A Cross-sectional, Observational Study to Characterise Long-COVID in an Urban Sample of South African Adults
Conditions: Long COVID
Sponsor: University of Witwatersrand, South Africa

                                           
 
 
 
 
 
 
 
EZ-FV-028 
 
 
A cross-sectional, observational study to characterise Long-COVID in an urban sample of South 
African adults 
 
 
 
Short Title: 
ChaLoC 
Protocol Version and Approval Date: 
Version 02, 27 May 2022 
Sponsor: 
Ezintsha, Faculty of Health Sciences 
University of the Witwatersrand  
Building C, Sunnyside Office Park 
32 Princess of Wales Terrance 
Parktown, Johannesburg 2193 
Funder: 
South African Medical Research Council 
Principal Investigator: 
Prof WD Francois Venter 
Ezintsha, Faculty of Health Sciences  
University of the Witwatersrand 
Building C, Sunnyside Office Park 
32 Princess of Wales Terrace 
Parktown, Johannesburg, 2193 
 
 
 
CONFIDENTIAL 
Property of Ezintsha, University of the Witwatersrand  
May not be used, divulged, published, or otherwise disclosed without consent 

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PRINCIPAL INVESTIGATOR SIGNATURE PAGE 
A cross-sectional, observational study to characterise Long-COVID in an urban sample of South 
African adults 
 
I, the undersigned, am responsible for the conduct of the trial at this site and affirm that: 
• 
I agree to personally conduct or supervise the study. 
• 
I have read the protocol and agree that it contains all necessary details for carrying out the study 
as described. I will conduct this protocol as outlined therein, as per any approved protocol 
amendments, as per ICH Good Clinical Practice (GCP) and all applicable national requirements and 
laws. 
• 
I will not deviate from the protocol without prior review and written approval from the 
Institutional Review Board or Ethics Committee, except where necessary to prevent immediate 
danger to the participant. 
• 
I will ensure that the requirements related to obtaining informed consent are in accordance with 
ICH Guidelines for GCP Section and local requirements. 
• 
I agree to document adverse events that occur during the study, to maintain adequate and 
accurate records and make those records available, in accordance with ICH Guidelines for GCP, 
South African GCP and other local requirements. I agree to promptly report to the Ethics 
Committee all changes in the research activity and all unanticipated problems involving risk to the 
participants. 
• 
I have sufficient time to properly conduct and complete the trial within the agreed trial period, 
and I will ensure that any qualified staff at my site(s) who are involved in the trial conduct are 
adequately trained regarding the protocol, and their responsibilities for the foreseen duration of 
the trial to conduct the trial properly and safely. If I delegate any of my trial activities, I will 
document this on a Delegation of Activities Form. I agree to ensure that all associates, colleagues, 
and employees assisting in the conduct of the study are informed about their obligations in 
meeting the above commitments. 
• 
I understand that the study may be terminated, or enrolment suspended at any time by me if it 
becomes necessary to protect the best interest of the participants. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Prof WD Francois Venter 
 
 
 
Date 
 

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SPONSOR SIGNATORY APPROVAL PAGE 
A cross-sectional, observational study to characterise Long-COVID in an urban sample of 
South African adults 
 
I, the undersigned have read this protocol and I approve the design of this trial: 
 
 
 
 
 
 
 
 
 
________________________________  
___________________ 
Nonkululeko Mashabane 
 
 
Date 
Head of Research Operations 
Ezintsha, University of the Witwatersrand  
 
  
 
 
 
 
 
 

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TABLE OF CONTENTS 
PRINCIPAL INVESTIGATOR SIGNATURE PAGE ................................................................................................ 2 
SPONSOR SIGNATORY APPROVAL PAGE ....................................................................................................... 3 
ABBREVIATIONS AND TERMS ........................................................................................................................ 6 
SYNOPSIS 
8 
1. 
SCHEDULE OF ASSESSMENTS ................................................................................................................ 14 
2. 
INTRODUCTION .................................................................................................................................... 15 
2.1 
Background ........................................................................................................................................... 15 
2.2 
Long-COVID ........................................................................................................................................... 17 
2.3 
Study Rationale ..................................................................................................................................... 18 
2.3.1 
Rationale for specific sub-study focus areas ............................................................................... 18 
3. 
OBJECTIVES AND ENDPOINTS ............................................................................................................... 19 
4. 
STUDY DESIGN ..................................................................................................................................... 20 
4.1 
Overall Design ....................................................................................................................................... 20 
4.2 
Study Treatments .................................................................................................................................. 22 
4.3 
Study Duration ...................................................................................................................................... 22 
4.4 
Individual Participant Withdrawal ........................................................................................................ 22 
5. 
STUDY POPULATION ............................................................................................................................ 22 
5.1 
Cohorts .................................................................................................................................................. 22 
5.2 
Recruitment .......................................................................................................................................... 23 
5.3 
Main Study Eligibility Criteria (Baseline Visit) ....................................................................................... 24 
5.3.1 
Inclusion criteria .......................................................................................................................... 24 
5.3.2 
Exclusion criteria .......................................................................................................................... 24 
5.4 
Sub-Study Eligibility Criteria (Subsequent Optional Visit(s)) ................................................................. 25 
5.4.1 
Inclusion criteria .......................................................................................................................... 25 
5.4.2 
Exclusion criteria .......................................................................................................................... 25 
5.5 
Justification of the Inclusion and Exclusion Criteria .............................................................................. 25 
5.6 
Participant Identification ...................................................................................................................... 25 
5.7 
Co-enrolment Guidelines ...................................................................................................................... 25 
6. 
STUDY VISITS ........................................................................................................................................ 26 
7. 
STUDY ASSESSMENTS, QUESTIONNAIRES, AND PROCEDURES .............................................................. 26 
7.1 
Main Study ............................................................................................................................................ 26 
7.1.1 
Screening assessments (Day 1) .................................................................................................... 26 
7.1.2 
Baseline assessments, questionnaires, and procedures (Day 1 [+7 days]) ................................. 27 
7.1.3 
Telephonic follow-up (up to 28 days after completion of baseline visit) .................................... 30 
7.2 
Sub-Studies ........................................................................................................................................... 30 
7.2.1 
Screening and informed consent ................................................................................................. 30 
7.2.2 
Sub-study visit(s) ......................................................................................................................... 30 
7.2.3 
Telephonic follow-up ................................................................................................................... 32 
7.3 
Biohazard Containment ........................................................................................................................ 32 
8. 
SAFETY MONITORING .......................................................................................................................... 33 
8.1 
Responsibilities for Ensuring the Safety of Study Participants .............................................................. 33 
8.1.1 
Principal Investigator ................................................................................................................... 33 
8.1.2 
Study Sponsor .............................................................................................................................. 33 
8.1.3 
Medical Monitor .......................................................................................................................... 33 
8.2 
Adverse Events ...................................................................................................................................... 33 
8.2.1 
Definitions ................................................................................................................................... 33 
9. 
STATISTICAL CONSIDERATIONS ............................................................................................................ 33 
9.1 
Sample Size Determination ................................................................................................................... 34 
9.2 
Analysis and Presentation of Data ........................................................................................................ 34 
9.2.1 
Disposition, demographic, and background data ........................................................................ 34 
9.2.2 
Primary and secondary endpoints ............................................................................................... 34 
9.2.3 
Safety data ................................................................................................................................... 34 
9.2.4 
Exploratory analyses .................................................................................................................... 34 
10. STUDY MONITORING ........................................................................................................................... 34 

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11. ETHICAL CONSIDERATIONS................................................................................................................... 35 
11.1 Ethical Considerations ........................................................................................................................... 35 
11.2 Informed Consent Process .................................................................................................................... 35 
11.3 Blood Volume ........................................................................................................................................ 35 
11.4 Study Records and Confidentiality ........................................................................................................ 35 
11.5 Participant Remuneration ..................................................................................................................... 36 
12. ADMINISTRATIVE CONSIDERATIONS .................................................................................................... 36 
12.1 Protocol Amendments .......................................................................................................................... 36 
12.2 Clinical Data Records ............................................................................................................................. 36 
12.3 Record Retention .................................................................................................................................. 36 
12.4 Discontinuation of the Study ................................................................................................................ 36 
12.5 Publication Policy .................................................................................................................................. 37 
12.6 Study Audits .......................................................................................................................................... 37 
13. REFERENCES ......................................................................................................................................... 38 
APPENDICES 
41 
APPENDIX 1: 
STUDY GOVERNANCE ........................................................................................................ 41 
 
 
 

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ABBREVIATIONS AND TERMS 
Term 
Definition 
ACTH 
Adrenocorticotropic hormone 
AE 
Adverse event 
ALP 
Alkaline phosphatase 
ALT 
Alanine aminotransferase 
AST 
Aspartate aminotransferase 
BMI 
Body mass index 
CK-MB 
Creatine kinase MB fraction 
COVID-19 
Coronavirus disease 
CPM 
Conditioned pain modulation 
CST 
Cosyntropin sensitivity test 
CT 
Computed tomography 
CTCAE 
Common Terminology Criteria for Adverse Events 
cTnT 
Cardiac troponin T 
DLCO 
Diffusing capacity of the lungs for carbon monoxide 
DNA 
Deoxyribonucleic acid 
DSM-5 
Diagnostic and Statistical Manual of Mental Disorders (Fifth Edition) 
ECG 
Electrocardiogram 
eCRF 
Electronic case report form 
EVDS 
Electronic Vaccination Data System 
GCP 
Good Clinical Practice 
GGT 
Gamma-glutamyl transferase 
HbA1C 
Glycosylated haemoglobin 
HIV 
Human immunodeficiency virus 
HREC 
Human Research Ethics Committee 
hs-CRP 
High sensitivity C-reactive protein 
IATA 
International Air Transport Association 
ICF 
Informed consent form 
ICU 
Intensive care unit 
IL-1 
Interleukin 1 beta 
IL-6 
Interleukin 6 
IL-8 
Interleukin 8 
LDH 
Lactate dehydrogenase 
Long-COVID 
Post-COVID-19 syndrome / Post-acute sequelae of COVID-19 / Chronic COVID 
syndrome 
MCH 
Mean corpuscular haemoglobin 
MCHC 
Mean corpuscular haemoglobin concentration 
MCP-1 
Monocyte chemoattract protein-1 
MCV 
Mean cellular volume 
MRC 
Medical Research Council 
MRI 
Magnetic resonance imaging 
OGTT 
Oral glucose tolerance test 
PCR 
Polymerase chain reaction 

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POPIA 
Protection of Personal Information Act 
pro-BNP 
Prohormone brain natriuretic peptide 
PSG 
Polysomnography 
PTSD 
Post-traumatic stress disorder 
QST 
Quantitative sensory testing 
RDW 
Red cell distribution width 
SAMRC 
South African Medical Research Council 
SAP 
Statistical Analysis Plan 
SARS-CoV-2 
Severe acute respiratory syndrome coronavirus 2 
SBIMB 
Sydney Brenner Institute for Molecular Bioscience 
SpO2 
Peripheral oxygen saturation 
TNF- 
Tumour necrosis factor alpha 
TNFR1 
Tumour necrosis factor alpha receptor 1 
WHO 
World Health Organization 
WHO-DD 
WHO Drug Dictionary 
Wits 
University of the Witwatersrand 
 

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SYNOPSIS 
Title 
A cross-sectional, observational study to characterise Long-COVID in an urban sample of 
South African adults 
Short Title 
ChaLoC 
Background 
“Long-COVID’’ (also known as post-COVID-19 syndrome, post-acute sequelae of COVID-
19, or chronic COVID syndrome, used here as ‘Long-COVID’ for brevity), is a complex array 
of postconvalescence symptoms following SARS-CoV-2 infection. The syndrome, common 
in COVID-19 survivors, can affect every organ system through as-yet uncharacterised but 
presumed immunological mechanisms. Prevalence depends on the definition used and 
time-period of follow-up, as well as the population being studied. The syndrome has been 
associated with significant and persistent disability in some survivors but has been 
hampered, until recently, by lack of a clinical definition, diagnostic criteria, and objective 
measures of disease or disability [1]. A Delphi-informed initial WHO clinical definition was 
released in early October 2021 but has attracted much criticism from both clinicians and 
survivors for a host of reasons, ranging from a lack of precision to a lack of inclusion [2]. 
 
Further complicating the syndrome is the context in which the SARS-CoV-2 epidemic 
occurred, which was associated with severe lockdowns in many countries (including South 
Africa) with social isolation, widespread fear and disinformation, widespread economic 
hardship, and loss of family and acquaintances, all of which contribute to symptoms 
(psychiatric and sleep disturbances, pain, and other syndromes) reported to be associated 
with Long-COVID. Finally, many Long-COVID symptoms overlap with those seen in 
patients hospitalised for any severe illness, especially those admitted to intensive care 
and ventilated. However, the proliferation of literature reporting associations of Long-
COVID symptoms with more severe COVID-19 disease, and objective immunological, 
radiological, and organ-specific dysfunction in those reporting symptoms, suggests that 
the entity is real. The pathogenesis of Long-COVID is poorly understood, but this 
association with more severe disease - where immune dysregulation plays a major role in 
those with hospitalization, respiratory failure, and death - suggests an immune-mediated 
inflammatory dysfunction that may impact all organs [3-14]. 
 
The sheer rapidity of four major infection waves in South Africa, the initial focus on 
containing the hospital burden of those with severe illness, and subsequent emphasis on 
the roll-out of a mass vaccination program, has left little space for studying SARS-COV-2 
sequalae in survivors. This group, loosely and inaccurately termed “recovered’’ in South 
African reporting, were largely unvaccinated or partly vaccinated at the time of infection, 
leaving them at risk of developing Long-COVID.  
 
Long-COVID reported symptoms are extraordinarily broad. Over 50 symptoms were 
reported in a recent review, with over 80% of COVID-19 survivors having at least one 
symptom after infection [4]. The diagnosis is one of exclusion, and symptoms may 
fluctuate although tend to resolve with time. It is unclear whether disability and organ 
damage may be permanent in some patients. No specific treatment exists, and 
interventions are typically symptom-directed. SARS-CoV-2 vaccination appears to benefit 
many symptomatic survivors [15]. At the time of writing, no African data characterising 
the clinical features of Long-COVID have been published. 
Rationale 
Characterizing Long-COVID and related disability in local populations is an important first 
step to understanding the burden of morbidity and potential requirements of the health 
system in addressing symptoms and disability. 
 
Almost all studies on Long-COVID have been undertaken in higher income countries, and 
largely in hospitalised patients, with almost no data emerging yet from Africa. This study 
aims to characterise Long-COVID in South African patients by leveraging an existing 

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patient base (including asymptomatic outpatients, symptomatic outpatients, hospitalised 
patients with severe COVID-19, and those who were vaccinated prior to widespread 
community exposure) and local research expertise. 
 
The study is anticipated to add significantly to understanding Long-COVID, and to 
contribute African data to current efforts to develop syndrome diagnostic criteria 
(including the WHO case definition) and an understanding of the therapeutic needs of 
symptomatic survivors, thus informing South African health care. 
Design  
This is a single centre, follow-up, observational, cross-sectional study of four distinct, 
longitudinal cohorts. Extensive clinical history will be obtained from each participant, and 
symptom questionnaire characterisation of Long-COVID (with a strong focus on organ-
specific dysfunction, psychiatric, sleep, and pain parameters – all of which appear to be 
major features of Long-COVID), as well as laboratory and genetic characterisation will be 
performed. A subset of each cohort will be randomly selected for more specific syndrome 
characterisation related to sleep and pain, respiratory, cardiology, renal and glucose 
metabolism. 
 
The consequences of Long-COVID will be described and compared in four large, well-
described clinical cohorts of African patients surviving SARS-CoV-2: 
• Cohort 1: asymptomatic subjects found to be PCR/antigen/antibody-positive during 
routine screening for SARS-CoV-2 infection 
• Cohort 2: symptomatic outpatients who were confirmed to have COVID-19 through a 
positive PCR/antigen test 
• Cohort 3: inpatients surviving hospitalisation for severe COVID-19 and who were 
PCR/antigen-positive 
• Cohort 4: participants vaccinated in clinical trials in 2020 prior to widespread 
community exposure to SARS-CoV-2, and hence protected from severe COVID-19 
(and possibly Long-COVID) if subsequently infected. 
 
After obtaining informed consent from potential participants, a single cross-sectional, 
baseline visit will be conducted for each participant. Demographic data, clinical history 
(including COVID-19 history, targeted symptoms, and risk factors), COVID-19 vaccination 
dates (if administered), and details of previous and concomitant medications will be 
collected. Multiple questionnaires related to psychiatric screening, psychosocial factors, 
work function assessment, sleep quality, and pain assessment will be administered. 
Respiratory and cardiac function will be evaluated through a dyspnoea scale, walking test 
and an electrocardiogram (ECG). Laboratory evaluations will include a full blood count, 
serum chemistry, liver function tests, renal function assessment, inflammatory markers, 
and DNA extraction for genotyping. Blood and urine samples will be stored locally for 
possible future analysis. HIV testing will be performed for participants consenting to this 
optional assessment. 
 
After the baseline visit, participants with Long-COVID will be identified using the WHO 
clinical definition and general health assessments [2]. Randomly selected sub-groups of 
participants with, and without, Long-COVID will be selected from each of the four cohorts 
for additional investigations through participation in the following sub-studies: 
• Respiratory evaluation: dyspnoea assessment, high-resolution CT scan, lung function 
studies including spirometry and diffusion capacity (DLCO) 
• Cardiac evaluation: clinical history and examination, serial blood pressure, six minute 
walk test (distance), ECG, echocardiogram including speckle tracking, cardiac 
magnetic resonance imaging (MRI), creatine kinase MB fraction (CK-MB), cardiac 
troponin T (cTnT), prohormone brain natriuretic peptide (pro-BNP), and possible 

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coronary angiography in patients with acute coronary syndromes and unstable 
angina 
• Psychiatric and neuroendocrine evaluation: questionnaires/surveys, semi-structured 
interview, home visit, saliva cortisol analysis, collection of diary data, actigraphy, 
adrenocorticotropic hormone (ACTH) challenge (cosyntropin sensitivity test [CST]), 
cellular immunity assessment 
• Sleep evaluation: questionnaires, actigraphy with sleep diaries, polysomnography 
• Pain evaluation: quantitative sensory testing (QST) and conditioned pain modulation 
(CMT) assessments 
• Glucose metabolism evaluation: oral glucose tolerance test (OGTT) including 
assessment of glucose, insulin, and c-peptide to estimate insulin sensitivity and beta-
cell function [16]. 
 
Abnormalities detected in the assessments (including undiagnosed mental health issues) 
will be managed by on-study medical personnel with referral as appropriate. 
Population 
Adults of at least 18 years of age with previous confirmed SARS-CoV-2 infection 
(symptomatic or asymptomatic) will be invited to participate. 
 
The following eligibility criteria will be used to select study participants for the main 
study (baseline visit only): 
• Inclusion criteria (baseline visit/main study): 
1. Able and willing to provide written or electronic informed consent for the 
baseline visit prior to any study-specific assessment or procedure. 
2. Age at least 18 years at the time of signing the informed consent form. 
3. Previous asymptomatic SARS-CoV-2 infection, confirmed through a 
documented positive PCR, antigen, or antibody test, at least six months prior to 
the baseline visit [Cohort 1] 
or, 
previous symptomatic SARS-CoV-2 infection for which hospitalisation was not 
required, confirmed through a documented positive PCR or antigen test at the 
time, at least six months prior to the baseline visit [Cohort 2 only] 
or, 
previous hospitalisation for management and treatment of COVID-19 
confirmed through a documented positive PCR or antigen test at the time, at 
least six months prior to the baseline visit [Cohort 3 only] 
or, 
previous asymptomatic or symptomatic SARS-CoV-2 infection, confirmed 
through a documented positive PCR, antigen, or antibody test, at least six 
months prior to the baseline visit and received a COVID-19 vaccine in a non-
placebo arm of a COVID-19 vaccine study during 2020 [Cohort 4 only]. 
4. Willing to consent to verification of vaccination status on the national 
Electronic Vaccination Data System (EVDS). 
5. Access to a reliable telephone or other device permitting information transfer. 
• Exclusion criteria (baseline visit/main study): 
1. Symptomatic SARS-CoV-2 infection at any stage prior to the baseline visit 
[Cohort 1 only]. 
2. Known SARS-CoV-2 infection, confirmed through a documented positive PCR 
test, prior to vaccination in a non-placebo arm of a COVID-19 vaccine study 
during 2020 [Cohort 4 only]. 
3. COVID-19 within three months of the baseline visit. 
4. Personnel (e.g., investigator, sub-investigator, research assistant, pharmacist, 
study coordinator or anyone mentioned in the delegation log) directly involved 
in the conduct of the study. 
5. Any physical, mental, or social condition, that, in the Investigator’s judgment, 
might interfere with the completion of the baseline assessments and 

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evaluations. The Investigator should make this determination in consideration 
of the volunteer’s medical history. 
6. Participant is judged by the Investigator to be at significant risk of failing to 
comply with the provisions of the protocol as to cause harm to self or seriously 
interfere with the validity of the study results. 
 
Additional eligibility criteria will be used to identify study participants who are eligible 
for random selection for any of the sub-studies: 
• Inclusion criteria (sub-studies): 
1. Enrolled into main study and completed baseline visit. 
2. Able and willing to provide written or electronic informed consent for the 
relevant sub-study. 
• Exclusion criteria (sub-studies): 
1. Pregnant women [Respiratory sub-study only]. 
2. Current or previous smokers [Respiratory sub-study only; refer to Respiratory 
Sub-Study Plan for definition of “previous”]. 
3. A well-characterised history of household exposure to biomass fuel used in the 
dwelling for heating and/or cooking, which preceded the acute COVID-19 
illness [Respiratory sub-study only]. 
4. A well-characterised underlying comorbid respiratory illness being treated on a 
continuous basis, which preceded the acute COVID-19 illness [Respiratory sub-
study only]. 
5. A well-characterised history of having had tuberculosis, appropriately 
diagnosed and treated, prior to the acute COVID-19 illness [Respiratory sub-
study only]. 
6. A history of diabetes and/or use of treatment for diabetes mellitus (Type 1 or 2) 
prior to COVID-19 or asymptomatic SARS-CoV-2 infection [Glucose metabolism 
sub-study only]. 
7. Current, known diabetic or on treatment for diabetes mellitus (Type 1 or 2) 
[Glucose metabolism sub-study only]. 
 
Participants may be enrolled into more than one sub-study. 
Treatment 
No treatment will be administered during this observational study. 
Objectives and 
Endpoints 
Primary Objective 
Primary Endpoint 
To characterise Long-COVID in four 
cohorts of patients with previous SARS-
CoV-2 infection (asymptomatic, 
symptomatic, severely symptomatic 
requiring hospitalisation, and those who 
were vaccinated prior to widespread 
community exposure). 
Incidence, severity, and duration of Long-
COVID symptoms. 
 
Secondary Objectives 
Secondary Endpoints 
 
To describe the following characteristics in 
four cohorts of patients with previous 
SARS-CoV-2 infection (asymptomatic, 
symptomatic, severely symptomatic 
requiring hospitalisation, and those who 
were vaccinated prior to widespread 
community exposure): 
 

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• Inflammatory markers 
• High sensitivity C-reactive protein (hs-
CRP) 
• Interleukin-1 (IL-1) 
• Interleukin-6 (IL-6) 
• Interleukin-8 (IL-8) 
• Tumour necrosis factor alpha (TNF) 
• Tumour necrosis factor alpha receptor-
1 (TNFR1) 
• Monocyte chemoattractant protein-1 
(MCP-1) 
 
• Psychological profiles 
• Headache Impact Test-6 
• Patient Health Questionnaire-9 
• Generalised Anxiety Disorder 7 
• PTSD Checklist for DSM-5 - Civilian 
Version 
• Mood Disorder Questionnaire 
• Montreal Cognitive Assessment 
• Daily Fatigue Impact Scale 
 
• Psychosocial exposures 
• COVID-19 related stress questionnaire 
• Multidimensional Scale of Perceived 
Social Support 
• Perceived Stress Scale 
• Adverse Childhood Experiences tool 
 
• Work performance in employed 
participants 
• Normal activities and work productivity 
questionnaire 
 
• Sleep quality and disorders 
• Pittsburgh Sleep Quality Index 
• Epworth Sleepiness Scale 
• Berlin Questionnaire for risk of sleep 
apnoea 
• International Restless Legs Syndrome 
Severity Scale 
• Sleep quality and mood visual 
analogue scale 
 
• Pain experience 
• Brief Pain Inventory 
 
• Cardiorespiratory function 
• Modified Medical Research Council 
Dyspnoea Scale 
• Six-minute walk test (distance) 
• ECG parameters and morphology 
 
• Standard laboratory parameters 
• Full blood count 
• Serum chemistry 
• Liver function tests 
• Glucose, HbA1C 
 
• Renal function 
• Creatinine clearance 
• Cystatin-C 
• Urine dipstick parameters 
• Urine albumin-to-creatinine ratio 
 
• Host genetic factors that may be 
associated with Long-COVID 
• Genotyping results 
• DNA sequencing results 
 
 

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Exploratory Objectives 
 
• The association between various specialised respiratory, cardiac, psychiatric and 
neuroendocrine, sleep and inflammation, pain, and glucose metabolism parameters 
and Long-COVID symptomatology will be explored in patients with Long-COVID from 
four cohorts of participants previously infected with SARS-CoV-2 compared to those 
with no signs or symptoms of Long-COVID (controls) from the same cohorts. 
Sample Size 
One hundred (100) participants will be enrolled into each cohort (400 participants 
overall). 
 
Enrolled participants with and without Long-COVID will be assessed for their suitability 
for various sub-studies. From the eligible participants, up to 30 from each cohort will be 
randomly selected to participate in each sub-study (while ensuring the requisite ratio of 
participants with Long-COVID to those without is maintained as applicable for each sub-
study). The same participant may participate in more than one sub-study. 
 
No formal sample size calculation was performed for this observational study. 
Duration 
The duration of participation for each participant will either be a single visit (of 
approximately 6 hours), or two or more visits depending on whether they are enrolled 
into any sub-studies. 
 
Participants selected for the sleep study will have an overnight admission at the Wits 
Faculty of Health Sciences sleep laboratory. 
 
Enrolment to the study is expected to complete within one year. 
Statistical 
Analysis 
Participants enrolled in the main, and various sub-studies, will be summarised. 
Demographic data and other baseline characteristics will be summarised overall, per 
cohort, and per disease status (Long-COVID or no Long-COVID) as relevant. 
 
All primary and secondary endpoints will be summarised descriptively overall, per cohort, 
and per disease status (Long-COVID or no Long-COVID) as relevant, and for various risk 
factors as deemed appropriate in this exploratory study. Associations between outcomes, 
COVID-19 history, vaccination status prior to and since infection, and COVID-19 risk 
factors for severe disease will be explored. Co-factors such as time since initial SARS-CoV-
2 infection, and possible confounders such as vaccination, exposure to potential disease-
modifying repurposed drugs, and undisclosed vaccination or prior infections, will be 
accounted for as relevant. 
 
Raw genetic genotyping results will be quality controlled by experienced SBIMB bio-
informaticists. Association and fine-mapping will be used to investigate links between 
host genetics and Long-COVID variables generated by the broader study. 
 
 
 

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1. 
SCHEDULE OF ASSESSMENTS 
All participant enrolled will form part of the main study. Main study assessments, questionnaires, and 
procedures may be performed or administered in any order. Specific sub-study questionnaires and 
assessments are detailed in Sections 7.2 and in the sub-study-specific plans. 
 
Main Study 
Sub-Study(ies) 
 
Day 1 (+281) 
Day 2 to 180 (+281) 
Procedure / assessment / questionnaire 
Screening 
Baseline 
FU#1 
Sub-Study 
FU#2 
Informed consent2 
X 
 
 
 
 
SARS-CoV-2 infection and vaccination history3 
X 
 
 
 
 
Main study eligibility review 
X 
 
 
 
 
Demographic data 
 
X 
 
 
 
Medical and surgical history4 
 
X 
 
 
 
Long-COVID symptom-directed history5 
 
X 
 
 
 
Previous and concomitant medications6 
 
X 
 
 
 
Symptom-directed physical examination7 
 
X 
 
 
 
Vital signs8 
 
X 
 
 
 
Psychological and Somatic Symptoms of 
COVID-19 Survey 
 
X 
 
 
 
Headache Impact Test-6 (HIT-6) 
 
X 
 
 
 
Patient Health Questionnaire-9 (PHQ-9) 
 
X 
 
 
 
Generalised Anxiety Disorder 7-item (GAD-7) 
 
X 
 
 
 
PTSD Checklist – Civilian Version (PCL-C) 
 
X 
 
 
 
Mood Disorder Questionnaire (MDQ) 
 
X 
 
 
 
Montreal Cognitive Assessment (MOCA) 
 
X 
 
 
 
Daily Fatigue Impact Scale (D-FIS) 
 
X 
 
 
 
COVID-19 Related Stress Survey 
 
X 
 
 
 
Multidimensional Scale of Perceived Social 
Support (MSPSS) 
 
X 
 
 
 
Perceived Stress Scale-10 (PSS-10) 
 
X 
 
 
 
Adverse Childhood Experiences (ACEs) 
Questionnaire 
 
X 
 
 
 
Normal Activities and Work Productivity 
Questionnaire 
 
X 
 
 
 
Pittsburgh Sleep Quality Index (PSQI) 
 
X 
 
 
 
Epworth Sleepiness Scale (ESS) 
 
X 
 
 
 
Berlin Questionnaire 
 
X 
 
 
 
International Restless Legs Syndrome Study 
Group Severity Rating Scale (IRLS) 
 
X 
 
 
 
Sleep quality and mood visual analogue scale 
 
X 
 
 
 
Brief Pain Inventory (BPI) 
 
X 
 
 
 
Modified MRC Dyspnoea Scale 
 
X 
 
 
 
Six-minute walk test (distance) 
 
X 
 
 
 
ECG9 
 
X 
 
 
 
Blood sample for laboratory tests10 
 
X 
 
 
 
Blood and urine sample storage11 
 
X 
 
 
 
Optional HIV test 
 
(X) 
 
 
 
Selection of participants for sub-study(ies)12 
 
X---------------X 
 
 
 
 

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Main Study 
Sub-Study(ies) 
 
Day 1 (+281) 
Day 2 to 180 (+281) 
Procedure / assessment / questionnaire 
Screening 
Baseline 
FU#1 
Sub-Study 
FU#2 
Telephonic follow-up 
 
 
X13 
 
X14 
Sub-study informed consent 
 
 
 
X15 
 
Participation in one, or more, sub-study 
 
 
 
X---------------X 
Collection of adverse event data16 
 
X 
 
X 
 
FU#1/2 = Telephonic follow-up number 1 or 2; ECG = electrocardiogram; HIV = human immunodeficiency virus 
1 Time window for the follow-up telephonic contact is +28 days from completion of the assessments for the relevant 
part of the study. 
2 Written or electronic informed consent to be provide prior to any screening or study-specific assessments, 
questionnaires or procedures are conducted. 
3 Including dates of diagnosis through SARS-CoV-2 PCR or antigen testing, or date of positive antibody testing, COVID-19 
vaccination dates (if administered), COVID-19 hospitalisation dates (if applicable). 
4 Including information related to specific risk factors associated with poor COVID-19 outcomes and a detailed history of 
previous COVID-19 episodes (if applicable). 
5 Targeting symptoms not covered in other administered surveys and questionnaires.  
6 Details regarding previous and concomitant medications from the time of the participant’s confirmed SARS-CoV-2 
infection will be collected. 
7 Including height and weight, and at least a general, cardiorespiratory, and skin examination and other systems as 
indicated through reported symptoms. 
8 Vital signs (blood pressure, pulse rate, respiratory rate, body temperature, and SpO2) will be measured after 5 min 
resting. 
9 A standard 12-lead ECG will be conducted after the participant has rested in the supine position for 10 minutes. 
10 A blood sample will be collected for full blood count, serum chemistry, liver function, renal function, glucose, HbA1C, 
and inflammatory markers assessments, and for DNA extraction for genotyping and sequencing. 
11 Blood (50 mL) and urine (100 mL) samples will be stored for possible future analysis pending emerging information. 
12 Study team personnel will review each participant’s baseline data and identify participants with and without Long-
COVID who are eligible for participation in one, or more, sub-study. Invited participants will be randomly selected from 
the set of eligible participants for each sub-study. Participants who decline the invitation to participate in one, or more, 
sub-study, will be replaced by a randomly selected, matched-cohort participant where possible. 
13 Participants will be contact telephonically to inform them of the results of their baseline assessments, and to arrange 
for follow-up clinical consultations as required for any identified medical or mental health issues. Participants that have 
been selected for sub-study participation will be invited to return for consent and enrolment into these sub-studies. 
14 Participants will be contact telephonically to inform them of the results of their sub-study assessments, and to arrange 
for follow-up clinical consultations as required for any identified medical or mental health issues. 
15 Written or electronic informed consent to be provide prior to any sub- study-specific assessments, questionnaires or 
procedures are conducted. 
16 Adverse events associated with study assessments or procedures will be recorded in the eCRF for each participant. 
2. 
INTRODUCTION 
2.1 
Background 
“Long-COVID’’ (also known as post-COVID-19 syndrome, post-acute sequelae of COVID-19, or chronic 
COVID syndrome, used here as ‘Long-COVID’ for brevity), is a complex array of postconvalescence 
symptoms following SARS-CoV-2 infection. The syndrome, common in COVID-19 survivors, can affect 
every organ system through as-yet uncharacterised but presumed immunological mechanisms. 
Prevalence depends on the definition used and time-period of follow-up, as well as the population 
being studied. The syndrome has been associated with significant and persistent disability in some 
survivors but has been hampered, until recently, by lack of a clinical definition, diagnostic criteria, and 
objective measures of disease or disability [1]. A Delphi-informed initial World Health Organisation 
(WHO) clinical definition was released in early October 2021 but has attracted much criticism from 
both clinicians and survivors for a host of reasons, ranging from a lack of precision to a lack of inclusion 
[2]. 

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Further complicating the syndrome is the context in which the SARS-CoV-2 epidemic occurred, which 
was associated with severe lockdowns in many countries (including South Africa) with social isolation, 
widespread fear and disinformation, widespread economic hardship, and loss of family and 
acquaintances, all of which contribute to symptoms (psychiatric and sleep disturbances, pain, and 
other syndromes) reported to be associated with Long-COVID. Finally, many Long-COVID symptoms 
overlap with those seen in patients hospitalised for any severe illness, especially those admitted to 
intensive care and ventilated. However, the proliferation of literature reporting associations of Long-
COVID symptoms with more severe COVID-19 disease, and objective immunological, radiological, and 
organ-specific dysfunction in those reporting symptoms, suggests that the entity is real. The 
pathogenesis of Long-COVID is poorly understood, but this association with more severe disease - 
where immune dysregulation plays a major role in those with hospitalization, respiratory failure, and 
death - suggests an immune-mediated inflammatory dysfunction that may impact all organs [3-14]. 
The sheer rapidity of four major infection waves in South Africa, the initial focus on containing the 
hospital burden of those with severe illness, and subsequent emphasis on the roll-out of a mass 
vaccination program, has left little space for studying SARS-COV-2 sequalae in survivors. This group, 
loosely and inaccurately termed “recovered’’ in South African reporting, were largely unvaccinated or 
partly vaccinated at the time of infection, leaving them at risk of developing Long-COVID. 

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2.2 
Long-COVID 
 
Figure 1: 
Long-term effects of coronavirus disease 2019 (COVID-19). The meta-analysis of the 
studies included an estimate for one symptom or more reported that 80% of the patients with COVID-
19 have long-term symptoms. 
CRP = C-reactive protein, CT = computed tomography, IL-6 = Interleukin-6, NT-proBNP = (NT)-pro 
hormone BNP, OCD = Obsessive Compulsive Disorder, PTSD = Post-traumatic stress disorder. 
This figure was created using Biorender.com (Reproduced under a Common Creation Licence, from 
Lopez-Leon, S., Wegman-Ostrosky, T., Perelman, C. et al [4]) 
 
Long-COVID reported symptoms are extraordinarily broad. Over 50 symptoms were reported in a 
recent review, with over 80% of COVID-19 survivors having at least one symptom, sign, or abnormal 

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laboratory parameter after infection [4]. Fatigue (58%), headache (44%), attention disorder (27%), 
hair loss (25%), and dyspnoea (24%) were the most reported symptoms in this review, with abnormal 
chest x-ray or computed tomography (CT) scan findings detected in 34% of patients. Other symptoms 
and signs were related to lung disease (cough, chest discomfort, reduced pulmonary diffusing 
capacity, sleep apnoea, and pulmonary fibrosis), cardiovascular disorders (arrhythmias, myocarditis), 
neuropsychiatric diseases (dementia, depression, anxiety, obsessive–compulsive disorders), and 
others were non-specific such as tinnitus and night sweats. 
The diagnosis of Long-COVID is one of exclusion, and symptoms may fluctuate although tend to 
resolve with time. It is unclear whether disability and organ damage may be permanent in some 
patients. No specific treatment exists, and interventions are typically symptom-directed. SARS-CoV-2 
vaccination appears to benefit many symptomatic survivors [15]. 
At the time of writing, no African data characterising the clinical features of Long-COVID have been 
published. 
2.3 
Study Rationale 
Characterizing Long-COVID and related disability in local populations is an important first step to 
understanding the burden of morbidity and potential requirements of the health system in addressing 
symptoms and disability. 
Almost all studies on Long-COVID have been undertaken in higher income countries, and largely in 
hospitalised patients, with almost no data emerging yet from Africa. This study aims to characterise 
Long-COVID in South African patients by leveraging an existing patient base (including asymptomatic 
outpatients, symptomatic outpatients, and hospitalised patients with severe COVID-19) and local 
research expertise. 
The study is anticipated to add significantly to understanding Long-COVID, and to contribute African 
data to current efforts to develop syndrome diagnostic criteria (including the WHO case definition) 
and an understanding of the therapeutic needs of symptomatic survivors, thus informing South 
African health care. 
2.3.1 
Rationale for specific sub-study focus areas 
• 
Psychiatric, pain and sleep assessments: All are major features of reported Long-COVID and are 
associated with impact on quality of life [4,5,7,8,11]. Sleep disturbances are associated with 
chronic inflammation and immune activation. 
• 
Respiratory assessment: SARS-CoV-2 is a respiratory virus, and most of the severe morbidity and 
mortality seen with COVID-19 is due to lung involvement. Respiratory symptoms, parenchymal, 
and functional abnormalities have been well-described and are common in survivors [9,13]. 
• 
Cardiac assessment: Significant myocarditis, both acute and chronic, has been described with 
COVID-19 infection, and concern expressed about long term effects on function [12,17]. 
• 
Renal assessment: Kidney injury is common with acute SARS-CoV-2 infection, and subclinical 
inflammation and injury may persist for many months, with progressive decline in kidney function 
[10]. 
• 
Glucose metabolism assessment: Diabetes is a strong risk factor for severe COVID-19 disease, as 
well as for Long-COVID and B-cell dysfunction, often irreversible, in survivors [11,18]. 
• 
Genetic assessment: SARS-CoV-2, COVID-19, and vaccination immune responses have notable 
clinical heterogeneity and interindividual differences, whether susceptibility to the virus, severity 
of COVID-19, or vaccine response, and may be partly ascribed to host genetic factors. Early host 

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genetic investigations have been limited to acute COVID-19 in European-ancestry individuals, with 
almost no attention to genetic correlations with Long-COVID [19,20,21]. 
3. 
OBJECTIVES AND ENDPOINTS 
Primary 
Objectives 
Endpoints 
To characterise Long-COVID in four cohorts of 
patients with previous SARS-CoV-2 infection 
(asymptomatic, symptomatic, severely 
symptomatic requiring hospitalisation, and those 
who were vaccinated prior to widespread 
community exposure). 
Incidence, severity, and duration of Long-COVID 
symptoms. 
Secondary 
Objectives 
Endpoints 
To describe the following characteristics in four 
cohorts of patients with previous SARS-CoV-2 
infection (asymptomatic, symptomatic, severely 
symptomatic requiring hospitalisation, and those 
who were vaccinated prior to widespread 
community exposure): 
 
• Inflammatory markers 
• High sensitivity C-reactive protein (hs-CRP) 
• Interleukin-1 (IL-1) 
• Interleukin-6 (IL-6) 
• Interleukin-8 (IL-8) 
• Tumour necrosis factor alpha (TNF) 
• Tumour necrosis factor alpha receptor-1 
(TNFR1) 
• Monocyte chemoattractant protein-1 (MCP-1) 
• Psychological profiles 
• Headache Impact Test-6 (HIT-6) 
• Patient Health Questionnaire-9 
• Generalised Anxiety Disorder 7 
• PTSD Checklist for DSM-5 - Civilian Version 
• Mood Disorder Questionnaire 
• Montreal Cognitive Assessment 
• Daily Fatigue Impact Scale 
• Psychosocial exposures 
• COVID-19 related stress questionnaire 
• Multidimensional Scale of Perceived Social 
Support 
• Perceived Stress Scale 
• Adverse Childhood Experiences tool 
• Work performance in employed participants 
• Normal activities and work productivity 
questionnaire 

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• Sleep quality and disorders 
• Pittsburgh Sleep Quality Index 
• Epworth Sleepiness Scale 
• Berlin Questionnaire for risk of sleep apnoea 
• International Restless Legs Syndrome Severity 
Scale 
• Sleep quality and mood visual analogue scale 
• Pain experience 
• Brief Pain Inventory 
• Cardiorespiratory function 
• Modified Medical Research Council (mMRC) 
Dyspnoea Scale 
• Six-minute walk test (distance) 
• Electrocardiogram (ECG) parameters and 
morphology 
• Standard laboratory parameters 
• Full blood count 
• Serum chemistry 
• Liver function tests 
• Glucose, glycosylated haemoglobin (HbA1C) 
• Renal function 
• Creatinine clearance 
• Cystatin-C 
• Urine dipstick parameters 
• Urine albumin-to-creatinine ratio 
• Host genetic factors that may be associated 
with Long-COVID 
• Genotyping results 
• DNA sequencing results 
Exploratory Objectives 
• Various specialised respiratory, cardiac, psychiatric and neuroendocrine, sleep and inflammation, 
pain, and glucose metabolism parameters will be explored in patients with Long-COVID from four 
cohorts of participants previously infected with SARS-CoV-2 compared to those with no signs or 
symptoms of Long-COVID (controls) from the same cohorts. 
 
4. 
STUDY DESIGN 
4.1 
Overall Design 
This is a single-centre, follow-up, observational, cross-sectional study of four distinct, longitudinal 
cohorts. Extensive clinical history will be obtained from each participant, and symptom questionnaire 
characterisation of Long-COVID (with a strong focus on organ-specific dysfunction, psychiatric, sleep, 
and pain parameters – all of which appear to be major features of Long-COVID), as well as laboratory 
and genetic characterisation will be performed. A subset of each cohort will be randomly selected for 
more specific syndrome characterisation related to sleep and pain, respiratory, cardiology, renal and 
glucose metabolism. 
The consequences of Long-COVID will be described and compared in four large, well-described clinical 
cohorts of African patients surviving SARS-CoV-2: 
• 
Cohort 1: asymptomatic subjects found to be PCR/antigen/antibody-positive during routine 
screening for SARS-CoV-2 infection 

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• 
Cohort 2: symptomatic outpatients who were confirmed to have COVID-19 through a positive 
PCR/antigen test 
• 
Cohort 3: inpatients surviving hospitalisation for severe COVID-19 and who were PCR/antigen-
positive 
• 
Cohort 4: participants vaccinated in clinical trials in 2020 prior to widespread community 
exposure, and hence protected from severe COVID-19 (and possibly Long-COVID) if subsequently 
infected. 
After obtaining informed consent from potential participants, a single cross-sectional, baseline visit 
will be conducted for each participant. Demographic data, clinical history (including COVID-19 history, 
targeted symptoms, and risk factors), COVID-19 vaccination dates (if administered), and details of 
previous and concomitant medications will be collected. Multiple questionnaires related to psychiatric 
screening, psychosocial factors, work function assessment, sleep quality, and pain assessment will be 
administered. Respiratory and cardiac function will be evaluated through a dyspnoea scale, walking 
test and an ECG. Laboratory evaluations will include a full blood count, serum chemistry, liver function 
tests, renal function assessment, inflammatory markers, and DNA extraction for genotyping. Blood 
and urine samples will be stored locally for possible future analysis. Human immunodeficiency virus 
(HIV) testing will be performed for participants consenting to this optional assessment. 
After the baseline visit, participants with Long-COVID will be identified using the WHO clinical 
definition and general health assessments [2]. Randomly selected sub-groups of participants with, and 
without, Long-COVID will be selected from each of the four cohorts for additional investigations 
through participation in the following sub-studies: 
• 
Respiratory evaluation: dyspnoea assessment, high-resolution computed tomography (CT) scan, 
lung function studies including spirometry and diffusion capacity (DLCO) [Section 7.2.2.1] 
• 
Cardiac evaluation: clinical history and examination, serial blood pressure, six minute walk test 
(distance), ECG, echocardiogram including speckle tracking, cardiac magnetic resonance imaging 
(MRI), creatine kinase MB fraction (CK-MB), cardiac troponin T (cTnT), prohormone brain 
natriuretic peptide (pro-BNP), and possible coronary angiography in patients with acute coronary 
syndromes and unstable angina [Section 7.2.2.2] 
• 
Psychiatric and neuroendocrine evaluation: questionnaires/surveys, semi-structured interview, 
home visit, saliva cortisol analysis, collection of diary data, actigraphy, adrenocorticotropic 
hormone (ACTH) challenge (cosyntropin sensitivity test [CST]), cellular immunity assessment 
[Section 7.2.2.3] 
• 
Sleep evaluation: questionnaires, actigraphy with sleep diaries, polysomnography (PSN) 
[Section 7.2.2.4] 
• 
Pain evaluation: quantitative sensory testing (QST) and conditioned pain modulation (CPM) 
assessments [Section 7.2.2.5] 
• 
Glucose metabolism evaluation: oral glucose tolerance test (OGTT) including assessment of 
glucose, insulin, and c-peptide to estimate insulin sensitivity and beta-cell function [16]. 
[Section 7.2.2.6] 
Abnormalities detected in the assessments (including undiagnosed mental health issues) will be 
managed by on-study medical personnel with referral as appropriate. 

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4.2 
Study Treatments 
No treatment will be administered during this observational study. Participants taking medication(s) 
at the time of screening and enrolment, will continue to take this throughout their participation in the 
study. 
4.3 
Study Duration 
The duration of participation for each participant will either be a single visit (of approximately 6 hours), 
or two or more visits depending on whether they are enrolled into any sub-studies. 
Total duration for each participant will be up to 6 months depending on sub-study enrolment. 
Participants selected for the sleep study will have an overnight admission at the University of the 
Witwatersrand (Wits) Faculty of Health Sciences sleep laboratory. 
4.4 
Individual Participant Withdrawal 
A participant may be withdrawn from the study for any of the following reasons: 
• 
At the request of the participant (withdrawal of informed consent), irrespective of the reason for 
this 
• 
At the request of the primary care provider if he or she thinks the study is not in the best interest 
of the participant 
• 
At the discretion of the Investigator if he or she believes that continuation in the study would be 
detrimental to the participant’s well-being in any way, or 
• 
At the discretion of the Institutional Review Board/Ethics Committee if they believe that 
continuation in the study would be detrimental to the participant’s well-being in any way. 
Participants will be considered withdrawn if they state an intention to withdraw, fail to return for 
visits, or are lost to follow-up for any other reason. For participants who are lost to follow-up, the 
Investigator will attempt to trace the participant, and will demonstrate "due diligence" by 
documenting all steps taken to contact the participant (e.g., dates of telephone calls, home visit, etc.) 
in the source documents. 
If a participant withdraws consent for disclosure of future information, the Sponsor may retain and 
continue to use any data collected before such withdrawal of consent. 
If a participant withdraws from the study, he or she may request destruction of any samples taken 
and not tested. This must be documented by the Investigator in the site study records. 
Participants who withdraw or are withdrawn will not be replaced. 
5. 
STUDY POPULATION 
Adults at least 18 years of age with previous confirmed SARS-CoV-2 infection (symptomatic or 
asymptomatic) will be invited to participate. Approximately 400 participants will be enrolled. 
5.1 
Cohorts 
The following well-described cohorts of participants will be enrolled (approximately 100 participants 
in each): 
• 
Cohort 1: participants who were asymptomatic but were found to be SARS-CoV-2 
PCR/antigen/antibody-positive during routine screening previously 

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• 
Cohort 2: participants who were previous outpatients with symptomatic COVID-19 confirmed 
through a positive SARS-CoV-2 PCR/antigen test 
• 
Cohort 3: participants who were previous inpatients and survived hospitalisation for severe 
COVID-19 confirmed through a positive SARS-CoV-2 PCR/antigen test 
• 
Cohort 4: participants who were previously infected with SARS-CoV-2 (symptomatic or 
asymptomatic) confirmed through a positive PCR/antigen/antibody test after being vaccinated in 
a non-placebo arm of a SARS-CoV-2 vaccine trial in 2020, with a vaccine demonstrated to be 
effective against severe COVID-19. 
Participants enrolled in early vaccine trials were screened out if they were concurrently infected with 
SARS-CoV-2 as detected through a minimum of PCR testing. Given that they were only exposed to the 
first wave of COVID-19 infections in South Africa during which severe lock-down measures were in 
place, the probability of them being infected with SARS-CoV-2 prior to vaccination would have been 
low. 
5.2 
Recruitment 
Potential participants may volunteer in response to advertised clinical trial information, or will be 
contacted in compliance with POPIA regulations and invited to participate in the informed consent 
process (Section 11.2) and attend the baseline visit. Written informed consent will be obtained prior 
to any screening, and study-specific assessments and procedures. The following platforms will be used 
to identify potential participants: 
• 
Cohort 1: 
o 
Volunteers who had previous asymptomatic SARS-CoV-2 infection confirmed by a 
documented positive PCR/antigen/antibody test during routine screening (e.g. for travel, 
elective surgery or as part of contact tracing) 
o 
Participants enrolled into prophylaxis studies who had a documented PCR/antigen/antibody-
positive test during routine screening but no symptoms of infection 
o 
Participants enrolled in self-sampling and antigen evaluation protocols with documentation 
of a positive PCR/antigen test during routine screening but no symptoms of infection. 
• 
Cohort 2: 
o 
Volunteers who had previous symptomatic COVID-19 confirmed by a documented positive 
PCR/antigen test, and who were managed as outpatients 
o 
Participants enrolled in large, early treatment, antiviral studies in symptomatic outpatients 
o 
Participants enrolled into prophylaxis studies who developed symptoms of infection 
confirmed to be COVID-19 through a documented positive SARS-CoV-2 PCR/antigen test at 
the time 
o 
Participants enrolled in self-sampling and antigen evaluation protocols who developed 
symptoms of infection confirmed to be COVID-19 through a documented positive SARS-CoV-
2 PCR/antigen test. 
• 
Cohort 3: 
o 
Age- and gender-matched patients treated at the Helen Joseph Hospital and Charlotte 
Maxeke Johannesburg Academic Hospital Infectious Diseases department who were infected 
at approximately the same time as participants recruited for Cohorts 1 and 2. 

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• 
Cohort 4: 
o Participants enrolled in a non-placebo arm of a COVID-19 vaccine trial who received a 
vaccine that has subsequently been proven to be effective, and who were subsequently 
confirmed to be infected with SARS-CoV-2 (symptomatic or asymptomatic) through a 
documented positive PCR/antigen/antibody test. 
5.3 
Main Study Eligibility Criteria (Baseline Visit) 
The following eligibility criteria will be used to select study participants for the main study (baseline 
visit only). 
5.3.1 
Inclusion criteria 
1. Able and willing to provide written or electronic informed consent for the baseline visit prior to 
any study-specific assessment or procedure. 
2. Age at least 18 years at the time of signing the informed consent form. 
3. Previous asymptomatic SARS-CoV-2 infection, confirmed through a documented positive PCR, 
antigen, or antibody test, at least six months prior to the baseline visit [Cohort 1 only] 
or, 
previous symptomatic SARS-CoV-2 infection for which hospitalisation was not required, 
confirmed through a documented positive PCR or antigen test at the time, at least six months 
prior to the baseline visit [Cohort 2 only] 
or, 
previous hospitalisation for management and treatment of COVID-19 confirmed through a 
documented positive PCR or antigen test at the time, at least six months prior to the baseline 
visit [Cohort 3 only] 
or, 
previous asymptomatic or symptomatic SARS-CoV-2 infection, confirmed through a documented 
positive PCR, antigen, or antibody test, at least six months prior to the baseline visit and received 
a COVID-19 vaccine in a non-placebo arm of a COVID-19 vaccine study during 2020 [Cohort 4 
only]. 
4. Willing to consent to verification of vaccination status on the national Electronic Vaccination Data 
System (EVDS). 
5. Access to a reliable telephone or other device permitting information transfer. 
5.3.2 
Exclusion criteria 
1. Symptomatic SARS-CoV-2 infection at any stage prior to the baseline visit [Cohort 1 only]. 
2. SARS-CoV-2 infection, confirmed through a documented positive PCR, antigen, or antibody test, 
prior to vaccination in a non-placebo arm of a COVID-19 vaccine study during 2020 [Cohort 4 only]. 
3. COVID-19 within three months of the baseline visit. 
4. Personnel (e.g., investigator, sub-investigator, research assistant, pharmacist, study coordinator 
or anyone mentioned in the delegation log) directly involved in the conduct of the study. 
5. Any physical, mental, or social condition, that, in the Investigator’s judgment, might interfere with 
the completion of the baseline assessments and evaluations. The Investigator should make this 
determination in consideration of the volunteer’s medical history. 

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6. Participant is judged by the Investigator to be at significant risk of failing to comply with the 
provisions of the protocol as to cause harm to self or seriously interfere with the validity of the 
study results. 
5.4 
Sub-Study Eligibility Criteria (Subsequent Optional Visit(s)) 
The following additional eligibility criteria will be used to identify study participants who are eligible 
for random selection for any of the sub-studies. Participants may be enrolled into more than one sub-
study. 
5.4.1 
Inclusion criteria 
1. Enrolled into main study and completed baseline visit. 
2. Willing to provide written or electronic information consent for the relevant sub-study. 
5.4.2 
Exclusion criteria 
1. Pregnant women [Respiratory sub-study only]. 
2. Current or previous smokers [Respiratory sub-study only; refer to Respiratory Sub-Study Plan for 
definition of “previous”]. 
3. A well-characterised history of household exposure to biomass fuel used in the dwelling for 
heating and/or cooking, which preceded the acute COVID-19 illness [Respiratory sub-study only]. 
4. A well-characterised underlying comorbid respiratory illness being treated on a continuous basis, 
which preceded the acute COVID-19 illness [Respiratory sub-study only]. 
5. A well-characterised history of having had tuberculosis, appropriately diagnosed and treated, 
prior to the acute COVID-19 illness [Respiratory sub-study only]. 
6. A history of diabetes and/or use of treatment for diabetes mellitus (Type 1 or 2) prior to COVID-
19 or asymptomatic SARS-CoV-2 infection [Glucose metabolism sub-study only]. 
7. Current, known diabetic or on treatment for diabetes mellitus (Type 1 or 2) [Glucose metabolism 
sub-study only]. 
5.5 
Justification of the Inclusion and Exclusion Criteria 
The eligibility criteria have been selected to enable evaluation of participants with and without Long-
COVID in clearly defined, real-life, cohorts with previous SARS-CoV-2 infection. Additional eligibility 
criteria have been selected to enable evaluation of specific organ systems in the context of Long-
COVID in selected sub-groups of the enrolled cohorts. 
5.6 
Participant Identification 
All volunteers who provide informed consent to participate in the study will be allocated a unique 
sequential screening number which will be used as the primary identifier for the duration of the study 
for enrolled participants. 
5.7 
Co-enrolment Guidelines 
Participants may be co-enrolled in other research studies if these are observational in nature or 
include behavioural interventions only. Other co-enrolments require approval by the Principal 

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Investigator after consideration of possible confounding effects and participant safety and well-being 
regarding blood draw volumes and exposure to multiple assessments. 
6. 
STUDY VISITS 
A brief screening assessment will be conducted for all study participants who provide informed 
consent for participation in the study. Those who are eligible for enrolment will continue into a 
baseline visit where all main study questionnaires will be administered, and assessments and 
procedures conducted. 
Participants who are eligible for various sub-studies will be identified by the study team after review 
of their baseline visit results. Sub-study participants (up to 30 per cohort per sub-study) will be 
selected from these eligible participants and will be invited to provide separate informed consent for 
each sub-study. An additional visit or visits will be conducted for each sub-study. The number of study 
visits for each participant will depend on the number and type of sub-studies in which they participate. 
All study visits (including the screening/baseline visits and sub-study visits) will be completed for each 
participant within approximately 6 months. 
Participants will be contacted telephonically within 28 days of the baseline visit, and again within 28 
days of their last sub-study visit (if relevant) to inform them of the results of their assessments. For 
participants taking part in the overnight sleep study, however, feedback may be delayed until 
6 months from the overnight visit. Abnormalities detected in assessments (suggesting undiagnosed 
medical, mental health, or sleep issues) will be managed by on-study medical personnel with referral 
as appropriate. 
Questionnaires to be administered, and assessments and procedures to be conducted at each study 
visit (main and sub-studies) are detailed in Section 1. Details regarding each questionnaire, assessment 
and procedure are described in Section 7. 
7. 
STUDY ASSESSMENTS, QUESTIONNAIRES, AND PROCEDURES 
No study-specific assessments will be performed, or information gathered, until the potential 
participant has given written, informed consent (Section 11.2) for screening assessments and (if found 
to be eligible) for study participation. 
The timing of all assessments and procedures is detailed in the Schedule of Assessments (Section 
Error! Reference source not found.). 
7.1 
Main Study 
Screening and baseline assessments are expected to be performed on the same day but may be spread 
over two or more separate days given the expected duration of the visit (approximately 6 hours). 
7.1.1 
Screening assessments (Day 1) 
SARS-CoV-2 infection and vaccination history will be collected. This will include: 
• 
dates of positive SARS-CoV-2 PCR, antigen, and/or antibody tests 
• 
dates of symptomatic COVID-19 
• 
date and duration of hospitalisation for COVID-19 (if relevant) 
• 
date(s) of COVID-19 vaccine(s) administration. 

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Review of the main study eligibility criteria will be performed for all participants providing informed 
consent for the main study (baseline visit). Participants may be re-screened if they may later become 
eligible for participation (due to, e.g., age or interval between SARS-CoV-2 infection and screening). 
7.1.2 
Baseline assessments, questionnaires, and procedures (Day 1 [+7 days]) 
The following main study (baseline) evaluations will be performed. These may be performed in any 
sequential order during the baseline visit. All questionnaires, surveys and rating scales will be 
administered in accordance with the specific guidelines associated with validated versions of the tools 
and will be included in the Study Assessments Manual. 
Participants will be required to fast for 8 hours (water permitted ad libitum) prior to the drawing of 
blood for serum glucose assessments. If additional baseline assessments are scheduled for the same 
day as the blood draw, this will be performed as one of the first procedures of the visit and participants 
will be given a meal at the research unit before proceeding with the remaining assessments. 
7.1.2.1 
Demographics, social background and habits data 
The following will be collected: 
• 
Sex, age, race, and country of origin 
• 
Town of residence 
• 
Marital status, occupation, and educational attainment 
• 
Socioeconomic status, access to healthcare, financial support, and debt 
• 
Household conditions (including home type, water facility, toilet facility, assets), number of people 
in household, number of rooms in household used for sleeping, and number of people in 
household who have had COVID-19 
• 
Use of tobacco products, alcohol, and illicit/street drugs. 
7.1.2.2 
Medical and surgical history 
In addition to general information related to past and current relevant medical conditions and surgical 
procedures, and menstrual cycle, pregnancy and lactation status (for female participants only), the 
following specific information will be collected: 
• 
COVID-19 history (for participants with previous symptomatic infection(s)) including: 
o 
Symptoms experienced (including cough, fatigue, fever, malaise, shortness of breath, 
ageusia, anosmia, and other symptoms) 
o 
Whether or not the participant was pregnant at the time of SARS-CoV-2 infection (female 
participants only) 
o 
Whether or not the participant required supplemental oxygen at the time of COVID-19 
o 
Whether or not the participant required intensive care unit (ICU) admission for COVID-19 
(Cohort 3 only) 
o 
Whether or not the participant required mechanical ventilation for COVID-19 (Cohort 3 only) 
• 
Long-COVID symptom-directed history to gather information about symptoms not assessed 
through the detailed questionnaires and surveys listed in Sections 7.1.2.5 to 7.1.2.6. 

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7.1.2.3 
Previous and concomitant medications 
Details of previous and ongoing medications taken from the time of the participant’s confirmed SARS-
CoV-2 infection will be collected. 
7.1.2.4 
Physical examination and vital signs 
Height and weight (as measured in light indoor clothing or underwear only, but without shoes) will be 
measured, and body mass index (BMI) derived. 
A symptom-directed physical examination will be performed (including at least a general, 
cardiorespiratory, and skin examination and other systems as indicated through reported symptoms). 
Vital signs (blood pressure, pulse rate, respiratory rate, body temperature, and oxygen saturation 
[SpO2]) will be measured after 5 min resting. 
7.1.2.5 
Psychological/psychosocial/sleep/work function evaluation 
The following questionnaires/surveys will be administered to screen for general health and 
psychological issues: 
• 
Psychological and Somatic Symptoms of COVID-19 Survey: a questionnaire assessing psychological 
symptoms of COVID-19 sequelae including fatigue, brain fog, headache, muscle weakness, 
delusions, hallucinations, disorganized thoughts/behaviours, slurred speech, and somatic 
symptoms 
• 
Headache Impact Test-6 (HIT-6): a tool for assessing the impact of headaches in patients’ day-to-
day lives, including six main domains affected by headaches (pain, social functioning, role 
functioning, cognitive functioning, vitality, and psychological stress) [22] 
• 
Patient Health Questionnaire-9 (PHQ-9): a multipurpose instrument for screening, diagnosing, 
monitoring, and measuring the severity of depression [23] 
• 
Generalised Anxiety Disorder 7-item (GAD-7): a short screening tool evaluating 7 symptoms of 
anxiety in adults [24] 
• 
PTSD Checklist – Civilian Version (PCL-C): a 20-item self-report measure that assessed the 20 DMS-
5 symptoms of PTSD [25] 
• 
Mood Disorder Questionnaire (MDQ): a short screening tool evaluating the symptoms of bipolar 
disorder [26] 
• 
Montreal Cognitive Assessment (MOCA): a cognitive screening test designed to accurately and 
quickly assess short term memory, visuospatial abilities, executive functions, attention, 
concentration and working memory, language, and orientation to time and place [27] 
• 
Daily Fatigue Impact Scale (D-FIS): a survey tool that assesses physical, cognitive, and psychosocial 
dimensions of fatigue in everyday life [28]. 
Psychosocial factors will be evaluated through the: 
• 
COVID-19 Related Stress Survey: a survey assessing psychosocial stress experienced during the 
COVID-19 pandemic and lockdown [29] 
• 
Multidimensional Scale of Perceived Social Support (MSPSS): a brief research tool designed to 
measure perceptions of support from 3 sources – family, friends, and a significant other; the scale 
is comprised of a total of 12 items, with 4 items for each subscale [30] 

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• 
Perceived Stress Scale-10 (PSS-10): a survey that assesses the degree to which situations in one’s 
life are appraised as stressful; items are designed to query how unpredictable, uncontrollable, and 
overloaded respondents find their lives [31] 
• 
Adverse Childhood Experiences (ACEs) Questionnaire: a short questionnaire assessing presence 
or absence of a pre-defined list of childhood traumas from the ages of 0 to 18 years [32]. 
Work performance in participants who are employed will be evaluated using the: 
• 
Normal Activities and Work Productivity Questionnaire. 
Sleep quality and associated disorders will be evaluated using the: 
• 
Pittsburgh Sleep Quality Index (PSQI): a self-rated questionnaire which assesses sleep quality and 
disturbances over a 1-month time interval [33] 
• 
Epworth Sleepiness Scale (ESS): a self-administered questionnaire consisting of 8 questions used 
to assess daytime sleepiness [34] 
• 
Berlin Questionnaire: a self-administered questionnaire designed to identify subjects at high risk 
for obstructive sleep apnoea [35] 
• 
International Restless Legs Syndrome Study Group Severity Rating Scale (IRLS) [36] 
• 
Visual analogue scale: 10 cm lines with 2 anchors will be used to assess sleep quality and mood 
(best sleep ever had – worse sleep ever had; feeling happy and motivated, wanting to do things – 
feeling down and low, not wanting to do things). 
7.1.2.6 
Pain assessment 
Pain experience will be assessed using: 
• 
The Brief Pain Inventory (BPI): a widely used questionnaire for the assessment of pain intensity, 
location, and interference with activities of daily living in clinical and research settings [37]. 
7.1.2.7 
Cardiorespiratory assessment 
Basic cardiorespiratory function will be evaluated using: 
• 
The mMRC Dyspnoea Scale [38] 
• 
A standard 12-lead ECG performed after 10 minutes resting in the supine position 
• 
A six-minute walk test: a sub-maximal exercise test used to assess aerobic capacity and endurance 
[39]. 
7.1.2.8 
Laboratory assessments 
Blood sampling will be performed to collect samples for the following laboratory assessments: 
• 
Full blood count: red cell count, haemoglobin, haematocrit, mean cellular volume (MCV), mean 
corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), red cell 
distribution width (RDW), white cell count and differential (neutrophil count, lymphocyte count, 
monocyte count, eosinophil count, and basophil count), and platelet count 
• 
Serum chemistry: serum sodium, potassium, chloride, bicarbonate, calcium (corrected), 
magnesium, phosphate, urate 

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• 
Liver function: total protein, albumin, total bilirubin, direct bilirubin, alkaline phosphatase (ALP), 
gamma-glutamyl transferase (GGT), alanine aminotransferase (ALT), aspartate aminotransferase 
(AST), lactate dehydrogenase (LDH) 
• 
Renal function: urea, creatinine, estimated creatinine clearance (Cockcroft-Gault method), 
cystatin C 
• 
Glucose metabolism: serum glucose, HbA1C 
• 
Inflammatory markers: a multiplex cytokine panel will be run using Luminex technology at the 
Wits School of Physiology. Markers investigated will include hs-CRP, IL-1, IL-6, IL-8, TNF-, 
soluble TNFR1, and MCP-1, but additional inflammatory markers may be included based on 
emerging reports in scientific literature 
• 
DNA extraction for genotyping and sequencing at the Sydney Brenner Institute for Molecular 
Bioscience (SBIMB) 
• 
HIV testing for participants consenting to this (optional). 
A urine sample will be collected for: 
• 
urinalysis via urine dipstick 
• 
determination of the urine albumin-to-creatinine ratio 
• 
pregnancy test for woman of reproductive potential. 
Additional blood (50 mL) and urine (100 mL) samples will be stored for possible future analysis. 
Details regarding blood and urine sample collection, handling and processing, and the assays (where 
applicable) will be documented in the Laboratory Manual. 
7.1.3 
Telephonic follow-up (up to 28 days after completion of baseline visit) 
Participants will be contacted telephonically within 28 days of completion of their baseline visit to 
inform them of the results of their assessments. Abnormalities detected in assessments (suggesting 
undiagnosed medical or mental health issues) will be managed by on-study medical personnel with 
referral as appropriate. 
7.2 
Sub-Studies 
Participants may be invited to take part in more than one sub-study, or none. Up to 30 participants 
from each cohort will be enrolled into each sub-study. Participants with and without Long-COVID will 
be age- and gender-matched where possible. 
7.2.1 
Screening and informed consent 
Separate informed consent will be obtained for each sub-study for participants invited to take part in 
these. 
7.2.2 
Sub-study visit(s) 
7.2.2.1 
Respiratory evaluation 
Participants who are selected and provide consent for participation in the respiratory sub-study, will 
attend a single visit where the following will be performed: 
• 
mMRC Dyspnoea Scale 

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• 
High-resolution CT scan of the chest 
• 
A full set of lung function studies, including full spirometry and DLCO. 
Depending on findings at this visit, participants may continue to be followed up for a further 3- to 6-
month period. 
Further details can be found in the Respiratory Sub-Study Plan. 
7.2.2.2 
Cardiac evaluation 
Participants who are selected and provide consent for participation in the cardiac sub-study, will 
attend up to three additional visits where the following will be performed: 
• 
Clinical history and examination 
• 
Serial blood pressure measurements 
• 
Six minute walk test (distance) 
• 
ECG 
• 
Echocardiogram including speckle tracking 
• 
Cardiac MRI 
• 
Blood sampling for: 
o CK-MB 
o cTnT 
o pro-BNP. 
Coronary angiography may be performed at the discretion of the treating cardiologist and dependent 
upon the clinical setting in patients with a clinical indication for diagnostic coronary angiography (e.g., 
acute coronary syndromes, unstable angina). 
Further details can be found in the Cardiac Sub-Study Plan. 
7.2.2.3 
Psychiatric and neuroendocrine evaluation 
Participants who are selected and provide consent for participation in the psychiatric and 
neuroendocrine sub-study, will have the following additional assessments performed: 
• 
Repeated administration of questionnaires related to the psychological impact of COVID-19 
• 
4-day home collection of cortisol samples, sleep measurements and diary data 
o Serial collection of passive drool saliva samples for evaluation of cortisol concentrations 
o Objective measurements of sleep timing and quality through a wearable, accelerometry 
device (actigraphy) 
o Morning and evening daily diary completion to collect data related to sleep quality, health 
behaviours and stressors 
• 
ACTH challenge (CST) 
• 
Ex vivo cell culture analysis to evaluate cellular immunity through assessment of leukocyte 
inflammatory responses to a microbial challenge 
• 
Semi-structured interview to assess perceptions of, and experiences related to, COVID-19 

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• 
A home visit to further understand the lived experience of long-COVID. 
Further details can be found in the Psychiatric and Neuroendocrine Sub-Study Plan. 
7.2.2.4 
Sleep evaluation 
Participants who are selected and provide consent for participation in the sleep sub-study, will have 
the following additional assessments: 
• 
Repeated administration of questionnaires related to sleep and psychosocial factors 
• 
Objective measurements of sleep performed within 60 days after the baseline visit: 
o 
Actigraphy with sleep diary for a week-long period 
o 
Overnight polysomnography (PSG) at the Wits Faculty of Health Sciences Sleep Laboratory at 
the end of the week-long actigraphy period. 
Further details can be found in the Sleep Sub-Study Plan. 
7.2.2.5 
Pain evaluation 
Participants who are selected and provide consent for participation in the pain sub-study, will attend 
a single visit where the following will be performed: 
• 
QST (using the protocol developed by the German Network for Neuropathic Pain [40,41]) 
• 
CMT (using the protocol published by Lie et. al., 2017 [42]) 
Further details can be found in the Pain Sub-Study Plan. 
7.2.2.6 
Glucose metabolism evaluation 
Participants who are not known diabetics, are not on treatment for diabetes mellitus (Type 1 or 2), 
and who have no history of diabetes or receiving treatment for diabetes mellitus (Type 1 or 2) prior 
to COVID-19 or asymptomatic SARS-CoV-2 infection, will be randomly selected for participation in the 
glucose metabolism sub-study. Those who provide consent for participation will attend a single visit 
where an OGTT, including measures of glucose, insulin, and c-peptide to estimate insulin sensitivity 
and beta-cell function, will be performed [16]. 
Further details can be found in Glucose Metabolism Sub-Study Plan. 
7.2.3 
Telephonic follow-up 
Participants taking part in sub-studies will be contacted telephonically within 28 days of completion 
of their last sub-study visit to inform them of the results of their assessments. The exception is 
participants taking part in the overnight sleep study for whom feedback may be delayed until 
6 months from the overnight visit. Abnormalities detected in assessments (suggesting undiagnosed 
medical or mental health issues) will be managed by on-study medical personnel with referral as 
appropriate. 
7.3 
Biohazard Containment 
Precautions will be employed by all personnel in the handling of blood and urine specimens collected 
during this study. 

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All biological specimens will be transported using packaging mandated by national and regional 
regulations. Details of these procedures will be described in the Laboratory Manual and will comply 
with relevant IATA Dangerous Goods Regulations. 
8. 
SAFETY MONITORING 
8.1 
Responsibilities for Ensuring the Safety of Study Participants 
8.1.1 
Principal Investigator 
The Principal Investigator has a personal responsibility to closely monitor study participants and an 
inherent authority to take whatever measures necessary to ensure their safety, including ensuring 
that procedures and expertise are available to cope with medical emergencies during the study. 
8.1.2 
Study Sponsor 
The Sponsor has an institutional responsibility to ensure participant safety and undertakes to 
promptly notify the Wits Human Research Ethics Committee (HREC) of findings that could adversely 
affect the safety of participants included in the study, impact the conduct of the study, or alter the 
HREC’s approval of, or favourable opinion to continue, the study.  
8.1.3 
Medical Monitor 
The Medical Monitor will provide medical review during the execution of the study. This oversight will 
include the review of safety information and the provision of applicable recommendations to both the 
Investigator and the Sponsor. This review is intended to facilitate protection of the study participants. 
Medical review of data will be specified in the medical section of the Monitoring Plan to be finalised 
prior to enrolment of the first participant. 
In addition to routine review of medical data, the Medical Monitor will also support the site and 
provide advice on protocol clarifications, assessment of eligibility and required medical follow-up of 
participants as and when indicated. 
8.2 
Adverse Events 
8.2.1 
Definitions 
Some of the assessments and procedures used in this study may be associated with rare discomfort 
or adverse events (AEs), e.g., blood sampling, six-minute walk test, injection of contrast media, skin 
irritation from electrodes used in polysomnography. 
For this study, AEs associated with study assessments or procedures will be recorded in the eCRF for 
each participant. The following information be recorded for each AE: 
• 
a description of the AE 
• 
the dates of onset and resolution of the event 
• 
the characteristics of the event (seriousness, severity in accordance with CTCAE Version 5.0, Nov 
2017 [43]) 
• 
the action taken in response to the event (including treatment required) 
• 
the outcome of the event. 

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9. 
STATISTICAL CONSIDERATIONS 
Details of the statistical analyses and their presentation will be documented in the Statistical Analysis 
Plan (SAP). 
9.1 
Sample Size Determination 
No formal sample size was calculated for this exploratory study. On-study findings may suggest that 
larger sample sizes are required to further analyse endpoints in specific groups, and further planning 
for this may be performed. 
9.2 
Analysis and Presentation of Data 
9.2.1 
Disposition, demographic, and background data 
Participants enrolled in the main, and various sub-studies, will be summarised. Demographic data and 
other baseline characteristics will be summarised overall, per cohort, and per disease status (Long-
COVID or no Long-COVID) as relevant.  
9.2.2 
Primary and secondary endpoints 
All primary and secondary endpoints will be summarised descriptively overall, per cohort, and per 
disease status (Long-COVID or no Long-COVID) as relevant, and for various risk factors as deemed 
appropriate in this exploratory study. Associations between outcomes, COVID-19 history, vaccination 
status prior to and since infection, and COVID-19 risk factors for severe disease will be explored. Co-
factors such as time since initial SARS-CoV-2 infection, and possible confounders such as vaccination, 
exposure to potential disease-modifying repurposed drugs, and undisclosed vaccination or prior 
infections, will be accounted for as relevant. 
Raw genetic genotyping results will be quality controlled by experienced SBIMB bio-informaticists. 
Association and fine-mapping will be used to investigate links between host genetics and Long-COVID 
variables generated by the broader study. 
9.2.3 
Safety data 
All AEs will be listed. 
9.2.4 
Exploratory analyses 
Details of exploratory endpoints and analyses to be performed will be included in the SAP. 
10. 
STUDY MONITORING 
Study conduct will be monitored by a research site monitor. Review of individual participant records, 
including consent forms, electronic case report forms (eCRFs), supporting data, questionnaire 
responses, and laboratory specimen records will be performed as detailed in the Clinical Monitoring 
Plan, to ensure protection of study participants, compliance with the protocol, and accuracy and 
completeness of records. The monitors also will inspect the site files to ensure that good clinical 
practice requirements are being followed. 
The Clinical Monitoring Plan will describe these activities and will take into consideration necessary 
adaptations to be made if physical access to the site is limited at any stage due to pandemic 
restrictions. 

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11. 
ETHICAL CONSIDERATIONS 
11.1 
Ethical Considerations 
The study will be conducted according to GCP (including South African GCP Guidelines [44]), the 
Belmont Report, the Declaration of Helsinki, and South Africa legal requirements regarding clinical 
research. The study protocol and relevant supporting documents will be submitted for review and 
approval by the Wits HREC responsible for oversight of research conducted at the study site. The study 
protocol will be registered with the South African National Clinical Trial Registry (www.sanctr.gov.za), 
National Human Research Ethics Committee (www.ethicsapp.co.za) and www.ClinicalTrial.gov. Six-
monthly progress reports will be submitted to the HREC for the duration of the study, and as 
requested. Upon completion or premature termination of the study, the Investigator will provide the 
HREC with a summary of the study’s outcome, and any reports required. 
11.2 
Informed Consent Process 
The principles of informed consent in the current edition of the Declaration of Helsinki will be 
implemented before any protocol-specified procedures or interventions are carried out. The consent 
form will describe the purpose of the study, the assessments, questionnaires, and procedures to be 
performed, and the risks and benefits of participation. Separate consent forms will be available for 
the main study and all sub-studies. Participation in the sub-studies is voluntary and consent will be 
sought for these after completion of the baseline visit of the main study for participants who are 
eligible for further enrolment into sub-studies. Separate consent will be required for each sub-study 
in which a participant is enrolled. 
HIV testing at the baseline visit is voluntary and will only be performed if participants provide explicit 
consent for this testing. Pre- and post-test counselling will be provided in accordance with local 
standard of care. 
Potential participants will have the opportunity to have any questions answered before and after 
signing the informed consent forms (ICFs). The informed consent process and all questions raised will 
be documented.  
The study staff who conduct the informed consent process will also sign the ICFs. A copy of the consent 
form(s) will be given to the participant, and this fact will be documented in the participant’s record. 
Any participant who is rescreened should be reconsented and eligibility for the study must be re-
checked prior to enrolment. 
11.3 
Blood Volume 
The volume of blood to be drawn from participants taking part in the main study is less than 100 mL. 
For a participant taking part in all additional sub-studies, the total blood volume across the entire 
study will not exceed 250 mL over 60 days. 
11.4 
Study Records and Confidentiality 
The study site will establish a standard operating procedure for confidentiality protection. The site will 
ensure that study records, including ICFs, locator forms, case report forms, notations of all contacts 
with the participant, and all other source documents are stored in a secure manner. 
All laboratory specimens, evaluation forms, reports, and other records will be identified in a manner 
designed to maintain participant confidentiality. 

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All records will be kept in a secure storage area with limited access. Clinical information will not be 
released without the written permission of the participant, except as necessary for monitoring, and 
auditing by the HREC. 
The Principal Investigator or designee and all employees and co-workers involved with this study may 
not disclose or use, for any purpose other than performance of the study, any data, record, or other 
unpublished confidential information disclosed to those individuals for the purpose of the study. 
All computers used during the study conduct will be password-protected, and records will only be 
accessible to authorised study staff. 
11.5 
Participant Remuneration 
For each day of protocol-related study procedures, the participants will receive compensation for 
travel costs to and from the clinic and inconvenience incurred, as per local regulating body 
recommendations. 
12. 
ADMINISTRATIVE CONSIDERATIONS 
12.1 
Protocol Amendments 
Any protocol amendments will be prepared by the Sponsor and submitted to the HREC in accordance 
with their requirements. 
Approval must be obtained from the HREC before the implementation of any changes, except for 
changes necessary to eliminate an immediate hazard to participants, or changes that involve logistical 
or administrative aspects only (e.g., change in contact information). 
12.2 
Clinical Data Records 
A log of names, signatures and initials of all staff authorised to enter data into a participant’s clinic file 
and eCRF will be kept. 
The Investigator will maintain paper or electronic source documentation for all study participants. 
Protocol-specific participant information will be captured in an eCRF. The Clinical Data Management 
System will comply with guidelines and requirements for electronic systems used in clinical research. 
Data validation and quality control procedures will be detailed in the Data Management Plan. 
All deviations from this study protocol will be documented in the Trial Master File and included in the 
final study report. An assessment of the significance of each protocol deviation will be presented in 
the clinical study report. 
12.3 
Record Retention 
All source data, clinical records and laboratory data relating to the study will be archived for a 
minimum period after completion of the study in accordance with South African GCP guidelines, 
Sponsor and Funder requirements. Data will be available for retrospective review or audit by 
arrangement with the appropriate representative at the archiving organisation (e.g., Sponsor Head).  
12.4 
Discontinuation of the Study 
The Sponsor, the Funder, the Principal Investigator, and the HREC independently reserve the right to 
discontinue the study at any time for safety or other reasons. This will be done in consultation with 
the Funder, Sponsor and Principal Investigator where practical. In the event of premature termination 
or suspension of the study, the above-mentioned parties will be notified in writing by the 

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terminator/suspender stating the reasons for early termination or suspension. Following such a 
decision, the Sponsor and the Investigator will ensure that adequate consideration is given to the 
protection of the participants’ interest and safety. Ongoing participants will be followed up 
telephonically to ensure that feedback related to the results of their assessments, questionnaires and 
procedures is provided. 
12.5 
Publication Policy 
A dissemination plan will be developed with all project partners prior to study completion. After study 
completion, results will be disseminated using the following strategies: written methods (i.e., 
publications in peer reviewed scientific journals), presentations at scientific conferences and 
workshops, in person dissemination of results to the research participants, and using electronic 
methods such as the project website and electronic media to publish results. 
12.6 
Study Audits 
Audits may be carried out by the SAMRC, Sponsor, or HREC quality assurance representatives. All 
documents pertinent to this study must be made available for such inspections after adequate notice 
of the intention to audit is provided. 

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13. 
REFERENCES 
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2. WHO. A clinical case definition of post COVID-19 condition by a Delphi consensus. 2021. 6 October 
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3. Venter WDF, Nel J. COVID-19: First data from Africa. Clin Infect Dis. Aug 31 2020. 
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More than 50 long-term effects of COVID-19: A systematic review and meta-analysis. Sci Rep. 2021 
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NeuroRehabilitation. 2021;48(4):469-480. doi:10.3233/nre-210025. 
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Hishmeh L, Attree E, Heightman M, Banerjee R, Banerjee A. Multiorgan impairment in low-risk 
individuals with post-COVID-19 syndrome: A prospective, community-based study. BMJ Open. 
Mar 30 2021;11(3):e048391. doi:10.1136/bmjopen-2020-048391. 
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APPENDICES 
APPENDIX 1: 
STUDY GOVERNANCE 
The following Investigators and Institutional Affiliations are established. Designees may be provided, 
as appropriate. 
Name 
Role  
Institution 
Prof Francois Venter 
Principal 
Investigator 
Ezintsha, University of the Witwatersrand 
Building C 
Sunnyside Office Park 
32 Princess of Wales Terrace 
Parktown, Johannesburg 
South Africa 
Dr Simiso Sokhela 
 
Investigator 
(Clinical Lead) 
Building C 
Sunnyside Office Park 
32 Princess of Wales Terrace 
Parktown, Johannesburg 
South Africa 
Dr Samanta Lalla-
Edwards 
Investigator 
(Research Lead)