4.1% in Ghana2 and 3.1% in Mozambique.3 
These failing practices are possibly due to poor health systems, education or current 
antihypertensive treatment not being as effective in black populations.4 Despite calls to increase 
awareness and education regarding hypertension in Africa, awareness does not translate into 
control, with awareness figures as low as 38% in South Africa,2 23% in Ghana2 and 14.8% in 
Mozambique.3  
Hypertension additionally enforces an immense economic burden upon SA. The SA Demographic 
and Health Survey5,6 states that from all participants taking chronic disease drugs, the most frequent 
condition being treated was hypertension. Some of the main consequences of hypertension are 
hypertensive heart disease and stroke. Apart from the additional economic burden of caring for 
stroke survivors, hypertension – especially in poverty-stricken communities – clearly results in lower 
quality of life. Due to the frequent underdiagnosis, the poor control, and the immense economic 
burden7,8 regarding the treatment of hypertension in Africa, the status quo cannot be maintained in 

 
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Africa concerning current practices to manage hypertension. It may have significantly greater impact 
if tailored population- and individual targeted prevention strategies are employed to early detect, 
prevent and delay hypertension onset. This should not only relieve the financial implications of 
treatment, but more importantly, dramatically improve the quality of life of black populations. 
South Africa, like several other developing countries, has been highlighted as experiencing a unique 
demographic moment to focus on introducing policies that will reduce the future impact of chronic 
disease, and to minimise the rise in CVD in particular.9 Hypertension is largely preventable. Less than 
100 years ago it was nearly impossible to find a single patient with hypertension in a hospital in 
Kenya.10 Today, the health behaviours consequential to urbanisation reveal the susceptibility of 
black populations to rapidly develop hypertension. We have demonstrated this in a black SA 
population in transition, where nearly 1 in 4 of black participants with optimal BP (≤120 and 80 
mmHg) developed hypertension over 5 years.11  
Among the reasons for the vulnerability of black individuals to develop hypertension are several 
social (socio-economic status, health disparities limiting access to care, experience of stress) and 
pathophysiological reasons (salt sensitivity, suppressed renin-angiotensin system, sympathetic nerve 
activity),12,13 but there is consensus amongst scientists that there is a severe lack of longitudinal data 
in Africa,14 and especially a lack of knowledge on the early pathophysiological development of 
hypertension of young black individuals.13 A better understanding of pathophysiological disease 
mechanisms involved in cardiovascular (CV) deterioration prior to hypertension development, will 
allow the identification of novel approaches to prevent and delay hypertension onset, and may 
identify new ethnic-specific targets for treatment. 
4. Research Aims, Objectives and Hypotheses 
4.1 
Aims 
The African-PREDICT study aims to 
(i) 
generate new knowledge on the early pathophysiology accompanying hypertension 
development in black South Africans; and 
(ii) 
to identify early novel markers or predictors for the development of hypertension and 
cardiovascular outcome. By employing also in Africa the latest cutting-edge scientific 
technologies to measure single and multiple biomarkers proven to predict hypertension 
and cardiovascular outcome (such as multiplex analyses, proteomics15-18 and 
metabolomics19-23), precision medicine may have the potential to lead to novel strategies 
in preventing and treating hypertension in Africa.  
4.2 
Objectives 
Short-term objectives (based on baseline data): To assess, compare and describe young, 
normotensive and apparently healthy black and white individuals in terms of 
a) traditional CV risk assessment techniques (office BP and conventional risk scores, such as 
age, sex, waist circumference); 
b) using advanced technologies to measure CV parameters of microvascular and macrovascular 
structure and function (retinal, brachial, carotid, aortic); 

 
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c) behavioural and biopsychosocial measures (tobacco, alcohol and dietary intake, 24-h sodium 
excretion, physical activity; body composition, personality and psychological well-being); 
d) conventional and novel biomarkers from biological samples (single, multiple biomarkers, 
including multiplex analyses and the – omics); 
e) subclinical organ damage (echocardiography, carotid intima-media thickness, arterial 
stiffness, renal function; retinal calibres and peak dilation); 
f) to integrate conventional and advanced biomarkers to establish and evaluate ethnic-specific 
risk scores. 
Medium to long term objectives: 
a) To track young black and white individuals (mean age 25 yrs) and perform similar detailed 
assessments (identical to baseline) after 5 and 10 years to identify both incident 
hypertension and early changes in cardiovascular structure and function; 
b) and whether specific predictors are apparent when these individuals were still in a 
normotensive and healthy condition – when compared to those who remained 
normotensive; 
c) to test and evaluate the additive predictive utility of novel biomarkers against existing low-
cost non-laboratory and laboratory-based risk scores; 
d) once detailed analyses are done with available data in the total sample, attempt to validate 
new findings in other African settings to determine the wider applicability and scalability of 
the findings. 
4.3 
Hypotheses 
a) At baseline young healthy black and white groups will exhibit similar brachial, central and 
24-h BP as well as level of end-organ functioning (retina, heart, arteries, kidneys); 
b) Health behaviours, and the biochemical biomarker profile (including markers of 
inflammation and nitric oxide (NO) bioavailability, as well as microvascular structure and 
function) will be more favourable in white than black participants; 
c) During follow-up, black individuals will develop higher 24-h BP and arterial stiffness at 
younger ages, accompanied by a more pro-arteriogenic (arterial stiffness) biochemical 
profile as reflected by an enhanced inflammatory state, altered NO metabolism, 
extracellular matrix alterations, attenuated glucose handling, but a favourable lipid profile 
compared to the white group; 
d) Biomarkers and biosignatures of the pro-arteriogenic biochemical profile (including 
biomarker and –omic profiles of increased elastin degradation, increased collagen 
production, endothelial dysfunction and inflammation) will be evident in black individuals 
and predict the development of hypertension, and associated end-organ damage. 
5. Research Design 
The African Prospective study on the Early Detection and Identification of Cardiovascular disease 
and Hypertension (African-PREDICT) employs a prospective study design to longitudinally 
characterise and monitor the early stages of hypertension development in 1200 young healthy black 
and white individuals (aged 20-30 years) over a follow-up period of 10 years. Healthy is defined here 
as brachial BP <140 and 90 mmHg; HIV uninfected; no previous diagnosis or medication for chronic 

 
5 
 
disease; not pregnant or breastfeeding. Our goal is to identify and understand early 
pathophysiological changes in CV function, and specific predictors contributing to the development 
of hypertension and target organ damage. To achieve this, we will use the latest technology in 
performing detailed CV and novel biomarker measurements (including multiplex measurements and 
–omics), as well as behavioural and biopsychosocial assessments every 5 years. The study is 
registered on ClinicalTrials.gov with identifier number NCT03292094 
(https://clinicaltrials.gov/ct2/show/NCT03292094). 
 
6. Target Population and Endpoints 
To balance out the known confounding effects of age, sex, ethnicity and socio-economic status 
(SES)2,4 on hypertension development, we employ a stratified study design that takes age, sex, 
ethnicity and SES into account. We used the stratification in Table 1 as a participant selection 
approach. The inclusion criteria are: aged 20-30 years; apparently healthy; equal distribution of 
men/women, black/white and socio-economic status (low/mid/high); brachial blood pressure < 140 
and 90 mmHg; HIV uninfected; no previous diagnosis of any chronic disease; not using any 
medication for chronic diseases; not pregnant or breastfeeding. Based on this stratification, we will 
recruit and follow-up for 10+ years a total of 1200 individuals. Participants will be contacted 
periodically to update contact details and any changes in residential address. 
Endpoints/Outcomes: Since the prospective study sample comprises of participants in their 
twenties, we anticipate few hard endpoints (e.g. stroke) in the 10 years of follow-up. However, in 
preliminary analyses we found that 60% of black and 50% of white individuals of the first 403 
participants, already present prehypertension (SBP>120 and/or DBP>80 mmHg).4 As primary 
endpoint, we thus expect a substantial proportion with incident hypertension as endpoint during the 
first 5-year follow-up, and an even higher proportion at 10-year follow-up. As secondary endpoints, 
the focus will also be on ‘soft’ surrogate outcomes such as intra-individual changes from baseline in 
CV measures, biomarker profiles and early target organ damage. 
Due to the sensitivity of detailed CV measurements we expect to be able to detect statistically 
significant changes from baseline as part of the (early) vascular aging process. All CV, biochemical 
and metabolic changes will be closely monitored, and as appropriate % change will be used in 
statistical analyses (e.g. % change in SBP or % change in urinary albumin-to-creatinine ratio, and 
shift-tables for categorical measures will be generated). 

 
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Table 1: Participant stratification.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
A participant diagnosed with hypertension or CVD at follow-up will continue to be followed, but 
pharmacologic treatment will be recorded. At each visit, identical clinical and laboratory evaluations 
will be completed in order to allow within-individual statistical analyses. 
7. Sample Size and Power Calculations 
Sample size and Power calculations were performed in collaboration with the biostatistician and 
collaborator of the African-PREDICT study (A/Prof. Phil Gona, Boston). Our ethnicity/age/sex/SES 
stratified sampling design will be a hybrid one with two distinct components. The first one is a cross-
sectional design to evaluate and compare the detailed characteristics and CV profiles of apparently 
healthy 1200 black and white men and women (aged 20-30 yrs). The second aspect of the design 
includes longitudinal monitoring, every 5 years for 10+ years of systematic follow-up of the same 
detailed characteristics of participants. The sample size for the groups is set to 200 black and 200 
white in each of the three SES categories of the 20-30 year old age group (for a total of 600 black and 
600 white participants, Table 1 above). Cross-sectional data analysis will focus on frequencies, 
proportions p1 and p2, means, and other basic statistical measures by ethnicity.  
Hypothesis Testing Approach: Table 2 shows exact statistical power of two-sided tests for 
comparing two binomial proportions p1 and p2 with an alpha level of 0.05 and a sample size of 100 
Low socio-economic status 
Sex 
20-30 years 
Black 
Men 
100 
Women 
100 
White 
Men 
100 
Women 
100 
Mid socio-economic status 
 
 
Black 
Men 
100 
Women 
100 
White 
Men 
100 
Women 
100 
High socio-economic status 
ES 
 
 
Black 
Men 
100 
Women 
100 
White 
Men 
100 
Women 
100 

 
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subjects per stratum for estimating proportions ranging from 10% to 50%. Power calculations were 
performed using a two-group Z test with pooled variance with a two-sided significance level of 0.05, 
using PASS 11 software. Highlighted cells are underpowered. Cells not highlighted indicate sufficient, 
i.e., >80% power. Power improves with increasing stratum size. Power also is higher when the 
distance p1 - p2 is large, and power is reduced when the distance p1 - p2 is small. If the prevalence of a 
condition in white participants is rare (2%, 4% or 6%), we will have adequate power to detect 
proportion differences larger than 18%, 16%, and 14%, respectively when n=100. Hypotheses which 
collapse across strata (e.g., comparing all black to all white participants, n=200 in each group) will 
have higher power to detect smaller differences in prevalence p1 and p2.  
Table 2: Exact power of two-sided tests for comparing two binomial proportions p1 and p2 with an 
alpha level of 0.05 and a sample size of 100 in each stratum. 
White group, % abnormality, 
p2, N=100 
Black group,% Abnormality p1, N=100 
10% 
20% 
30% 
40% 
50% 
60% 
70% 
Rare 
2% 
0.69 
1.00 
1.00 
1.00 
1.00 
1.00 
1.00 
4% 
0.39 
0.96 
1.00 
1.00 
1.00 
1.00 
1.00 
6% 
0.18 
0.86 
1.00 
1.00 
1.00 
1.00 
1.00 
 
Medium 
10% 
- 
0.52 
0.96 
1.00 
1.00 
1.00 
1.00 
15% 
0.19 
0.16 
0.73 
0.98 
1.00 
1.00 
1.00 
20% 
0.52 
- 
0.37 
0.88 
1.00 
1.00 
1.00 
 
High 
30% 
0.96 
0.37 
- 
0.32 
0.83 
0.99 
1.00 
40% 
1.00 
0.88 
0.32 
- 
0.31 
0.83 
0.99 
50% 
1.00 
1.00 
0.83 
0.31 
- 
0.31 
0.83 
60% 
1.00 
1.00 
0.99 
0.83 
0.31 
- 
0.32 
For example if the prevalence in whites is rare (2%, 4% or 6%), we will have power to detect 
proportion differences larger than 8%, 16%, and 14%, respectively when n=200. This will of course 
further increase if all black and white (n=600 each) are collapsed. Therefore, we will have high 
confidence that the sample sizes will have sufficient power (>80%) to address the hypotheses. In 
most instances more sensitive continuous outcome variables (which yield more statistical power 
than binary outcome variables) will be used when comparing characteristics, as well as in cross-
sectional and longitudinal regression models. Finally, published studies employing proteomics and 
metabolomics included total samples sizes ranging from N=25,22 N=49,24 N=6015 and N=62316 
reporting significant and useful findings. 

 
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8. Human Research Participants 
The research programme in itself entails detailed monitoring of human participants over many 
years.  
8.1 
Approval Health Research Ethics Committee (HREC) 
Prior to commencement of data collection in November 2012, the project was approved by the 
Ethics Committee of the North-West University in 2012 (NWU-00001-12-A1). After screening 
commenced in 2012, eligible research participants started entering the study from 6 February 2013. 
As procedures in terms of approval and review of ethics applications were improved at the North-
West University, a newly formed Health Research Ethics Committee (HREC) again reviewed the 
project and approved continuation of the study annually for continuation. A more detailed 
application for the full study was submitted again to the HREC in April 2016 and in 2017, to ensure 
fully that all of the latest ethics requirements are met. 
The running of the full study is done mainly by trained research staff under the supervision of the 
Head of the Hypertension Clinic, who is a registered research nurse and acts as a mediator between 
the Principal Investigator and research participants. The risks to participants within this monitoring 
process falls within the category of medium risk, and participants are more likely to derive benefit 
from participation since measurement results are immediately made available to them. Should a 
health problem be identified, action is taken to immediately refer the participant for further medical 
treatment. Furthermore, the benefit to society from the long-term results of this study may have 
wide-ranging impact that may also benefit these individuals. 
Risks:  
Physical:  
 
Physical discomfort: venepuncture during blood sampling; performing eye pressure test 
with a tonometer, applying an eye drop may cause a slight burning sensation, and 
performing light flicker test may be slightly uncomfortable. After the eye measurement 
some discomfort may be experienced (similar to a visit to an ophtalmologist) while waiting 
for the pupil to dilate. Placing the hand in an ice water bucket for 1 minute may cause some 
physical discomfort. 
Psychological: 
 
Inconvenience, embarrassment, emotional discomfort: participants may experience some 
emotional discomfort when having to undress for anthropometric measurements, ECG and 
echocardiography/heart sonar measurements. Furthermore as part of psychological 
questionnaires may experience some self-disclosure e.g. as part of depression information, 
or stress. HIV testing as part of screening procedures may cause anxiety or fear for 
participants anticipating or worried about the outcome of the test. This is also partly 
applicable to other health outcomes that are disclosed to participants during all 
assessments, e.g. indication of hypertension or diabetes. 
Social harm: 

 
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 
Stigmitization: as HIV testing is part of the test measurements there is a risk that the 
outcome of the test may become known to other participants or researchers involved. 
Economic harm: 
 
Loss of income not being on the job, time spent in the research: as measurements take 
place during the working week (Monday to Friday), participants getting daily wages may 
suffer loss of income, or may get into trouble for not being at work due to time spent in the 
research. 
Precautions by the research team to address the above risks are described below: 
Physical: 
 
A trained, SANC registered research nurse perform all blood sampling and regularly undergo 
GCP training. She also performs the eye pressure test and apply the eye drop. To ensure 
correct procedures and minimum participant discomfort she undergone training at an 
Ophtalmologist to ensure correct procedures are undertaken to make it a swift procedure. 
The light flicker test may cause discomfort but the researcher is highly experienced and 
ensures that the measurement is done quickly and accurately. It does not cause any long 
term harm and is comparable to standard ophtalmologist measures. As the pupil is dilated, 
an eye patch is provided and all lights of the clinic turned off when these assessments start 
(at the end of the day’s measurements). Furthermore, participants are informed of these 
measurements beforehand and are also encouraged to bring sunglasses. Transport is also 
provided afterwards and participants discouraged to drive prior to the pupil returning to 
normal. Placing the hand in ice water causes physical discomfort and some pain due to the 
very cold water. The time is only for 1 minute to reduce discomfort to a minimum, and a 
small electric blanket provided afterwards to heat up the hand and ensure comfort. 
 
Psychological: Having to undress for some measurements may be experienced as 
uncomfortable – hence private temperature controlled rooms are available for every 
measurement. For sensitive measurements a female scientist is trained to perform 
measurements to ensure especially comfort of female participants, e.g. only female 
scientists perform ECG, ambulatory blood pressures and anthropometric measurements. 
Echocardiography is performed by a medical technologist with registration by the Health 
Professions Council of South Africa, in a semi-dark room. For heart sonars, the clinical 
technologist has vast experience in performing the sonars (also perform echocardiography 
on a daily basis in hospitals in Potchefstroom and Klerksdorp) and also provides a blanket 
should the participant require this. All staff are also trained in these aspects to be highly 
professional and discreet and to ensure maximum comfort of participants and to avoid any 
embarrassment. For psychological questionnaires a psychologist is trained by a registered 
clinical psychologist to complete the questionnaires in a private area. All necessary aspects 
are adhered to make sure it is done in a professional and comfortable manner. If any 
abnormality is detected, the psychologist informs the Head of the Hypertension Clinic, who 
will then refer the participant for consultation at the Potchefstroom Campus Institute for 
Psychology and Wellbeing (Dr. Annelize Bonthuys) at no cost – or to the participant’s 
preferred psychologist. HIV testing is done by trained counsellors in a private room, and 

 
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include pre and post counselling. During the informed consent stage all participants are also 
very clearly informed that HIV testing forms part of the screening procedures and they are 
therefore prepared beforehand. Pre and post-counselling, and also support by the research 
nurse afterwards are done in positive cases, to ensure that the participant receives 
treatment immediately (reference, and follow-up done). For other health outcomes, such as 
giving feedback that the young participant is hypertensive may be quite stressful. Thus all 
participants with an identified abnormality individually receive their health feedback 
information, and are supported to make sure that they receive appropriate medical care and 
are taken care of. 
 
Social harm: With HIV stigmatisation being a reality in South Africa, specific care is taken to 
ensure highly private testing and counselling before and after testing in a private room. 
 
Economic harm: If a participant is losing wages due to his/her participation in the study, 
direct communication from the Principal Investigator or Project Leader is made with the 
employer. Generally the employer is contacted before participants are screened to get 
his/her support, namely to provide a service to give health information to his/her employees 
as part of the research project (results are not disclosed to the employer, but only to the 
participant). Once an employer agrees that the participant can attend the study during 
normal working hours without having to take leave or lose any wages, the participant will 
join the study. 
Benefits to participants: The most significant benefit to participants is receiving high-level health 
measurements at no cost, with immediate feedback on their health status. 
After selection of the participants, they are invited to attend the screening phase of the study, and if 
they comply to the inclusion criteria they will also be invited to take part in the advanced research 
project. In both instances, participation is voluntary and participants may withdraw at any time. 
They are also fully informed about the study prior to participation, after which they complete an 
informed consent form. 
8.2 
Recruitment and Eligibility Criteria 
Recruitment: 
Active participant recruitment takes place on a continuous basis until the full baseline sample of 
1200 individuals have been included. Participants are invited from Potchefstroom and surrounding 
areas in South Africa via different routes. Centrally a research nurse was appointed to manage the 
recruitment of participants, and act as gatekeeper. As this is the central task of this nurse, she is not 
directly involved in the further research project measurements. The different routes of recruitment 
are described below in more detail: 
a) Active contact via field workers, 
Field workers are appointed, and trained by the research nurse on the requirements and 
eligibility criteria of the project. This takes place on an ad hoc basis, when the study 
population is reviewed and when it is noticed that participants from a particular group 
should be recruited. They are further trained on how to approach possible candidates, and 
to target these specific groups of participants that would be suitable for the study, especially 

 
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as the study aims to include individuals from low, medium and high socio-economic status. 
Field workers access potential research participants within an area where they are known 
and knowledgeable, clearly explaining what will be required from them, and that 
participation will be voluntary. Those individuals expressing interest to undergo screening 
are then contacted, and an appointment is made. Transport to and from the Hypertension 
Clinic is provided. 
b) Access through the workplace, 
The research nurse may also target specific workplaces for recruitment, also in light of the 
attempt to include participants from low to high socio economic status. An appointment is 
made with the employer to gain permission to speak to employees. Once permission was 
obtained, it is arranged with the employer to make an appointment to discuss the project 
with a large group of employees. During this engagement, details of the screening and 
research project are discussed, and interested individuals are invited to contact the nurse 
should they be interested to participate. Once they contact the research team/nurse, an 
appointment is scheduled for screening. In many instances, and if agreed by the employer, 
the screening phase of the study is conducted at the workplace within a private area made 
available by the employer. This ensures that employees are only briefly absent from their 
workplace, as the screening procedures takes about 30 minutes. The process also differs 
depending on the specific workplace. In some instances, individuals from a particular 
employer were contacted by a field worker (see section (a) above), and then notifies the 
research nurse that there may be other colleagues interested to participate. Such individuals 
are then sometimes contacted directly by the field worker or research nurse, and not via the 
employer. 
c) Advertisements by means of radio, noticeboards and local newspapers, 
Advertisements, originally approved by the Health Research Ethics Committee, are placed in 
the local newspapers, on noticeboards in the city or using radio stations. The recruitment 
team also performs health screening in public places, where potential participants are 
invited should they adhere to the study eligibility criteria. Contact details of the research 
team are provided and interested individuals are invited to contact the research nurse 
should they wish to participate. Once contact was established, an appointment for screening 
is made. 
Eligibility: 
Participants that are recruited firstly undergo screening to determine eligibility for participation in 
the African-PREDICT study (Table 3). The participants therefore constitute a convenience or 
availability sample, stratified into different ethnic (black and white), sex and socio-economic class 
groups (low, mid, high). 
Table 3: Eligibility criteria for the African-PREDICT study. 
Inclusion criteria: 
1. Self-reported black or 
white ethnicity 
2. Aged 20-30 years 
3. Men and Women 
(equally distributed) 
Exclusion criteria: 
1. Self-reported Indian, Asian, mixed origin ethnicity 
2. Not permanent resident of Potchefstroom or surrounding areas or not 
intending to return regularly to this area 
3. Inability to read or understand English 
4. Previously diagnosed with Type 1 or 2 Diabetes Mellitus 

 
12 
 
4. Apparently healthy 
5. Normotensive or pre-
hypertensive (SBP<140 
and DBP<90mmHg) 
based on the average of 
4 BP measures in one 
day 
 
5. Elevated glucose >5.6 mmol/L (confirmed glycated haemoglobin 
(HbA1c) ≥ 6.5%) 
6. HIV or other known infectious disease 
7. Fever (ear temperature > 37.5°C on the research day) 
8. Previously diagnosed liver disease, cancer, tuberculosis or renal 
disease 
9. Microalbuminuria > 30 mg/ml in spot morning urine or proteinuria 
10. Medication use for chronic disease, i.e. antihypertensive, anti-
diabetic, antiretroviral or anti-inflammatory medication 
11. Self reported pregnancy or women who breastfeed 
12. Recent surgery or trauma (within the past three months) 
13. Self-reported previous history of stroke, angina pectoris or myocardial 
infarction 
14. Phobia for needles (used during blood sampling) 
All participants receive feedback on their health measures and some basic health education at the 
end of the screening day. When eligible, participants are invited to join the research project or 
where screening results indicate necessary, participants are given onward referral for medical care.  
Those participants meeting the eligibility criteria for the research project are invited to join the study 
and given the detailed participant information leaflet detailing what is involved. If they are willing to 
participate, an appointment is made for them in the Research Clinic (Building F12).  
No additional follow-up visits are required for screening participants not meeting eligibility criteria or 
eligible participants who decline to participate, and referrals are made as appropriate by the 
Research Nurse.  
8.3 
Screening for Eligibility to Participate 
Health screening takes place continuously and in parallel with the research project in order to 
identify eligible participants for the research study. This will take place until all participants for 
baseline measures are included. In addition, the health screening provides a valuable and much 
needed service to the local community.  
Health screening will continue throughout the recruitment phase of the research project, continuing 
to provide a resource to the local community and a potential opportunity to follow up participants 
not eligible for research studies. The purpose is primarily for the selection of participants (based on 
inclusion/exclusion criteria) for the research protocol while providing appropriate care for screen 
failures. Screening takes place by a research nurse or permanent qualified research staff member in 
the Hypertension Clinic (F11) of the Potchefstroom Campus of the North-West University and also 
externally at other locations (e.g. participant’s workplace) to increase accessibility for participants. 
The measures done as part of screening are detailed in Table 4. The research support staff of the 
Hypertension Clinic have all undergone training to act as HIV counsellors (VCT training). With regards 
to HIV testing, the testing is done by trained counsellors in a private room, and include pre and post 
counselling, using a specific VCT informed consent form. During the recruitment and information 
sessions (and when informed consent is done) all participants are also very clearly informed that HIV 
testing forms part of the screening procedures and they are therefore prepared beforehand. Pre and 
post-counselling, and also support by the research nurse after the screening is done (follow-up 
contact) to ensure that participants receive treatment. 

 
13 
 
Once screening was performed, each participant will meet individually with the research nurse in a 
private room for a feedback session. He/she will receive their feedback on their health 
measurements orally, and on a report that is placed in a sealed envelope. Also discussed is their 
eligibility to continue for the measurements of the larger study. If an individual was found not to be 
eligible for the study, due to any of the exclusion criteria, this is explained to them. If they have any 
health abnormalities they are referred for further testing or treatment and the referral letter is also 
included in the sealed envelope. If a participant is eligible for participation in the larger study, the 
details of the larger study are discussed and the informed consent form provided to them. Each item 
of the informed consent form is also discussed in detail, and this takes place at least 48 hours to 1 
week before the participation in the larger study.  
 
Table 4 Screening measures (normal range & referral range indicated) 
Measure: 
Optimal Range: 
Referral indicated if: 
Clinical Biochemistry: 
 
 
Fasted Blood (finger prick) 
 
 
*Total cholesterol20 
<5.2 mmol/l 
>6 mmol/l 
Fasting Blood glucose 
4-5.6 mmol/l 
>6 mmol/l  
HbA1c# 
<6% 
>7% 
HIV test 
 
Positive result 
Urine (spot sample) 
 
 
Specific gravity & pH 
1.002-1.030, pH5-7 
 
Protein 
negative-trace 
1+ (>20mg/dl) 
Glucose 
negative 
1+ (& blood glucose >5.6mmol/l) 
Ketone 
negative 
 
Blood 
Negative  
Repeat measurement first; if 1+ (>5ery/ul 
if not menstruating) 
Nitrite 
negative 
1+ (if leukocytes elevated) 
Leukocyte 
negative 
3+ (>10/ul = significant Pyuria) 
Physiological measures: 
 
 
Brachial Blood Pressure 
<120/80 mmHg 
>140/90 mmHg 
Anthropometry: 
 
 
Height cm 
 
 
Weight kg 
 
 
Waist circumference cm19 
 
 
  South Asian male 
<89.9 cm 
 
  South Asian female 
<79.9 cm 
 
  All other males  
<93.9 cm 
 
  All other females 
<79.9 cm 
 
Hip circumference cm 
 
 
Neck circumference cm 
 
 
BMI (weight kg/height m2) 
18.5-25 
 
*Friedwald equation cannot be used at TG >4.5 mmol/l, Framingham risk score cannot be calculated at TG >5 mmol/l. 
#HbA1c is only measured if participant has a fasting blood glucose of >5.6 mmol/l 

 
14 
 
8.4 
Informed Consent 
To protect the safety and rights of participants, it is critical that informed consent be obtained 
appropriately. This applies to both the screening and the African-PREDICT research study with two 
separate informed consent forms. Furthermore, informed consent for African-PREDICT will be 
obtained from each participant at baseline, and during each phase of follow-up. 
Once a specific workplace, individual or area for recruitment is identified, the research nurse make 
contact with the participant before screening, either telephonically, sending an email, or meeting in 
person. The community worker acts as independent mediator between the Principal Investigator 
and the participant. During this contact session a detailed oral explanation is given on the study, all 
measurements and the eligibility criteria for participation. Once an individual has then voluntarily 
expressed interest to participate in screening (according to the Recruitment procedures, Section 
8.2), an appointment is made for the screening procedures on a different day. When a participant 
meets with the research nurse and her assistants for the screening, the information contained in the 
informed consent form are discussed again in detail, the form is handed to the participant and they 
are given as much time as needed to decide on participation, and given sufficient opportunity to ask 
questions prior to deciding whether they wish to participate. 
As part of the exclusion criteria of the study, is the inability to read or understand English – this is 
due to several questionnaires, dietary, psychological and other questionnaires forming part of the 
larger study. Prospective participants are also ensured that participation is voluntary and that they 
can withdraw at any stage. If they voluntarily and verbally express their intent to participate – also 
after questions were answered – the form is signed by the participant. Also, the research nurse 
responsible for explaining the screening process and taking informed consent (and the witness to 
the consent) also signs the form after the participant signs, as well as another witness who is not 
part of the study team. No study specific measures are taken before the consent is signed by all 
parties. The informed consent covers all measures and procedures to be taken and these are 
outlined in the participant information sheet and explained to the participant before consent is 
requested. This must also include any sub-studies that are planned in future. The whole process of 
informed consent is performed in a private area dedicated for the study – whether the Hypertension 
Clinic or in a dedicated study area off site - with privacy given to the individual to ask questions. 
If the participant is found eligible (according to the inclusion/exclusion criteria) for the African-
PREDICT study, they are invited to participate. This information is provided in a private feedback 
session after screening, as described in detail in Section 8.3. If they express interest, the detailed 
informed consent form for the research study is provided to them. They therefore have the 
opportunity to review this information over a few days. An appointment is scheduled, and transport 
provided (if required) to come to the Hypertension Clinic on the Potchefstroom Campus of the NWU 
(Building F12). Upon arrival at the Clinic, similar procedures as with screening are followed by the 
mediator (a research nurse). In addition, a video describing all study information and procedures are 
shown to research participants thereby providing them with visual clips on what each study 
procedure entails. This video is available in English and Setswana: 
https://www.youtube.com/edit?o=U&video_id=_wTIu64tcLQ (English) 
https://www.youtube.com/edit?o=U&video_id=bire3RdOa5s (Setswana) 

 
15 
 
9. Detailed Research Methodology 
With reference to the aim and objectives of the study, a range of detailed measurements will be 
taken at baseline, and will be repeated every 4-5 years in order to track small changes in biomarkers, 
biopsychosocial measures, cardiovascular structure and function, and subclinical organ damage 
development. 
9.1 
Data Collection for the African-PREDICT study 
Data collection takes place within the Hypertension Clinic (Building F12) of the Potchefstroom 
Campus of the North-West University under the supervision of a registered Research Nurse.  
The Hypertension Clinic is equipped with an Emergency Kit in Building (with First Line Emergency 
Drugs). Also available in both Clinic buildings is an Automated External Defibrillator. 
Participants arrive at the Clinic at 8:00 in the morning and are familiarised with the surroundings. 
The procedures of the measurements are again explained to the participants and details captured in 
the informed consent form discussed. Participants also have the opportunity to ask questions. After 
written informed consent is obtained, the measurements commence. Measurements are done in 
temperature controlled private rooms to take into consideration the privacy of the participant. A 
maximum of four participants are accommodated per day to ensure detailed and quality 
measurements. A summary of the measurements are provided below, starting with biological 
sampling. After blood sampling, anthropometry, bio-impedance and a set of cardiovascular 
measurements are done, the participants are provided with a light meal (excluding caffeine). When 
all measurements are completed at approximately 13:00, transport is provided to all participants to 
go home. 
 
Table 4: Research measurements 
Measure: 
Vitals: 
Ear temperature 
Biological Sampling: 
Fasted Blood Sample (100ml) 
Urine (spot sample) 
Urine (24-hour sample + additional spot sample on a separate day) 
Cardiovascular Measures: 
Brachial blood pressure 
Pulse wave analyses (central BP, AI) 
Carotid-femoral pulse wave velocity 
ECG 
Echocardiography 
Carotid intima-media thickness and distensibility 
Cardiovascular reactivity during stress testing (cold pressor test and Stroop test) 
Retinal microvascular calibre and responses to a light flicker stimulus 
24-hour Ambulatory BP and ECG monitoring 
Anthropometry: 
Height & Weight 
Waist, hip and neck circumference 
Body mass index 

 
16 
 
Bio-electrical impedance (body fat, lean body mass) 
Physical Activity Monitoring: 
7-day Accelerometry 
Questionnaires: 
General Health Questionnaire 
GPAQ & Godin physical activity 
Psychological questionnaire battery 
24-hour dietary recall and salt frequency intake (on site and 2 within 7 days) 
Berlin Sleep Questionnaire 
In the detection of any abnormalities during measurements, participants are immediately referred. 
9.2 
Questionnaire Data 
Questionnaires are completed with the help of a research nurse, trained research assistant or 
trained postgraduate students, and are done one-on-one in the Clinic. Depending on the nature of 
the questionnaires, some are done in a private room (e.g. psychological questionnaires), whereas 
others are performed in a quiet area within the Clinic. Dietary questionnaires are completed by a 
trained dietician, whereas psychological questionnaires are completed by a trained psychologist or 
intern psychologist. 
General Health Questionnaire – completed online on a web-based program and involves 
demographic information, employment information, alcohol and tobacco use, medication use, and 
family history (15 min).  
Berlin Sleep Apnea Questionnaire – to assess or identify the risk of patients for developing the sleep 
apnea syndrome (5 min). 
Global Physical Activity Questionnaire (GPAQ) – This questionnaire was developed by the World 
Health Organization to collect information on physical activity participation in different countries. (5 
min) 
24-hour Dietary Recall Questionnaire and Salt Frequency Intake – dieticians administer this 
questionnaires on day 1 and on two further days within a week, using food models and photo books 
to estimate portion-size. Macro- and micronutrient intake are calculated in the appropriate units, 
using an algorithm developed by the Medical Research Council (South African Food Composition 
Tables). Factor analysis will be used to develop dietary clusters for use in statistical analysis. (30 min) 
Psychosocial Questionnaire Battery – For psychological questionnaires a psychologist is trained by a 
registered clinical psychologist to complete the questionnaires in a private area. All necessary 
aspects are adhered to, to make sure it is done in a professional and comfortable manner. Once 
questionnaires have been completed, the psychologist can detect any abnormalities on the same 
day. If any abnormality is detected, the psychology intern informs the research nurse, who will then 
privately debrief the participant (within her scope of practice as nurse with training in psychiatric 
nursing). If necessary, she will refer the participant for consultation at NWU Wellness (for NWU 
employees), INGRYP (for NWU students). All other participants are referred to their chosen clinic or 
psychologist.   
Psychologists obtain information on positive mental health (Keyes et al. 2002; 2008), stress overload 
(Amirkhan, 2012), personality (Taylor & De Bruin, 2005), depression (Kroenke, Spitzer & Williams, 

 
17 
 
2001), psychological distress (Goldberg & Hillier, 1979), and coping responses (Amirkhan, 1990) 
using validated psychological questionnaires. (30-45 min) Once the questionnaires are completed in 
hard copy, the data is entered by a statistical consultant on an anonymous database, and hard 
copies of the questionnaires are stored in privately locked room at the Subject Group Psychology, 
under the supervision of Dr. Werner de Klerk. 
 
Scale 
Abbreviation and 
number of items 
Authors / developers 
The measured 
construct 
1. 
Mental Health 
Continuum Short-
Form* 
MHC-SF (14 items) 
Keyes (2002) 
Keyes et al. (2008) 
Positive mental 
health (flourishing) 
2. 
Stress Overload Scale * 
SOS (30 items) 
Amirkhan (2012) 
Stress overload: Event 
load and personal 
vulnerability   
3. 
Basic Traits Inventory 
(Short Form: Research 
Version) 
BTI-S (77 items) 
Taylor & de Bruin (2005) 
Personality  
4. 
Patient Health 
Questionnaire – 9 * 
PHQ-9 (10 items) 
Kroenke, Spitzer & 
Williams (2001) 
Depression and it 
severity  
5. 
Subjective Vitality 
Scale 
SVS (7 items) 
Ryan & Frederick (1997) 
Bostic, McGartland-
Rubio & Hood (2000) 
Subjective vitality  
6. 
Meaning in life 
Questionnaire* 
MLQ (10 items) 
Steger, Frazier, Oishi & 
Kaler (2006).  
Meaning in life: 
Presence; search   
7. 
Coping Strategy 
Indicator* 
CSI (33 items) 
Amirkhan (1990) 
Coping responses: 
Problem-solving; 
Seeking social 
support; Avoidance  
8. 
General Health 
Questionnaire* 
GHQ (28 items) 
Goldberg & Hillier 
(1979) 
Psychological distress: 
Anxiety and insomnia; 
depression; social 
dysfunction; somatic 
symptoms 
9. 
New General Self-
efficacy scale* 
NGSES (8 items) 
Chen, Gully, & Eden 
(2001) 
Self-efficacy across 
life-domains 
10. 
Health-specific Self-
efficacy Scales 
N-SES (5 items) 
PE-SES (5 items) 
AR-SES (3 items) 
Schwartzer & Renner 
(nd) 
Self-efficacy for 
health-related 
behaviours: nutrition; 
physical exercise; 
alcohol resistance  
*For each follow-up phase, only these questionnaires will be repeated since the other 
questionnaires measure stable traits or include duplicate measures of the same aspect. 
9.3 
Cardiovascular Measurements 
Measurement of the brachial blood pressure is conducted using the Dinamap Procare 100 Vital 
Signs Monitor/Dinamap CARESCAPE V100 Vital Signs Monitor (GE Medical Systems, Milwaukee, USA) 
with appropriately sized GE Critikon latex-free Dura-Cuffs. Prior to the measurement being 
performed, participants are requested to not have smoked, exercised or eaten at least 30 minutes 

 
18 
 
beforehand and were required to be in a seated resting state with the arm supported at heart level. 
The first measurement is taken on the left arm after the participant was seated calmly for 5 minutes. 
Thereafter blood pressure is taken on the right arm in duplicate. A final measurement is made on 
the left upper-arm. Systolic blood pressure, diastolic blood pressure and heart rate are captured for 
each measurement.  
24hr Ambulatory blood pressure (ABPM) is measured using a 24hr ABPM and electrocardiogram 
(ECG) apparatus (Card(X)plore, Meditech, Budapest, Hungary, British Hypertension Society (BHS) 
validated). An appropriate sized cuff is fitted to the participant’s non-dominant arm and instructions 
given to participants on how to ensure successful inflations across the 24hr time period. The ABPM 
apparatus is programmed to measure blood pressure in 30 minute intervals during the day and 
hourly during the night, while the ECG records measurements continuously. An ambulatory diary 
card is distributed and completed by participants during the 24hr duration of the measurements. 
Cardiovascular reactivity is tested by making use of the validated25-27 Finometer device (FMS, 
Finapres Measurement Systems, Amsterdam, The Netherlands). The participant is requested to lie in 
the Fowler’s position with their arm at heart level. A finger cuff is placed on the middle phalanx of 
the left hand’s middle finger, and a brachial cuff is connected to the upper arm. A 2-minute 
calibration is performed to provide an individual subject-level adjustment of the finger arterial 
pressure with the brachial arterial pressure.26 The highest precision in cardiovascular measurement 
can be achieved only after this calibration,26 and blood pressure measurements complied with the 
requirements of the Association for the Advancement of Medical Instrumentation (AAMI).26 
Continuous measurement of resting cardiovascular variables is performed for a 5-minute period, 
after which the participant is exposed to the cold pressor test for 1 minute. It is performed by 
immersing the participant’s right hand in ice water with a temperature of 4C for 1 minute. After a 3-
min resting period, another baseline measurement is recorded after which the Stroop test 
commences. The Beatscope® software is used to calculate systolic (SBP) and diastolic blood pressure 
(DBP), heart rate (HR) and computed stroke volume (SV), total peripheral resistance (TPR), and 
“Windkessel” compliance of the arterial system (Cwk).28 Cwk is the “Windkessel” compliance of the 
arterial system also referred to as Windkessel compliance, or buffer compliance. As part of the 
nonlinear three-element model (aortic characteristic impedance, arterial compliance, and systemic 
vascular resistance), it is computed from an age-dependent, aortic pressure–area relationship and 
represents the lumped compliance of the entire arterial system.28. Cardiovascular reactivity is 
calculated for each participant as the percentage change from the resting value to the stressor 
value. All measurements are performed in a temperature controlled room.  
Arterial stiffness is assessed according to the manufacturer’s instructions to determine carotid-
femoral pulse wave velocity (PWV) non-invasively using the Sphygmocor® XCEL device (AtCor 
Medical Pty. Ltd., Sydney, Australia). Participants are requested to be in a supine position and in a 
relaxed state for approximately 5 minutes before commencement of the measurement. The device 
is firstly used to estimate central systolic blood pressure and the augmentation index by performing 
pulse wave analysis (PWA). A brachial cuff is placed on the right upper-arm. By using a general 
transfer function the device estimates central systolic pressure, and the measurement is performed 
in duplicate. Afterwards, the participant’s right carotid artery is located by means of palpation to 
identify the strongest pulse point. The carotid pulse is measured using a tonometer while the 
femoral pulse is measured by a femoral cuff placed around the thigh of the participant. The direct 

 
19 
 
distance between the carotid pulse point and upper femoral cuff is noted (transit-distance method), 
and 80% of the distance calculated and entered. The PWV is therefore measured along the 
descending thoracic-abdominal aorta, using the foot-to-foot velocity method. Measurements are 
taken in duplicate. 
9.4 
Body Composition and Physical Activity Assessments 
Anthropometric measurements: A trained female researcher uses standard procedures to obtain 
height (m) determined by the SECA 213 Portable Stadiometer (SECA, Hamburg, Germany), weight 
(kg) using the SECA 813 Electronic Scales with weighing capacity up to 200kg (SECA, Hamburg, 
Germany) and waist circumference (cm)(Lufkin Steel Anthropometric Tape; W606PM;Lufkin, Apex, 
USA). The body mass index (BMI) (weight (kg) / height (m2)) is then calculated. All anthropometric 
measurements are performed according to guidelines as described by ISAK (International Society for 
the Advancement of Kinanthropometry). Privacy of the participant is taken into consideration by 
doing the measurements in a private room, with temperature control. Bio-electrical impedance 
assessment (BIA) is applied to assess lean body mass and body fat percentage, using a Bodystat 
1500MDD dual-frequency analyser (Bodystat, Douglas, UK). Bodystat®1500MDD is a lightweight, 
battery operated bio-impedance analyser which is a non-invasive device, which measures the 
impedance value of the body providing quick and effective analysis of body composition. 
After being fitted with the ambulatory blood pressure apparatus, participants will also be fitted with 
an ActiHeart physical activity monitor (CamNtech Ltd., England, UK). This is a compact, chest-worn 
monitoring device that records heart rate, inter-beat-interval and physical activity in one combined 
unit. It is designed for capturing heart rate variability data and for calculating and measuring activity 
energy expenditure. The ActiHeart device is worn for a maximum of 7 days. 
9.5 
Assessment of Target Organ Damage (TOD) 
Electrocardiography (ECG) is performed by a trained female researcher registering a standard 12 
lead ECG (Norav Medical Ltd, PC 1200, v5.030, Israel), with the participant in a supine position. At 
least six cardiac cycles are recorded. We use standard formules, such as the Cornell Voltage Product 
to provide an estimation of left ventricular wall thickness.29-31 
Echocardiography: A standard transthoracic echocardiography procedure is followed by a medical 
clinical technologist (registered with the HPCSA) while each participant is in a partial left decubitus 
position with the head of the examining table modestly elevated. The General Electric Vivid E9 
device (GE Vingmed Ultrasound A/S, Horten, Norway) is used along with the 2.5 to 3.5 MHz 
transducer and a single ECG-lead for timing purposes. Standardised methods are employed to obtain 
high quality recordings according to the current recommendations as outlined in the guidelines of 
the European Association of Echocardiography and the American Society of Echocardiography.32,33 
Two trained sonographers obtain the data and the data is analysed by one experienced registered 
clinical technician.  
Prognostically validated gender-specific cut-off values are used to identify LV hypertrophy.34 LV mass 
is calculated by a standard formula32,35  and is normalized for both body surface area and height 
(meter2.7).34 Inappropriate LV mass is estimated using an equation previously developed in a 
separate population of normal-weight, normotensive adults between the ages of 18 to 85 years.34,36 
LV end-diastolic and end-systolic volumes are calculated using Teichholz’s formula.37 Stroke volume 

 
20 
 
is generated (mL/beat) and normalised for height in the power of 2.04 as the stroke volume index 
(SVi).38,39 Stroke work (in gram-meters/beat) is computed as previously described.34 Cardiac output is 
obtained by multiplying stroke volume with heart rate. The ratio between pulse pressure and SVi is 
used as a measure of arterial stiffness.40 
Global LV systolic function is derived from linear measurements obtained from 2D images, since the 
study population did not present with regional wall motion abnormalities or irregular heart 
rhythm.41 Standard methods are used to determine endocardial fractional shortening (fractional 
shortening).42,43 The mid-wall fractional shortening (mFS) is alternatively calculated by a validated 
method previously described by De Simone, et al.44 LV ejection fraction (EF) is calculated from LV 
end-diastolic and end-systolic volume estimates derived from 2D images according to the biplane 
method. LV EFs of <52% for men and <54% for women are suggestive of abnormal LV systolic 
function.45 
A pulsed-wave (PW) Doppler is performed in the apical 4-chamber view to obtain mitral inflow 
velocities for assessing LV filling.46 A 1-mm to 3-mm sample volume is then placed between the tips 
of the mitral valve leaflets with parallel alignment with the inflow. Parameters of left ventricular 
diastolic function include: peak velocities of both early (E) and atrial (A) diastolic filling; the E/A ratio 
and E-wave deceleration time (DT). We additionally perform Tissue Doppler imaging to calculate the 
velocity of myocardial tissue movement in relation to mitral valve blood flow. This is a clinically 
robust index of diastolic function in combining the assessment of transmitral flow and mitral annulus 
velocity. The E/E’ ratio most confidently separates normal filling pressures (<8) from elevated filling 
pressures (>12).47 
Carotid intima media thickness (cIMT) and plaque scores are made on the left and right common 
carotid artery, as well as the internal carotid (General Electric Vivid E9, GE Vingmed Ultrasound A/S, 
Horten, Norway). Various optimal angles on each side will be measured and recorded for similar 
future assessments. The images will be digitised and imported in the Artery Measurement Systems 
software for dedicated analyses (Gustavvson, Sweden).48 Carotid cross-sectional wall area (CSWA) is 
calculated to confirm structural and not functional changes in luminal diameter (CSWA = π(d/2 + 
CIMT)2 – π(d/2)2, where d denotes luminal diameter). A videoclip will also be taken to determine 
carotid distensibility. 
Retinal microvascular calibre, oxygen saturation and responses to flicker induced provocation: 
Retinal photography and the dynamic retinal vessel assessment of vessel responses following light 
flicker provocation is performed using the Dynamic Retinal Vessel Analyzer (Imedos, Jena, Germany) 
fitted with a Zeiss Fundus camera FF-450. No intake of food or fluid is permitted 1 hour prior to the 
measurement. Participants also refrained from smoking and exercise during this time. Prior to the 
measurement the research nurse determines the intraocular pressure in each eye with the 
Tonometer - Tonopen Avia (Reichert technologies) and Ocu-film sleeve. A local anaesthetic, Novesin 
Wander (0.4% eye drop) is instilled before this measurement can be made. If the eye pressure 
exceeds 24 mmHg no further ocular measurements are performed due to the possiblity of glaucoma. 
Fifteen to thirty minutes prior to the measurement a drop of Tropicamide (1% Alcon) is administered 
in the right eye to induce mydriatic conditions. To avoid inducing angle-closure glaucoma, an 
estimation of the depth of the angle of the anterior chamber is done by the research nurse prior to 
administering the Tropicamide. Should the participant indicate any adverse reactions from the 

 
21 
 
Tropicamide, Amazid (Acetazolamide 250 mg tablet) is kept on hand. The research nurse ensures 
that all substances (Novesin Wander, Tropicamide and Amazid) are kept within their expiry dates. All 
these procedures falls within the scope of practice of the research nurse (Sr. Adele Burger), and she 
was trained by Dr. Geldenhuys (ophthalmologist) to perform all procedures. 
The Dynamic Retinal Vessel Analyzer is used to obtain static retinal images. These images can be 
used to to evaluate retinal artery and vein calibres, , estimations of vessel oxygen saturations and 
signs of retinopathy . In addition, a dynamic measurement is performed where the device records 
data for 6 minutes in which time a light flicker provocation is used to elicit  retinal artery and vein 
reactivity/responses. 
Monochrome and colour retinal images are captured (using Visualis 2.81 software) at a 50° camera 
angle. The monochrome or colour image underwent vessel analysis using VesselMap2 software. 
Briefly all vessels located between 0.5 and 2 optic disc diameters from the outer margin of the optic 
disc are marked either as artery or vein. The central retinal artery equivalent (CRAE) and central 
retinal vein equivalent (CRVE) are subsequently calculated using the Knudtson formula, where only 
the six largest artery and 6 largest vein segments were included in the calculation.49 The arteriolar-
to-venular ratio was calculated as CRAE/CRVE. Signs of retinopathy will also be quantified from this 
image. Both CRAE and CRVE were measured in measuring units (MU) where 1MU is equivalent to 
1µM if the dimensions of the eye are similar to the normal Gullstrand eye. The reproducibility of the 
analysis was computed previously for a randomly selected cohort with a correlation coefficient of 
0.84. The intra-class correlation (ICC) analysis involved a mixed-model framework, whereby random 
effects were assumed for participants and fixed effects were assumed for the graders. The 
Cronbach's alpha-reliability index for the AVR was 0.91 for this randomized cohort. The VesselMap2 
software further enables the determination of estimated artery and vein oxygen saturation. Within 
the same measuring area as described above, artery and vein branches are selected, and the 
software determines the oxygen saturation of each vessel branch. At the end of vessel selection, the 
software averages are determined to yield an oxygen saturation value for arteries and veins. 
For the dynamic measurement, two to 6 segments from each of an artery and vein branch are 
selected. The fundus camera is set at an angle of 30°. A light flicker stimulus is induced after a 50 
second baseline phase, for 20 seconds followed by an 80 second recovery phase. The protocol is 
automatically repeated for a total of 3 cycles. Following the measurement, the software 
automatically generates a summary curve of the 3 cycles. From this curve, maximum flicker induced 
vessel (artery and vein) dilation and maximum arteriolar constriction is manually determined. 
Alternatively, the data is exported into an excel sheet with built in macros to determine the 
previously mentioned dilation and constriction parameters, but additionally determines time 
dependent parameters and information regarding the area under the curve to better describe these 
vessel dynamic respones.50 The raw data can also be processed further to obtain information on 
retinal vessel pulsations and pulse wave velocity.51 
Renal function: Urinary albumin-to-creatinine ratio is determined using spot urine samples and 
obtained early in the morning when participants arrive at the research facilities. It is also determined 
from 24-hour urine samples. Additionally the blood serum levels are used to determine Cystatin-C 
and creatinine concentrations, which are used to calculate the estimated creatinine clearance and 
estimated glomerular filtration rate (GFR). Methodology on estimation of urinary albumin and 

 
22 
 
creatinine, and serum Cystatin-C and creatinine are described in the Biochemical Measurements 
section. 
9.6 
Biological Sampling and Biochemical Measurements 
The inner ear temperature is measured to establish that the participant is not suffering from an 
underlying infection resulting in an increase of body temperature (fever). Biological sampling 
entails the collection and preparation of different blood and urine samples. Planning for these 
storage procedures were done based on (a) the ability to analyse known markers of hypertension 
and related conditions, but also in line with the study aims (b) to store samples for the longer term 
and to measure novel biomarkers that may only be discovered after data collection were completed. 
In the early morning fasting blood samples are taken by a registered research nurse, and participants 
provide a spot urine sample. The following samples are collected: 
 
early morning spot urine sampling, to be stored as 7 x 1.5 ml cryovials and 3 x 500 µL 
cryovials; 
 
24-hour urine sampling, to be stored as 4 x 1.5 ml cryovials and 1 x 2 ml cryovial; 
 
blood sampling (fasted), to be sampled as 5  x 9 ml red-top serum, 2 x 9 ml purple-top EDTA 
plasma, 3 x 4 ml purple-top EDTA full blood; 2 x 4.5 ml light-blue top citrate, 1 x 2ml gray-top 
sodium fluoride, 1 x 4.5 ml Stabilyte®; 
 
blood samples on absorbant paper Guthrie cards. 
In the event that a blood sample cannot be obtained, the research nurse will make an appointment 
with the participant to collect the blood sample where it is most convenient for the participant e.g. 
at their workplace.  
Once the research nurse has taken the samples, a research assistant, trained in the handling of 
biological samples (using latex gloves), collects the samples in the Hypertension Clinic and place the 
sample tubes in a closed container. The samples are taken immediately to the on-site temperature 
controlled laboratory. Samples are then aliquoted into cryovials for short- and long term (screw cap) 
storage in biofreezers at -80°C. This is done by trained postgraduate students under the supervision 
of the qualified laboratory manager (Prof. Carina Mels). All staff and students handling biological 
samples undergone extensive training by the laboratory manager to reduce any risks to the 
laboratory students. During all procedures protective laboratory coats as well as latex gloves are 
worn, and students were particulary trained on how to handle samples. When any abnormality or 
problem arises, the laboratory manager is immediately notified and the necessary steps are taken to 
ensure the safety of staff members. Staff and students working with biological samples are also part 
of a Hepatitis surveillance programme.  
The biofreezer room in the F12 Physiology building is protected by a secure alarm system, sending 
SMS notifications to the full time academic staff member responsible for the security of the samples 
(Dr. Wayne Smith). Samples are further protected by a UPS system, and emergency electricity from 
the NWU, should it be required. 
Samples will be stored in this facility for the duration of the study, i.e. at least until 2018. It is further 
possible that these samples may be stored for a longer term and until sufficient funding was 
obtained to ensure appropriate analyses in line with and as per the Informed Consent Form. 

 
23 
 
Biochemical measurements are wide-ranging, and include (amongst others) the following: 
 
immediate basic serum analyses (such as HDL-C, LDL-C, triglycerides, glucose, C-reactive 
protein, creatinine, liver enzymes, albumin, uric acid), glycated hemoglobin (HbA1c), vitamin 
D and cotinine 
 
full blood count 
 
24-hour urinary sodium, potassium, creatinine, marinobufagenin 
 
markers of the renin angiotensin aldosterone system, such as renin, angiotensin II, 
aldosterone, angiotensin converting enzyme 
 
stress hormones, such as cortisol, testosterone, estradiol, FSH, SHBG 
 
N-terminal pro-brain natriuretic peptide, troponin T 
 
interleukin-6, tumor necrosis factor-α, P-selectin, E-selectin, soluble urokinase plasminogen 
activator receptor (suPAR) 
 
vascular endothelial growth factor-A, angiopoietin-2 
 
intracellular adhesion molecules (ICAM) and vascular cell adhesion molecule (VCAM) 
 
L-arginine, asymmetric dimethyl arginine, NO metabolites 
 
insulin, leptin, adiponectin, resistin, C-peptide, pro-insulin 
 
growth hormone, insulin-like growth factor-1 (IGF-1), insulin-like growth factor binding 
protein-3 
 
extracellullar matrix: metalloproteinases 1 and 2, fibulin-1, elastin 
 
markers of oxidative stress, such as reactive oxygen species, superoxide dismutase, 3-
nitrotyrosine 
 
markers of hemostasis, such as fibrinogen, prothrombin 1 and 2, von Willebrand factor 
antigen and activity, D-dimer, global fibrinolytic assay, plasminogen activator inhibitor-1 
 
in line with the aim of the study, i.e. to find new ways to identify early predictors for 
hypertension development, this study will also investigate other novel biomarkers that are 
not yet identified but are found to be associated with hypertension and CVD development, 
and may be measureable in the biological samples stored. 
Omics: 
Dedicated blood samples from cell preparation tubes and spot urine samples are stored for genomic, 
proteomic and metabolomic analyses in collaboration with local and international collaborators 
according to the appropriate procedures. The proteomic and metabolomic analyses are an extention 
of the abovementioned biomarker analyses, are will be analysed whilst particularly viewing the 
relationship to cardiovascular disease development. For proteomic and metabolic analyses spot 
urine samples (as described under Biological Sampling, Section 9.6) are used as follow: 
Proteomic analyses 
In collaboration with Professor Christian Delles at the Institute for Cardiovascular and Medical 
Sciences (ICAMS) at the University of Glasgow, spot urine samples are analysed for detailed 
proteomic profiles to establish biosignature predictors of CV deterioration. Different technologies 
are available for proteomic analyses. In this study we make use of capillary electrophoresis-mass 
spectrometry (CE-MS) technology. Proteins are separated by capillary electrophoresis based on their 
electrophoretic migration through a buffer-filled capillary. The separated proteins are subsequently 

 
24 
 
detected by mass spectrometry. This technique provides fast separation with high resolution, the 
capillaries used are less expensive than liquid chromatography columns, and it is compatible with 
various buffers and analytes. All analyses and data processing are done at ICAMS in Glasgow using 
only anonymous participant numbers. For each participants approximately 2000 urinary peptides are 
identified. Based on previous experience, the peptides are classified according to specific panels as 
observed previously in patients with certain conditions (e.g. coronary artery disease, chronic kidney 
disease, heart failure etc.) and healthy controls. Specific peptides are grouped as the most significant 
ones into classifiers. These are usually 50-300 peptides that can define a condition. 
 
The classifiers are quite robust as they contain many features. Therefore, participants will be 
grouped and categories even if they do not show all e.g. 300 peptides differentially regulated but 
only e.g. 100. They also allow a degree of quantitation of disease severity, assuming that those with 
more differentially regulated peptides (higher classifier scores) are more affected than those with 
less (lower classifier scores). Participants with lower scores will usually exhibit asymptomatic 
disease, which falls within the direct scope of the African-PREDICT study, namely to early identify 
those at possible risk for future CVD development. Those individuals with low scores, and with the 
lowest scores are less likely to proceed to overt disease than people who are already at the higher 
end of the normal spectrum. 
 
Therefore the classifiers, in theory, can be used as biomarkers for early detection, diagnosis, 
assessment of severity of disease, and prediction of disease. 
 
The evidence for proteomics as clinically useful biomarker is not yet as robust as it is for other, 
traditional, biomarkers that are used in the African-PREDICT study. However, as the concept is 
convincing, and since there are already some evidence that it is effective, the participants from the 
study, and the larger South African population may benefit from the results of this study. Particularly 
if effective peptide panels can be identified as future prediction tool. Existing panels that will be 
evaluted in the study include: 
1. Coronary artery disease. A diagnostic panel has been developed (PMID 20811296), it has 
been shown that it predicts events (PMID 25786980) and that it can be modified, e.g. by 
dietary measures (PMID 25527749).  
2. Chronic kidney disease. Panel developed (PMID 20616184), further validated (PMID 
24589724), predictive value shown (23086559), clinical trial underway (http://eu-
priority.org/).  
3. Heart failure panel (PMID 22789915).  
Firstly the panels (classifiers) will be evaluated against other cardiovascular measurements in the 
study (arterial stiffnes, retinal microvasculature, echocardiography, ambulatory and central 
pressures), to determine if there are differences between appropriately defined cases and controls, 
between men and women, and between black and white groups. Once this was performed, further 
analyses will be done one the remaining 2000 peptides. 

 
25 
 
These baseline analyses will give insight into the differences in protein expression between the 
groups. Additional proteomic studies in the future will enable the detection of not only early 
changes (first follow-up after 5 years) within the urinary proteome but also long term changes over a 
period of 10+ years. As further indicated in a 2015 American Heart Association Position Statement 
on Proteomics, in the setting of CVD and health, proteomics is particularly useful for the early 
detection and identification of processes involved in several CVD states, including atherosclerosis, 
hypertension, and stroke.  
Metabolomic analyses 
The analysis of the urinary metabolome is the study of the metabolic phenotype of the particpants 
of the African-PREDICT study, and is also directly in line with the central aim of the study. Several 
platforms exist that can be applied to perform such analyses, and in collaboration with the Centre 
for Metabolomics at the North-West University (Prof. Carools Reyneke, Dr. Roan Louw, Prof. 
Francois van der Westhuizen), the following strategy will be used: 
NMR spectroscopy – as it is quantitative in nature an doffers precise structural information that 
enables relatively easy metabolite identification. As the technique is not destructive, the samples 
will then also be used for further metabolomic analyses. As it has a low sensitivity, it will mainly 
allow the detection of high-abundance metabolites. 
Mass spectrometry, coupled with separation techniques, namely liquid chromatography (LC), has a 
higher sensitivity than NMR spectroscopy, and will therefore also be employed to detect a significant 
number of very-low-abundance metabolites. Targetted LC-MS will be used for butylated compounds  
(amino acids and carnitines), whilst untargetted gas chromatography (GC-MS) will be used for 
organic acids, and untargetted LC-MS will be used for positive and negative ionisation. 
The final analyses of the results will be done using participant characterisation based on sex, 
ethnicity and cardiovascular and metabolic profiles of participants.  
Genetic analyses: Samples are presently being stored for genetic analyses at the Centre of Excellence 
for Nutrition, under the supervision of Dr. Wayne Towers. Future applications to the HREC and to 
funding bodies will be submitted to detail and specify these approaches. 
9.7 
Rationale/Justification for the Measurements 
The decision to perform the specific measurements as listed previously are based on the existing 
knowledge, available literature, or lack thereof with reference to specific biomarkers, psychosocial 
measurements, cardiovascular assessments and markers of target organ damage. 
Previous research performed by our group and others have found ethnic-specific differences with 
regards to the following measurements to be undertaken, and may potentially play a significant role 
in the development of cardiovascular disease: 
Office and ambulatory blood pressure measurements are undertaken to confirm previous reports 
that some of the highest blood pressures have been recorded in African populations.1,52 Our 
research has indicated significantly higher daytime and nighttime ambulatory BP in African school 
teachers compared to Caucasian school teachers.53 Insufficient data is available on these values in 
healthy normotensive individuals. Additionally the dietary data and 24-hour urine sodium 

 
26 
 
concentrations should provide more clarity on salt-sensitive hypertension which is reported to be 
highly prevalent in black populations.54 
Arterial stiffness is reported to be increased in African populations which has been confirmed in 
black populations across the globe.55-57 Whether stiffness is already elevated at younger ages or 
during healthier periods is expected, but this requires confirmation. This is also relevant to the 
biomarkers related to the arteriosclerosis and atherosclerosis processes – such as extracellular 
matrix markers, e.g. fibulin-1,58 markers of vascular calcification59,60 and growth factors.61,62 
Markers of NO bioavailability should be measured to establish during which stages of hypertension 
development these markers are dysregulated. We determined that Africans have lower L-arginine 
concentrations,63 and that asymmetric dimethylarginine values are only significantly linked to pulse 
wave velocity in older African men.64 Africans are also reported to present with a prothrombotic 
profile, with fibrinogen, plasminogen activator inhibitor-1 and von Willebrand factor being strongly 
linked to unfavourable cardiovascular and metabolic health.65-67 The first results to be generated 
from this study on retinal microvascular reactivity in healthy individuals should provide greater 
insight into the early development of endothelial deterioration, whether it is ethnic specific and 
whether it may be a significant predictor of the development of hypertension later in life. 
Renal function: Non-diabetic Africans with normal kidney function indicated significant associations 
between albumin-to-creatinine ratio and ambulatory blood pressures which was not present in 
Caucasian individuals.68 Furthermore, due to the importance of low-renin hypertension and 
aldosterone levels, the occurrence of which is highly prevalent in African populations, these markers, 
as well as possible genetic polymorphisms affecting these levels, need to be tested as potential 
predictors for hypertension.69-71 
Left ventricular hypertrophy: African populations are reported to have an increased risk of 
developing a left ventricular mass and a two- to three-fold higher prevalence of left ventricular 
hypertrophy than in the general population, with increased risk of developing a left ventricular mass 
already occurring from late childhood.72,73 By using echocardiography we hope to obtain a clear 
profile on the occurrence of left ventricular mass in normotensive Africans and Caucasians, with 
additional biomarkers, such as Nt-proBNP and troponin T, supporting this data.74 
Obesity and inflammatory markers: Globally, obesity is becoming an increasing trend, and even 
more so in black populations.75,76 Its role in the expression of adipokines, such as leptin and 
adiponectin, is significant in the development of cardiovascular disease, especially in Africans.77,78 
The associated pro-atherogenic inflammatory profile of obese individuals may contribute to the 
elevated cardiovascular risk of Africans, and we have determined that in general Africans exhibit 
significantly higher levels of inflammatory markers, such as C-reactive protein and soluble urokinase 
plasminogen activator receptor (suPAR).79,80 
Behaviour: smoking, alcohol and psychological distress: In Africans with lower socio-economic 
status, we have discovered the debilitating effects of the high prevalence of smoking, and alcohol 
abuse in these populations.11,81 The inability to cope with the effects of rapid urbanisation and a 
westernised environment in older African populations is also strongly linked to cardiovascular 
deterioration.82-84 By investigating these aspects inter-disciplinary in a bi-ethnic young population 

 
27 
 
with similar levels of socio-economic stress, we could obtain better insight into whether these 
behaviours may continue exerting their effects on cardiovascular disease development. 
Omics: The significant potential that polyomics (e.g. genomics, metabolomics and proteomics) pose 
for future prediction of cardiovascular disease development cannot be ignored, especially due to the 
significant ongoing technological advances in these fields. We therefore aim to perform these 
measurement in collaboration with local and international expertise, and as funding allows. It forms 
a crucial part of the study and allows specifically to address the aim to identify novel biomarkers for 
hypertension and cardiovascular disease development. 
10. 
Statistical Analyses Plan 
The statistical analyses plan was developed in collaboration with the biostatistician and collaborator 
of the African-PREDICT study, namely Prof. Phil Gona, Boston, USA. The participants will be 
longitudinally monitored, with subjects returning to the Hypertension Centre every 5 years for 10+ 
years. At each post-baseline clinic visit, the same parameters assessed at the baseline visit will be 
measured. Prior to conducting formal statistical analysis, exploratory data analysis will be conducted 
to detect outliers and assess normality assumptions as well as to identify any notable features that 
could strongly affect results and conclusions. The Kolmogorov-Smirnov test will be used to test 
normality, and logarithmic transformation applied to variables with a skewed distribution. Data will 
be expressed as mean ± SD if normally distributed or median with interquartile range or 5th and 95th 
percentiles for non-Gaussian distributed variables. Categorical variables will be presented as 
frequency counts and proportions will be compared using Chi-squared tests. Chi-squared and 
Fisher’s exact tests will be used to test univariate associations. Inference about the study hypotheses 
focusing on baseline ethnic comparisons will be made based on summarising all continuous variable 
outcome measures within strata to be compared across ethnicity using methods for continuous 
data. Independent T-tests, ANOVA, ANCOVA, as well as linear and partial regression models and 
multiple logistic and linear regression will be used to evaluate whether incidence rates or percentage 
change of each outcome variable vary across ethnicities and sub-groups. The analysis will include 
covariates to adjust for patient demographic characteristics. Stepwise regression will be used to 
select variables to include in the most parsimonious model. Weighted prevalence estimates 
accounting for the sampling design will be used if the data are collapsed across strata. Weights will 
be based on the population distribution of SA. Biomarker and –omics data will be natural log-
transformed or standardised (to mean=0 and SD =1), if indicated.  
To classify a protein signature sensitivity and specificity will be determined and ROC plots will be 
constructed to evaluate the area under the curve as measure of overall accuracy. Model fit will be 
assessed using R-squared, the overall F-test statistic, and Akaike information criterion (AIC), for 
continuous outcome linear regression models, and using the c-statistic, -2 log likelihood, the 
Bayesian information criterion (BIC), Pseudo R-squared for binary logistic regression models. 
For repeatedly-measured continuous measures, mixed effect models for longitudinal data will be 
used. The correlation structures (e.g., compound symmetry or unstructured) will be determined by 
the data variance covariance matrices. To account for repeated measures we will use longitudinal 
multivariate analyses using random effects regression models, which allow for simultaneous 
estimation of within-subject (person-specific) intercepts and slopes and their association with other 

 
28 
 
predictors/ biomarkers for CV outcomes of interest. For repeatedly measured categorical outcomes, 
analysis will be implemented using generalized estimating equations (GEEs). Both linear mixed 
effects models and GEEs provide a practical method with reasonable statistical efficiency to analyse 
these kinds of longitudinal data and the methods help to provide deeper understanding in studies of 
change over time while appropriately accounting for the multi-level correlations arising from high 
dimensional repeated measurements. Missing data can be easily accommodated in these models. 
These models will be implemented for continuous, ordinal and dichotomous response data using 
SAS procedures PROC MIXED and PROC GLIMMIX. Every effort will be made during the study to 
minimise missing data.  
It is recognised that analysis of such data is hindered by the presence of incomplete data. Prior to 
statistical analysis, all study variables will be reviewed for missing data by generating summary 
statistics by comparison groups (ethnicity, sex and age groups) to assess the degree of missing-ness. 
It’s important to understand the mechanism generating the missing-ness because it affects the 
extent to which the missing data biases the results. That understanding will inform the choice of 
how we are to deal with the missing data. Missing-ness pattern could either be missing at random 
(MAR) or missing completely at random (MCAR). MAR refers to the propensity for a data point to be 
missing which is not related to the missing data but it is related to some of the observed data, i.e., 
missing-ness is conditional on another variable (ethnicity, sex and age groups, or the limits of 
detection of technology generating the data). On the other hand, MCAR means there’s no 
relationship between missing-ness and any variable. 
We will employ multiple imputation techniques to account for missing data in regression models and 
also perform sensitivity analyses to compare results with and without imputation. Multiple 
imputation is widely used to cope with missing data. The procedure fills in missing values to 
generate multiple complete datasets to preserve the main characteristics of the original data. The 
procedure entails that plausible values for missing data are drawn randomly from the underlying 
joint distribution of the variables, and each dataset analysed using standard methods. Across the 
datasets, parameters estimates and their variances are combined, accounting for between- and 
within-imputation variability, to generate overall estimates and standard errors. This process of 
estimation, reflecting our uncertainty in missing values, is called “Rubin’s Rules”. Multiple 
imputation, therefore, will serve to preserve the statistical power as presented in the proposal. To 
respond to the reviewer comments, we are not concerned about compromised power as a result of 
missing data because we will use standard analysis techniques that preserve the study statistical 
power.  [References (1) Little, R. J. A., & Rubin, D. B. (2002). Statistical analysis with missing data. 
New York: John Wiley & Sons. To compare results with and without imputation. (2) Xiaoyan Yin, 
Daniel Levy, Christine Willinger, Aram Adourian,Martin G. Larson. Multiple imputation and analysis 
for high-dimensional incomplete proteomics data. Statistics in Medicine, Nov. 2015]. 
Regarding markers that are censored because their values are below or above the limit of detection 
or due to low concentrations, the first step is to generate distributions for each marker to determine 
if the distributions are truncated in the upper or lower tails. Our colleagues at the Framingham Heart 
Study, have confronted a similar problem. Pedigree-based linear mixed-effects models (see R 
package “lmekin” https://cran.r-project.org/web/packages/coxme/vignettes/lmekin.pdf) were used 
to analyse continuous values using logistic regression models to analyse binary values (i.e., below a 
cut-point vs above a cut-point). This was necessary because expression of the marker was not 

 
29 
 
universal, but varied from as low as 1.5% to as high as 99.9%. The observed marker values generally 
did not have a truncated normal distribution, which precluded Tobit regression modeling. 
[Reference: Tobin, J. (1958). Estimation of relationships for limited dependent variables. 
Econometrica 26: 24-36] Furthermore, imputation produced extreme bi-modal distributions and was 
not acceptable for response data in linear modeling. Therefore, they employed an adaptive 
approach as follows: for a given marker, if at least 90% of participants expressed it, they used the 
linear-model p value; if <10% of participants expressed it, they used the logistic-model p value; if 
between 10% and 90% of participants expressed the marker, they combined results from the two 
models. Specifically, they added their Chi-squared statistics and then calculated the p-value from the 
distribution of a Chi-squared variate with two degrees of freedom. [Reference: Professor Marty G. 
Larson, ScD, NHLBI Framingham Heart Study, personal communication to Phil Gona, 15 December, 
2015] Other statistical methods that can incorporate approaches have been used with similar data 
[Reference: MinJae Lee, et al. Statist. Med. 2012, 31 1838–1848; Multiple imputation for left-
censored biomarker data based on Gibbs sampling method. Statistics in Medicine, 2012, 31 1838–
1848]. 
11. 
Data Handling and Management 
The African-PREDICT study uses the REDCap (Research Electronic Data Capture, see http://project-
redcap.org) system to capture all data elements. REDCap is a free, secure web-based, and user-
friendly electronic database software which can be quickly developed and customized for studies for 
collecting and tracking information and data from research studies and scheduling patient visits.85 A 
specific Data Manager, Dr. Lisa Uys, was appointed and trained to fulfil this function. When using 
this system all laboratory specimens, evaluation forms, reports, data and other records are identified 
only by the participant number to maintain subject confidentiality using the RedCap system. Apart 
from this system, data are also backed up on password protected hard drives – done by the Data 
Manager. 
A six-digit ID number is used; the first 2 digits correspond with a particular examination where, for 
example "00" will refer to the baseline phase and "01" to the first follow-up; the next four digits 
denote the subject’s unique four-digit number. Prior to participation in the study (also as part of 
screening) detailed information is conveyed individually to participants, with opportunities to ask 
questions. As explained orally and in the participant leaflet attached to the informed consent form, 
voluntary participation includes consent that all personally identifiable information (PII) as well as 
sensitive PII (SPII) will be captured as part of the study with the understanding that this information 
will be stored and handled very securely, and that anonymisation will take place promptly and 
equally securely. Furthermore, the minimum amount of PII is being captured within the study and 
only as far as the information contributes towards the aims of the study. As part of informed consent 
the implications of biological sample storage, international sample shipment and analyses are 
included, with samples being shipped only identifiable by the six-digit ID number, and thus blinded 
to international investigators. Data generated in other locations (e.g. Glasgow and Vienna) will either 
be uploaded directly onto REDCap or sent via password protected files to SA to be uploaded by the 
Data Manager. 
All paper and computerised records are kept in a secured area (Hypertension Centre, Building F11 
office with locked file drawers). All subject identification are done with the ID numbers only with all 

 
30 
 
identifying information kept in a separate file. Only three individuals (Data Manager, Head of the 
Hypertension Centre, and Head of Screening) have access to information linking ID numbers to 
participant names. Data entered on REDCap is saved online via secure and password protected fire-
walls of the NWU computer systems. Clinical information will not be released without written 
permission of the subject, except as necessary for cleaning, monitoring, and statistical analysis by 
the authorised study team members or when the participant’s well-being is at risk. 
Although biological samples are shipped to international destinations for high-level biochemical 
analyses, the dataset is not accessed by international collaborators. Laboratories will receive 
anonymous data and send results linked to participant numbers to the Data Manager. Should it 
become necessary to share parts of the dataset with collaborators, appropriate material transfer 
agreements and confidentiality agreements will be signed beforehand. 
12. 
Timeframe 
 
 
Participant recruitment started in 2012 and will continue until the baseline measurements 
are completed, and this is anticipated by end of 2017. 
 
Screening for eligibility commenced along with recruitment and will cease once 1200 
participants have advanced through all data collection assessments of the study. 
 
Participant follow-up is a continuous process performed by clinic nurses, fieldworkers and 
research assistants. Participants are contacted twice annually via email, telephone or 
personal visits, and contact information updated. This will continue until completion of 2nd 
follow-up by 2027. 
 
Basic biomarker analyses including standard glucose, lipid, liver enzyme, full blood count 
analyse, done in-house at the Hypertension Research and Training Centre laboratory. It will 
be done for all three sampling sessions (baseline, 1st and 2nd follow-up). 
Recruitment
Screening
Data collection
Participant follow-up
Basic biomarker analyses
ELISA, RIA
Multiplex analyses
Proteomics
Metabolomics
RAS Fingerprint
1st follow-up
2nd follow-up
Biomarker analyses
Baseline
Participants 

 
31 
 
 
ELISA and RIA analyses for several novel but clear-cut cardiovascular biomarkers (such as 
renin and aldosterone) are performed, similarly to basic analyses in-house for all three 
sampling sessions. 
 
Multiplex analyses will be performed in Glasgow using Newton Funding and for the whole 
baseline sample (2016-2018). If funding permits, it will also be analysed for the two follow-
up sessions. 
 
Proteomics will similarly be performed in Glasgow using Seed Funds (N=160) and Newton 
Funding.  
 
Metabolomics will be performed in South Africa once all baseline sampling are completed, 
i.e. 2017-2018, utilising funding from the Technology Innovation Agency (TIA) for the 
Metabolomics Platform. 
 
RAS-Fingerprint multiplex analyses will be performed in Vienna. 
 
Data management and cleaning will take place continuously. 
13. 
Budget 
Sufficient funding has been obtained from different sources to continue with the study for the next 
three years. The budget is reviewed at a continuous basis, with possible further funding applications 
planned. Security for continuation of the longer term part of the study (follow-up in the future) is 
evident from the SARChI Chair and the MRC Unit Funding which are both available for a five year 
cycle (until 2018) and both being renewable for another 10 years. Funding from these grants would 
be sufficient to ensure continuing at least all data capturing (but not necessarily expensive 
biochemical analyses). 
Item
Description of  Item 
TOTAL 2016-2018 
Other Sources of Income: 
  
1 
MRC Flagship Seed Fund (AE Schutte) 
R 500,000
2 
NRF/DST SA Research Chair, Running Costs (AE Schutte) 
R 1,063,327
3 
NRF/DST SA Research Chair, student bursaries 
R 2,250,430
4 
MRC Unit for Hypertension and CVD  
R 1,550,000
5 
MRC SIR Seed funds, Proteomics (CMC Mels) 
R 600,000
6 
Technology Innovation Agency (TIA) Metabolomics Platform 
R 420,000
7 
Newton Fund (MRC UK, SAMRC, GlaxoSmithKline) 
R11,000,000
  
TOTAL Income from other sources 
R17,383,757
 
 

 
32 
 
14. 
Remuneration 
As participants arrive fasted for screening and research procedures, they receive healthy meals on 
these two occasions. After baseline phase measurements are completed they receive a R50 gift 
voucher from a supermarket as token of appreciation for their participation. For the first follow-up 
study the voucher is increased to R300. 
15. 
Data Dissemination Plan 
The REDCap system85 used in the African-PREDICT study, allows direct sharing of all or selected data 
to any collaborator. Publication plan: Scientific research papers in high-impact journals on 
preliminary cross-sectional, full baseline cross-sectional, as well as findings from the 5 and 10-year 
follow-up will be of the highest priority to the PI, co-PI and scientific team members. Depending on 
the findings, journals such as the EHJ, Hypertension, Circulation, JACC and the Lancet will be 
targeted. It is expected that first authors will in all instances be from African origin, with expert input 
from other local and international collaborators and biostatisticians as co-authors and senior 
authors. With many postgraduate students, postdoctoral fellows, young and senior scientists already 
working in the study, several early publications are already expected in the next 3-5 years. These 
papers will mainly focus on cross-sectional analyses between ethnicities, to determine whether 
divergent cardiovascular phenotypes are evident in black and white normotensives, and whether 
these are accompanied by unique biomarkers or biosignatures. The most impactful publications, 
however, are expected to appear after 5-year follow-up and onwards, focusing on using precision 
health to predict and prevent hypertension in Africa. In addition, innovative strategies to translate 
this knowledge into cost-effective screening measures will be of high priority (e.g. rapid testing). 
Meetings: Accompanying publications, findings from the study will be presented at scientific 
meetings taking place in SA (e.g. SA Hypertension Society, SA Heart Society), Africa (e.g. Pan-African 
Society of Cardiology), and internationally (e.g. ISH, ESH, ESC, AHA Meetings, etc.) to engage in 
dialogue with other experts in the field. Policymakers: With existing open channels of 
communication with the Provincial and National Department of Health, as well as recent 
collaboration in publishing a new Health Policymaker’s Booklet for Africa, there is no doubt that the 
findings of the study will be presented to these stakeholders on a continuous basis, with in-depth 
discussions. Public: Due to national and international leadership roles of the primary investigators, 
regular engagement with the media does take place especially to increase awareness of 
hypertension (e.g. radio, papers and television appearances, social media). Important findings from 
the study will therefore be easily conveyed to the public using these routes. The PI and others have 
also contributed to other successful online initiatives, such as The Conversation, where scientific 
findings are presented by scientists to the public, especially to increase impact and change in 
society. Research participants: After the screening procedures of the study the registered research 
nurse immediately makes the results available to the research participants, including results from 
blood pressure, HIV (with counselling prior/after HIV testing), lipids, glucose, body composition. If 
they have any health abnormalities they are referred for further testing or treatment and the 
referral letter is also included in the sealed envelope (see procedures below).  
For the larger African-PREDICT study, participants get immediate feedback on site from the 
researchers as measurements are being made. During the follow-up phases, participants will receive 
a report on their health status as measured during the previous data collection phase. If 

 
33 
 
abnormalities are detected, they are appropriately and privately informed by the research nurse (Sr. 
Adele Burger) and referred by her using an official referral letter. 
All referrals will depend on the abnormality:  
 
Psychological questionnaires: Once questionnaires have been completed, the psychologist 
can detect any abnormalities on the same day. If any abnormality is detected, the 
psychologist informs the research nurse, who will then privately debrief the participant 
(within her scope of practice as nurse with training in psychology) and if necessary, she will 
refer the participant for consultation at the Potchefstroom Campus Institute for Psychology 
and Wellbeing at no cost – or to the participant’s preferred psychologist. An agreement was 
made with the Institute for the referral of participants. 
 
Eye abnormalities: As the researchers performing retinal vessel analyses are not trained 
ophthalmologists, and as the purpose of this measurement is not to detect eye 
abnormalities, the measurements made are not similar to those done during a normal visit 
to an ophthalmologist. However, as the researchers are trained on normal anatomy and 
physiology of the retinal vascular area, abnormalities are noted, and participants with a 
medical aid are referred to an ophthalmologist with whom the researchers have discussed 
this arrangement. Participants without a medical aid are referred to their clinic of choice, as 
once a month the clinics are visited by an ophthalmologist. 
 
Cardiac and blood pressure abnormalities: participants with a medical aid are referred to 
their general practitioner of choice, but depending on the abnormality they could also 
referred to an internist with whom the research team has had discussions. If necessary 
further referral are then made to a cardiologist. For participants without a medical aid, they 
are referred to a general practitioner at Potchefstroom Hospital, who will then refer 
participants to an internist or cardiologist at the Baragwanath hospital, should it be 
necessary. 
 
HIV infection: Should a participant be diagnosed with HIV during the screening phase, the 
research nurse will refer participants with a medical aid to their general practitioner of 
choice, and those without a medical aid to their clinic of choice.  
 
Abnormal full blood counts: Should any abnormalities be detected in the laboratory, these 
are referred to the research nurse and she will then refer the participant to their general 
practitioner or clinic of choice, as appropriate. 
For all referrals, further contact with the participant is maintained to ensure that they have been 
to a healthcare facility and that treatment was initiated. The research team, particularly the 
Head of the Hypertension Clinic (Sr. Adele Burger), has communicated and has an agreement 
with all clinics from the Department of Health in the area that our research participants are 
referred to them. They also recognise our referral letter, and maintain communication with the 
research team. 
 

 
34 
 
16. 
Expected Outputs, Outcomes and Impact 
16.1 Expected Outcomes 
Although hypertension is generally considered to develop over a long period of time, we expect that 
of the original 600 young black and 600 young white participants, at least 10 % would have 
developed pre-hypertension or hypertension after four years (with possibly a higher percentage in 
the African sample).* Furthermore, a larger group will possibly develop smaller changes in vascular 
function, such as microvascular dysfunction and/or increased arterial stiffness, with possible changes 
in biomarkers. 
 
Based on the assumptions above, the first main outcome from this study will be the 
identification of markers (in any form such as behaviours, biomarkers, direct cardiovascular 
measurements or a biosignature based on e.g. proteomics) that may act as predictors for 
early vascular changes. As the project progresses over 10+ years in the same individuals, it 
would become even clearer which individuals are developing subclinical organ damage and 
which markers relate most strongly to these developments. Based on well-reported ethnic 
differences in hypertension development, this result will specifically address the 
development of hypertension in the black group (compared to white participants). Baseline 
data comparison should also shed light on the age-based differences in healthy 
normotensive black and white individuals. This advancement in knowledge on cardiovascular 
disease development will be imperative in understanding disease mechanisms and to 
develop strategies to curb the development of cardiovascular disease at young ages. 
 
Another outcome within this research programme, is the development of research capacity 
in all the disciplines participating in the project (BSc Honours, Masters, Doctoral students, as 
well as postdoctoral fellows and young emerging staff members). As with current research 
projects, it is impossible to perform such studies without the direct involvement of these 
students (for recruitment, cardiovascular measurements, laboratory analyses, feedback to 
the participants, etc.). Continuous mentoring needs to take place in order to develop these 
young individuals as scientists and to create the career pathways of especially those who are 
already appointed in permanent research positions.  
 
On a community level, an important outcome will be the greater awareness of hypertension 
as the “silent killer”. 
 
An indirect outcome of the study is that due to the study design and the advanced research 
methods used in this study, the results generated will be novel and highly competitive with 
international Hypertension Centers of Excellence. 
 
The final long-term outcome of this research will be to prevent the development of 
cardiovascular disease in individuals at increased cardiovascular risk based on identified 
predictors. The implementation of these specifically designed prevention programmes 
(based on the characteristics of the identified early predictor(s)) will take place in South 
Africa, and perhaps within other countries. 
*This assumption is based on previous research by our group.11,57 

 
35 
 
16.2 Expected Outputs 
During the first baseline phase of this prospective study, we expect that numerous publications in 
high-impact international journals will be generated. These manuscripts will typically focus on the 
baseline data generated, describing e.g. the methodology and cohorts collected in detail with 
respect to the various age, gender and ethnic reference groups included; and also the first data 
generated as part of the South African initiative to lower salt intake (based on the 24-h salt excretion 
data). The novel markers to be tested in this study have not been investigated in large bi-ethnic 
samples of young individuals, and the cross-sectional data collected will be highly publishable. 
Measures of cardiovascular structure and function and their association with different health 
exposures and biomarkers will be the focus of most papers, highlighting the different aspects with 
regard to ethnicity and gender. With respect to the first phase, we expect approximately 15-25 
publications. 
As the study progresses and the data from the first follow-up becomes available, the scientific 
impact will be significantly greater, and we expect to publish these results in journals with impact 
factors higher than 6. 
Capacity development (as described above) will also allow several PhD and MSc students to 
complete their studies in this timeframe (2-4 postdoctoral fellows, 4 PhDs, 6 Masters, 10 Honours). 
Results from this study are also expected to be disseminated at numerous national and international 
scientific conferences. 
16.3 Impact: Improvement in health outcomes 
Should this project be able to identify novel markers for the early identification of cardiovascular 
disease in young black South Africans, then these results could impact significantly on the ultimate 
prevention or delay in the development of CVD by identifying young individuals at risk prior to 
disease development. 
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