Malaria as a Protection Factor Against Severe COVID-19 in the Democratic Republic of Congo (Palu-COVID)
Conditions: Covid19, Paludism
Sponsor: Institute of Tropical Medicine, Belgium

 
 
 
 
 
RESEARCH STUDY PROTOCOL 
*THIS IS AN UNOFFICAL TRANSLATION MADE FOR CLINICALTRIALS.GOV AS THE STUDY ONLY 
WORKED WITH A FRENCH PROTOCOL. 
MALARIA AS A PROTECTIVE FACTOR AGAINST SEVERE COVID-19 IN THE 
DEMOCRATIC REPUBLIC OF CONGO  
 
Version 1.3 on 07/12/2021 
 
Sponsor 
Institute of Tropical Medicine 
Nationalestraat 155 
B-2000 Antwerp - Belgium 
Coordinating Investigator : 
Dr. Laurens Liesenborghs 
Financial backer 
Credo- Project Directorate General for 
Development Cooperation and Humanitarian 
Aid (DGD) - Belgian Federal Government 
 
 
 
Coordinating 
Investigator : 
Dr. Laurens Liesenborghs 
Service: 
Institute of Tropical Medicine 
Address: 
Nationalestraat 155 
B-2000 Antwerp - Belgium 
Phone : 
+32 475311904 

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E-mail : 
lliesenborghs@itg.be 
 
Partner institutions : 
National Biomedical Research Institute (INRB) 
Principal Investigator 
▪ 
Prof Dieudonné Mumba 
Co-investigator  
▪ 
Dr. Solange Mbamvu Ngwey  
Scientific Committee 
▪ 
Prof. Papy Mandoko 
▪ 
Prof. Placide Mbala 
▪ 
Dr. Peter Mukendi 
▪ 
Dr. Mimie Bitshi 
▪ 
Dr. Eddy Kakiesse 
▪ 
Dr. Flory Luzolo 
▪ 
Dr. Fidèle Kayembe 
Immunology investigator 
▪ 
Elysée Matungulu 
Project management/Monitoring 
▪ 
Jepsy Yango 
Data management 
▪ 
Cédric Nsingi 
Statistician 
▪ 
Dr. Armand Mutuadi 
 
Institute of Tropical Medicine 
Principal Investigator 
▪ 
Dr. Laurens Liesenborghs  
Scientific Committee 
▪ 
Prof. Wim Adriaensen 
▪ 
Prof. Eva Bartok 
▪ 
Prof. Emmanuel Bottieau 
▪ 
Prof. Lutgarde Lynen 
▪ 
Kaouther Chammam 
Project management/Monitoring 
▪ 
Emeline de Viron 
Data management 
▪ 
Harry van Loen 

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Data review  
▪ 
Diana Arango 
Statisticians 
▪ 
Achilleas Tsoumanis 
      Head of Clinical Research Unit (CTU) 
▪ 
Yven Van Herrewege 
 
 
PROTOCOL HISTORY 
Protocol / Amendment # 
Date 
Reason for amendment 
Protocol v1.0 
30/05/2021 
/ 
Protocol v1.2 
 
Protocol v1.3 
24/06/2021 
 
12/07/2021 
Minor amendment as suggested by 
the ITM IRB 
Minor amendment to replace "final 
outcome" with "clinical condition" as 
secondary objective 
 
 
 

 
 
DECLARATION OF CONFORMITY  
This protocol contains the information required to carry out the present research study. By 
signing this document, the Investigator agrees to conduct the study in compliance with the 
protocol, applicable ethical guidelines such as the Declaration of Helsinki, the European 
General Data Protection Regulation (GDPR), the ESF/ALLEA Code of Conduct for Research 
Integrity, as well as in compliance with international scientific standards and all applicable 
regulatory requirements. The Investigator will also make every reasonable effort to complete 
the study within the designated calendar deadlines. 
Once the protocol has been published and signed by the Investigator(s) and authorized 
signatories, it can no longer be modified informally. Amendments to the protocol have the 
same legal status, and must go through the mandatory review and approval stages before 
being implemented. 
Principal Investigator INRB 
 
Prof Mumba Dieudonné 
Date : 
07/13/2021  
 
Signed 
: 
 
 
 
 
 
Principal Investigator ITM 
 
Dr Liesenborghs Laurens 
Date : 
07/13/2021  
 
Signed 
: 
 
 
 
 
 
 
 

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TABLE OF CONTENTS 
Declaration of conformity ................................................................................................. 0 
Summary .......................................................................................................................... 3 
List of abbreviations and acronyms ................................................................................... 7 
1 
Introduction .............................................................................................................. 9 
2 
Study objectives ...................................................................................................... 10 
3 
Variables of interest ................................................................................................ 11 
3.1 
Primary variables of interest : .................................................................................... 11 
3.2 
Secondary variables of interest : ................................................................................. 11 
4 
Study design ........................................................................................................... 11 
5 
Methods ................................................................................................................. 12 
5.1 
Study environment and population. ........................................................................... 12 
5.2 
Procedures ................................................................................................................ 16 
6 
Data management .................................................................................................. 19 
6.1 
Data management responsibilities ............................................................................. 19 
6.2 
Data collection and management ............................................................................... 19 
6.3 
Data security and confidentiality ................................................................................ 20 
6.4 
Data sharing .............................................................................................................. 20 
6.5 
Recording retention ................................................................................................... 21 
7 
Data analysis .......................................................................................................... 21 
7.1 
Primary analysis .......................................................................................................... 21 
7.2 
Secondary analysis ...................................................................................................... 22 
7.3 
Subgroup analysis ....................................................................................................... 22 
7.4 
Multiplicity and missing data ...................................................................................... 24 
8 
Ethical considerations ............................................................................................. 24 
8.1 
Ethical (and regulatory) review ................................................................................... 24 
8.2 
Protocol changes ....................................................................................................... 24 
8.3 
Obtaining informed consent ....................................................................................... 25 

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8.4 
Privacy ....................................................................................................................... 26 
8.5 
Risks and benefits ...................................................................................................... 26 
8.6 
Sample storage .......................................................................................................... 27 
8.7 
Insurance ................................................................................................................... 27 
8.8 
Monitoring and quality control ................................................................................... 27 
9 
Dissemination of results and intellectual property ................................................... 28 
10 
Calendar ............................................................................................................. 29 
11 
References .......................................................................................................... 31 
 
 
 

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SUMMARY 
Justification 
Covid-19 has enormous socio-economic consequences 
worldwide. Africa remains the continent least affected, 
despite the poverty level of its population and the fragility 
of its health systems.  In inter-tropical African countries 
with high malaria prevalence, low prevalence of COVID-19 
is reported.  
According to some studies, the high prevalence of malaria 
in sub-Saharan Africa could play a role in protecting 
against the severity of COVID-19 in endemic areas. 
However, scientific evidence for the physiological role of 
this "trained" immunity has not been established. We 
wish to undertake this study to elucidate this hypothesis. 
Objectives 
The main objective is to assess whether the immunity 
conferred by Plasmodium protects against severe forms of 
COVID-19. This will be achieved by comparing anti-
plasmodium antibody levels in severe and non-severe 
COVID-19 cases. 
The secondary objectives are :  
▪ Comparing the cellular response to malaria 
between severe and non-severe COVID-19 
cases  
▪ Compare the proportions of acute malaria 
infection between severe and non-severe cases 
of COVID-19 using the Thick Gout (TG) and 
Rapid Diagnostic Test (RDT). 
▪ Describe the clinical features and status of 
severe and non-severe cases of COVID-19 with 
and without malaria exposure 
▪ Evaluate the modulatory impact of existing 
antimalarial immunity on the strength of 
humoral and cellular immune responses to 
SARS-COV-2 in severe and non-severe cases of 
COVID-19  

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▪ Compare the SARS-COV-2-specific cellular 
immune response between severe and non-
severe cases 
▪ Evaluate the modulatory impact of antimalarial 
immunity on trained immunity using specific 
stimulations of antiviral receptors in the innate 
immune system. 
Methodology 
Observational, multicenter, matched case-control study. 
The study will compare anti-plasmodium antibody levels 
between severe and non-severe cases of COVID-19.   
Study population 
The study will be carried out in four COVID-19 treatment 
centers for the recruitment of severe cases (cases). For 
each case, two controls will be recruited from the 
community based on the following concordance criteria: 
▪ Place of origin: same Health zone 
▪ Gender  
▪ Maximum 5-year age difference 
Inclusion criteria  
For cases :  
▪ Must be at least 18 years old  
▪ PCR-confirmed SARS-CoV-2 infection within 72 
hours of inclusion. 
▪ Have given informed consent to participate in 
the study. 
▪ Diagnosed as severe COVID-19 according to 
the following criteria: 
o Clinical signs of pneumonia: fever, 
cough, dyspnea or crepitations 
AND 
o With at least one of the following :  
▪ Respiratory rate > 30 cycles/min 
OR 
▪ Severe respiratory distress or 
SpO2 < 90% on room air 
▪ Admission to a care unit for COVID-19 

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▪ Have lived in the health zone for at least 6 
months  
 
For witnesses :  
▪ Must be at least 18 years old 
▪ PCR-confirmed SARS-CoV-2 infection within 72 
hours of inclusion. 
▪ Have given informed consent to participate in 
the study 
▪ Diagnosed as a non-severe COVID-19 case 
according to the following criteria: 
o Be asymptomatic 
OR 
o Be symptomatic, but without any signs 
of severe pneumonia: 
▪ Respiratory rate > 30 cycles/min 
OR 
▪ Severe respiratory distress or 
SpO2 < 90% on room air 
▪ Not to be admitted to a care unit for COVID-19. 
▪ Have lived in the study area for at least 6 
months 
 
Exclusion criteria  
 
▪ 
The subject has a contraindication to venipuncture, 
determined by clinical judgment 
▪ 
The subject is vaccinated against SARS-CoV-2 
▪ 
The subject has already been infected with SARS-
CoV-2 in the past and now presents with reinfection. 
Sample size 
300, including 100 cases and 200 controls: 
Power is calculated by simulation. Power is calculated for 
different effect sizes (i.e. difference in anti-plasmodium 
antibody levels between cases and controls). 
Calendar 
▪ Protocol drafting: February-May 2021 
▪ Ethics committee submission and approval: June 2021 
▪ Formation of study team: July 2021 

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▪ Data collection : July 2021 - December 2021 
▪ Data analysis: as soon as samples are received - March 
2022 
▪ Presentation of results: May 2022 
 
 
 
 
 

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LIST OF ABBREVIATIONS AND ACRONYMS  
 
AMA-1 
Apical Membrane Antigen 1 
COVID-19 
Corona virus disease 2019 
CSP 
Circum Sporozoite Protein 
CRF 
Case Report Form' (data collection form, observation notebook) 
CRC 
Clinical Research Center 
CTCo  
COVID-19 processing center 
ELISA  
Enzyme-Linked Immunosorbent Assay 
FM 
Thin smear 
GE  
Thick drop 
GLURP  
Glutamat Rich Protein 
IC  
Investigator Coordinator 
IC(F) 
Informed Consent (Form)' - Formulaire de Consentement Eclairé 
IC  
Confidence interval         
IFN-ꭚ 
Interferon gamma 
IgG  
Immunoglobulin G 
INRB  
National Institute for Biomedical Research 
HbA1 
glycated haemoglobin 
MSP-1  
Merozoite Surface Protein 1 
WHO 
World Health Organization 
Pf 
Plasmodium falciparum 
PBMC  
peripheral blood mononuclear cell 
GROUND FLOOR  
Democratic Republic of Congo 

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RT-PCR 
Reverse transcription polymerase chain reaction 
SARS-CoV-2    
Severe Acute Respiratory Syndrome Coronavirus 2 
TDR   
Rapid diagnostic test 
ZS  
Health Zone 
 
 
 
 

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1 
 INTRODUCTION   
Corona virus disease 2019 (COVID-19) is caused by a new virus, severe acute respiratory 
syndrome corona virus 2 (SARS-CoV-2), which was first reported in a group of pneumonia 
patients in the Chinese city of Wuhan on December 31, 2019 [1]. The epidemic that began in 
Wuhan then spread very rapidly worldwide and became a pandemic declared by the World 
Health Organization (WHO) on March 11, 2020 [2]. COVID-19 in its first wave and the current 
second wave is having enormous socio-economic consequences worldwide. The disease is 
therefore a major public health problem. According to data compiled by the American Johns-
Hopkins University, up to May 2, 2021, more than 150 million people have tested positive for 
Covid-19 worldwide, and more than 3 million have died from the disease [3].  
No continent is spared from COVID-19, although Africa remains the least affected despite its 
population's level of poverty and the fragility of its health systems, which predispose it to 
high mortality. However, the realities on the ground are proving to be contrary to all 
predictions [4]. Although there are many factors that can influence the transmission of 
COVID-19, including demographics, socio-cultural aspects, the young age of the population, 
the warm climate and genetics, this does not fully explain why the mortality rate is lower 
than those observed in Europe and the USA [5-7]. 
According to data compiled by the American Johns-Hopkins University on March 2, 2021, 
Africa has around thirty million confirmed cases of COVID-19 and 104,384 deaths, with 
proportions varying from region to region. There is a high prevalence of COVID-19 in regions 
with low malaria prevalence, such as the Maghreb region, notably Morocco with 513,314 
confirmed cases and Tunisia with 318,236 cases, and South Africa with 159,2626 cases. 
Conversely, in inter-tropical African countries with high malaria prevalence, low prevalence 
of COVID-19 has been reported, perhaps due in part to under-diagnosis. This is notably the 
case in Nigeria, with 9,7478 confirmed cases, and the Democratic Republic of Congo (DRC), 
with over 29,000 cases [8].  
The DRC is the second malaria-endemic country in Africa after Nigeria, and malaria remains 
the main cause of morbidity and mortality. At the time of writing on May 2, there are over 
18 million cases and more than 18,000 deaths per year [9].  
The prevalence of malaria in the DRC varies according to geographical location and depends 
on individual and community diversity; incidence is generally high in rural areas [10]. The DRC 
has a population of over 90,000,000, with a precarious health system and lax protective 
measures. However, according to the COVID-19 DRC epidemiological report of March 5, 
2021, there are around 712 deaths due to COVID-19, despite effective circulation of the virus 
in the population. 75% of cases are concentrated in the capital Kinshasa, which is the 

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epicenter of COVID-19, where malaria is meso-endemic, with a lower prevalence in urban 
areas compared with peri-urban areas [11]. 
Several recent studies have demonstrated that the "early" innate immune response can be 
shaped differently by prior exposure to immune stimuli. However, little is known about how 
endemic micro-organisms such as Plasmodiums and helminths shape the innate immune 
response and direct the reactivity and efficacy of the immune response as a whole [12, 13]. 
The high prevalence of malaria in sub-Saharan Africa has been assumed to confer possible 
protection against the severity of COVID-19 in endemic areas [14]. Our preliminary in vitro 
data indicate that prior and repetitive stimulation of anti-parasitic innate immune receptors 
induces an immune entrainment mechanism, stimulating the type I IFN response while 
attenuating the release of pro-inflammatory cytokines during SARS-CoV-2 infection (prof. Eva 
Bartok et al., unpublished results). According to several studies, high IFN type I release in 
conjunction with low induction of pro-inflammatory cytokines correlates with a less severe 
clinical course of COVID-19. This suggests a potential protective effect against COVID-19 virus 
in malaria-endemic areas [15, 16].  
However, scientific evidence for the physiological role of this "trained" immunity is not 
established. This is why we are undertaking this study to shed light on a possible protective 
role of malaria in patients testing positive for COVID-19. The aim of this study is to show 
whether the immunity conferred by repeated Plasmodium infections protects against severe 
forms of COVID-19. 
2 STUDY OBJECTIVES 
The main objective is to investigate whether the immunity conferred by Plasmodium protects 
against severe forms of COVID-19. This will be achieved by comparing anti-plasmodium 
antibody levels in severe and non-severe COVID-19 cases. 
 
 
The secondary objectives are :  
▪ Comparing the cellular response to malaria between severe and non-severe 
COVID-19 cases  
▪ Compare the proportions of acute malaria infection between severe and non-
severe cases of COVID-19 using the thick drop (GE) and rapid diagnostic test (RDT). 
▪ Describe the clinical features and status of severe and non-severe cases of COVID-
19 with and without malaria exposure 

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▪ Evaluate the modulatory impact of existing antimalarial immunity on the strength 
of humoral and cellular immune responses to SARS-COV-2 in severe and non-
severe cases of COVID-19  
▪ Compare the SARS-COV-2-specific cellular immune response between severe and 
non-severe cases 
▪ Evaluate the modulatory impact of antimalarial immunity on trained immunity 
using specific stimulations of antiviral receptors in the innate immune system.  
3 VARIABLES OF INTEREST  
3.1  PRIMARY VARIABLES OF INTEREST : 
The study's primary variable of interest is the level of anti-malarial antibodies in severe and 
non-severe COVID-19 cases. This level will be assessed by measuring the concentration of IgG 
against Circum Sporozoite Protein (CSP) using the Luminex- MAGPIX. 
3.2  SECONDARY VARIABLES OF INTEREST : 
▪ Anti-malarial IgG antibody levels against other antigens: AMAI, CSPM, GLURP, 
Pf/RESA55, as well as IgM 
▪ The cellular response against malaria between severe and non-severe COVID-19 cases, 
measured by IFN-y ELISPOT  
▪ Proportions of acute malaria infection between severe and non-severe cases of 
COVID-19 using the Thick Gout (TG) and Rapid Diagnostic Test (RDT). 
▪ Clinical characteristics and clinical status of severe and non-severe cases of COVID-19 
with and without malaria   
▪ SARS-COV-2 specific cellular and humoral immune response of severe and non-severe 
cases 
▪ Antiviral cytokine release levels, measured by ELISA and flow cytometry, of peripheral 
blood monocyte cells (PBMC) from controls and patients with acute malaria infection 
(according to GE and TDR). 
4 DESIGN OF THE STUDY 
An observational, multicenter, matched-case-control study. The study will compare the 
quality of malaria immunity in severe and non-severe cases of COVID-19.  

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The study will be carried out in four Treatment Centers (CTCo) to recruit severe cases (cases). 
Controls will be recruited from the Health Zone (ZS) on the basis of the same characteristics 
as the cases, taking into account age (upper and lower 5-year intervals), sex and ZS of origin.  
5 METHODS 
5.1  STUDY ENVIRONMENT AND POPULATION.  
This case-control study will be carried out in the Republic of Congo, in the city of Kinshasa, 
the epicenter of COVID-19. Kinshasa is a malaria-endemic zone, with a lower prevalence of 
malaria in urban areas than in peri-urban areas. In fact, the city of Kinshasa is structured into 
four health districts and sixteen hospital centers, spread across the thirty-five Health Zones 
for the management of COVID-19. 
The study will consist of cases and controls: 
▪ The cases will be severe COVID-19 cases, and will comprise hospitalized patients 
recruited from COVID-19 Treatment Centers (CTCOs).  
▪ Controls will consist of asymptomatic or symptomatic cases without signs of 
severity. For each case, two controls will be recruited after being matched in 
terms of age, sex and Health Zone of origin. 
 
5.1.1 INCLUSION CRITERIA 
To be eligible, study patients must meet the following criteria: 
 
For cases :  
▪ Must be at least 18 years old  
▪ RT-PCR-confirmed SARS-CoV-2 infection no more than 72 hours prior to inclusion. 
▪ Have given their informed consent or that of their guardian/representative to 
participate in the study. 
▪ Diagnosed as severe COVID-19 according to the following criteria: 
o Clinical signs of pneumonia: fever, cough, dyspnea or crepitations 
AND 
o Presenting at least one of the following signs:  
▪ Respiratory rate > 30 cycles/min 
OR 
▪ Severe respiratory distress or SpO2 < 90% on room air 

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▪ Being admitted to a care unit for COVID-19 
▪ Have lived in the health zone for at least 6 months  
 
For witnesses :  
▪ Must be at least 18 years old 
▪ PCR-confirmed SARS-CoV-2 infection within 72 hours of inclusion. 
▪ Have given informed consent to participate in the study 
▪ Diagnosed as a non-severe COVID-19 case according to the following criteria: 
o Be asymptomatic 
OR 
o Symptomatic, but no signs of severe pneumonia 
▪ Respiratory rate > 30 cycles/min 
OR 
▪ Severe respiratory distress or SpO2 < 90% on room air 
▪ Not to be admitted to a care unit for COVID-19. 
▪ Have lived in the study area for at least 6 months 
 
 
 
5.1.2 Exclusion criteria 
To be eligible, study patients must not meet the following criteria for cases and controls:  
▪ The subject has a contraindication to venipuncture, determined by clinical 
judgment 
▪ The subject is vaccinated against SARS-CoV-2 
▪ The subject has already been infected with SARS-CoV-2 in the past and now 
presents with reinfection. 
 
5.1.3 RECRUITING SEVERE CASES 
Severe cases will be recruited from four state CTCOs. The choice of CTCOs takes into account 
the frequency of severe cases and the geographical distribution of malaria in the city of 
Kinshasa. The centers are as follows: 
▪ Saint Joseph Hospital 
▪ Kinkole General Hospital 
▪ Cliniques Universitaires de Kinshasa  
▪ Ngaliema Clinics  

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After political and administrative authorizations have been obtained by a qualified member 
of the research team, all severe cases in the care units of the selected centers will be 
investigated for their eligibility for the study, and then invited to participate and give 
informed consent. 
5.1.4 RECRUITING WITNESSES 
For each case, two controls will be recruited in the Health Zone from which the severe cases 
originated. The Health Zone nurse assigned to the COVID-19 response and the investigator 
assigned to the site will record the witness information in the register of RT-PCR-positive 
cases for COVID-19. 
The first two controls on the linear list with a COVID-19 RT-PCR test result of 72 hours or less 
and with the same characteristics will be contacted and invited to take part in the study. If 
participation is refused, the next person on the list will be taken. Controls must be matched 
to the case on the basis of the following concordance criteria: 
▪ Age: +/- 5 years relative to case age 
▪ Gender: same as case  
▪ Place of origin: same health zone as the case  
During the follow-up home visit planned by the ZS for the non-severe COVID19 patient, the 
investigator will accompany the ZS nurse supervisor, and the study will thus be introduced to 
the patient. Once informed consent has been obtained, the questionnaire and blood sample 
will be taken during the same visit. In compensation for their time, the controls will receive 
masks and soap to limit the spread of the virus in their environment. 
 
5.1.5 SAMPLE SIZE AND STATISTICAL POWER  
The sample size is 300 participants, made up of 100 cases and 200 controls. Sample power is 
calculated according to the following assumptions: 
▪ Median antibody titers in controls are estimated at 898 mfi (mean fluorescence 
intensity). This estimate is based on a previous study in which antibody titres 
against CSP were determined by the same Luminex technology in a malaria-
endemic context [17].  
▪ The logarithm of the anti-malarial anti-CSP antibody levels of the controls is 
normally distributed, with a mean of 6.8 and a standard deviation of 1.  
▪ The logarithm of malaria antibody levels in cases is normally distributed with a 
standard deviation of 1. 
▪ For each case, two matched controls are recruited. 

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▪ The correlation between the logarithm of anti-malarial antibody levels in cases 
and matched controls is 0.7. 
The data collected will be analyzed using conditional logistic regression with a two-tailed test 
at the 5% significance level. 
Power is calculated for different effect sizes (i.e. difference in malaria antibody levels 
between cases and controls). The table below shows the power for different effect sizes, or 
rather design effects. With a sample size of 300, the study is able to detect a 17% difference 
in median antibody titres with 80% power, which is considered clinically relevant. 
 
 
 
 
 
Mean of logarithm 
of anti-malarial 
antibody levels in 
controls 
Average of the 
logarithm of anti-
malarial antibody 
levels in cases 
Median malaria 
antibody levels of 
controls 
Median malaria 
antibody levels of 
cases 
Power 
6.8 
6.7 
898 
812 
0.3 
6.8 
6.69 
898 
804 
0.35 
6.8 
6.68 
898 
796 
0.4 
6.8 
6.67 
898 
788 
0.48 
6.8 
6.66 
898 
781 
0.53 
6.8 
6.65 
898 
773 
0.6 
6.8 
6.64 
898 
765 
0.65 
6.8 
6.63 
898 
757 
0.7 
6.8 
6.62 
898 
750 
0.76 
6.8 
6.61 
898 
742 
0.81 

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6.8 
6.6 
898 
735 
0.83 
6.8 
6.59 
898 
728 
0.87 
6.8 
6.58 
898 
721 
0.9 
6.8 
6.57 
898 
713 
0.93 
6.8 
6.56 
898 
706 
0.94 
6.8 
6.55 
898 
699 
0.96 
 
In conclusion, with a sample size of 300, the study is able to detect a 17% difference between 
median antibody titers with 80% power. 
 
5.2 PROCEDURES 
5.2.1 OVERVIEW OF STUDY PROCEDURES 
Using the CRF, the investigator will interview both conscious cases and witnesses. For cases 
who are unconscious or unable to communicate, the medical file will serve as a source of 
information. Blood samples for cases and controls will be taken on D1 of the study. 
5.2.2 STUDY/VISIT SCHEDULE  
5.2.2.1 
Day 1: main study visit 
Participants will be informed of the study's objectives and design; if they agree, an informed 
consent form will be signed. After inclusion, the main study procedures will take place 
(questionnaire, sampling and follow-up).  
The following variables will be collected: 
▪ Socio-demographic variables : 
▪ Age 
▪ Gender 
▪ Profession 
▪ Civil status 
▪ Study level 
o Medical history 

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▪ Comorbidities: diabetes, tuberculosis, cancer, HIV, renal failure, chronic 
liver disease, chronic lung disease, heart disease 
▪ Medicines  
▪ Smoking 
▪ Pregnancy   
o Clinical variables : 
▪ Clinical status according to WHO scale 
▪ Duration of symptoms 
▪ Type of symptoms 
▪ Size  
▪ Weight 
▪ Blood Pressure 
▪ Heart Rate 
▪ Respiratory Rate (RR) 
▪ Temperature (T°) 
▪ Free Oxygen Saturation (SaO )2 
▪ Spleen size 
The following biological samples will be collected: 
▪ 30 ml of venous blood will be collected using Lithium-Heparin vaccuitenaire tubes 
for the various immunological analyses (described below). 
▪ 5 µl of capillary blood will be taken for the thick smear, two µl of blood for the 
thin smear (GE/FM) and one drop of blood for the TDR. 
The results of the thick drop and RDT will be communicated to the attending physicians for 
hospitalized patients (cases). For outpatients (controls), the results will be communicated to 
the ZS team in charge of malaria management, in accordance with national guidelines.  
Day 7 +- 2: follow-up 
For controls: a telephone contact on Day 7 with the treating clinician or the patient will be 
considered in order to obtain information on their clinical evolution. If, in the meantime, they 
have developed severe COVID-19, they will be excluded from the study and will no longer be 
considered as controls. 
During participation in the study, the telephone number of the control as well as that of a 
close relative and the attending physician will be recorded in order to monitor the 
participant's health status. The patient's telephone number will be recorded separately in 
the patient identification register, which will be accessible only to local investigators. This 

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number will not be included in the CRF or patient recruitment register. If no telephone 
contact is possible, a home visit by the control will take place at D7. 
 
5.2.3 
 LABORATORY PROCEDURES. 
 All analyses will be carried out by the INRB laboratory in Kinshasa.  
30 ml of venous blood will be collected using Lithium-Heparin vaccuitenaire tubes. After 
centrifugation, the plasma fraction will be stored at -80°C and subsequently used to 
determine antibodies against malaria and COVID-19 using Luminex. A Luminex test will be 
developed specifically to determine the following Plasmodium antigens: CSP, AMAI, CSPM, 
GLURP, Pf/RESA55 and SARS-CoV-2 antigens. In the unlikely event of it not being possible to 
install the Luminex test in Kinshasa, a commercial ELISA test will be used as an alternative. 
The PBMC fraction will be isolated for ELISPOT assays with malaria and SARS-CoV-2 antigens 
and in vitro experiments on entrained immunity, and the remainder cryopreserved 
afterwards. The potency of cellular immunity against Plasmodium and SARS-CoV-2 will be 
measured by IFN-y ELISPOT assays. In a subgroup of individuals, flow cytometry 
measurements may be performed to validate the functionality and phenotype of responding 
cells. 
For experiments on trained immunity, the PBMC fraction will be stimulated with ligands 
specific to antiviral receptors of the innate immune system (TLR3, TLR7-9, RIG-I, MDA-5, 
cGAS) [18]. The release of antiviral cytokines (e.g. type I IFN, CXCL10) will be measured by 
commercial ELISAs and multiplexed flow cytometry assays.  
In addition, 5 µl of capillary blood will be taken for the thick smear, two µl of blood for the 
thin smear (GE/FM) and one drop of blood for the TDR. 
5.2.4 WITHDRAWAL AND TERMINATION OF THE STUDY  
Participants may withdraw from the study at any time. This is the case if the participant or 
legal representative withdraws consent, or if the investigator considers that continuation of 
the study would have a negative effect on the patient's health.  
Data and biological samples collected up to the time of the participant's withdrawal may be 
used for analysis purposes. However, if the participant expressly requests that they not be 
used, the data and samples will be destroyed. 

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6 DATA MANAGEMENT 
6.1 DATA MANAGEMENT RESPONSIBILITIES 
Although the principal investigator assumes ultimate responsibility for data management, 
practical implementation and monitoring will be carried out by the INRB data manager in 
collaboration with study staff at the sites. This will be done in accordance with the study's 
data management plan, which broadly describes the aspects below. The ITM Data Manager 
and other ITM colleagues will help support the implementation of this DMP. 
 
6.2 DATA COLLECTION AND MANAGEMENT 
6.2.1 DATA TYPES 
Study data includes individual participant-level data at baseline and follow-up study visits. 
These include variables such as study participant identifiers, socio-demographic variables, 
medical history, symptoms, vital signs, laboratory results and final results status). See also 
5.2.2.1 for further details. 
6.2.2 COLLECTION AND MANAGEMENT METHODS 
Study data will be collected on paper and/or directly via electronic questionnaires in the 
study database using REDCap. Only data defined by the study protocol will be collected. 
REDCap (Research Electronic Data Capture) is a software application widely used in the 
academic research community to create and manage surveys, databases and research 
studies. It is a secure web application as well as a mobile application that can be used to 
collect, cleanse and manage subject information in compliance with various applicable 
standards and regulations (GCP, CFR 21 part 11, FISMA, HIPAA). Its mobile application 
enables offline data collection with smartphones or tablets. In addition, the software includes 
a query management system and capabilities for importing and exporting data in a variety of 
formats. 
Paper questionnaires will be used where possible and appropriate. In addition, electronic 
questionnaires on tablets or laptops will be used to collect data from participants at remote 
sites. Subsequently, when connected to the Internet or cellular networks, data from the 
mobile device can be synchronized with the study server. Edit controls and branching logic, 
programmed on the electronic forms, will validate at the point of data entry and support data 
quality. To ensure better data quality, appropriate data management training will be 
organized before the actual data collection (and processing) begins. In addition, data will be 

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reviewed and monitored during the course of the study by various study collaborators to 
achieve timely database lock. 
Because of the biological risk of COVID-19 contamination, any original written source 
document created at the patient's bedside (i.e. in the "red zone") should be treated as 
potentially infectious unless an effective means of decontamination can be instituted. 
Wherever possible, photographs, digital scans or other electronic data capture methods can 
be used.  
6.3 DATA SECURITY AND CONFIDENTIALITY 
The IT/data management system to be used incorporates a robust security architecture 
including encryption, firewalls, anti-virus software and controlled user access 
(tablet/computer authentication, REDCap username, personal password and authorized user 
role). Data backup will be provided at server, computer, mobile device and/or database level, 
where applicable. 
ITM and INRB will also ensure that the necessary measures are taken to ensure the 
safekeeping of all data management documents (Data Management Plan, procedures, 
completed questionnaires) and that IT equipment (tablets, computers, server) is secured 
(locked cupboards, locked offices and/or rooms and/or controlled by badge). 
Study participants' private data will be treated confidentially. Study participants' names and 
contact data will be replaced by an identification code specific to the study subject (= 
pseudonym). Only pseudonymized data will be used and distributed within the wider study 
team. See also 7.4 for further details. 
6.4 DATA SHARING 
The study will make its research data available for secondary research or to support the 
Congolese response to COVID-19 and considering the following key aspects: 
▪ ITM's data sharing policy will be followed, with the following key aspects mentioned 
below 
▪ The European FAIR data principles ("Findable"; "Accessible"; "Interoperable"; 
"Reusable") will also be respected ("as open as possible, as closed as necessary"). 
▪ Ensure confidentiality by anonymizing data (e.g. by replacing the study subject's 
identification code with a new code that can no longer be used to identify the patient; by 
generalizing and randomizing specific variables). 
▪ Ensure that ethical approvals have been obtained by ethics committees and study 
participants. 

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▪ Making data available, mainly medical, personal and sensitive data, through a regulated 
data access mechanism and along a transparent decision-making process, with :  
(i) Filling in a data request form,  
(ii) Evaluation through access to committee data,  
(iii) agreement to share data through this committee,  
(iv) secure transfer of anonymized data,  
 (v) additional metadata that clarify the research data. Metadata will include study 
protocol, questionnaire(s), data dictionary. 
 
6.5 RECORDING RETENTION 
The principal investigator is responsible for the retention of all essential documents listed in 
the Good Clinical Practice guidelines: 'Informed Consent (Form)' or IC(F) or Formulaire de 
Consentement Eclairé. All essential documentation for all study subjects must be retained by 
the principal investigator in a secure storage facility for at least twenty years in accordance 
with INRB policies or local country regulatory requirements (whichever is longer). The DRC 
requires that study files be kept for up to 2 years after publication. These records must also 
be retained in accordance with the ethics committee's medical record retention 
requirements, whichever is longer. All stored records must remain confidential to the extent 
required by national, state and local laws of the jurisdiction in which they are stored. 
7 DATA ANALYSIS 
A detailed statistical analysis plan will be developed prior to completion of the statistical 
analysis. The analysis plan will provide a full description of the variables of interest, statistical 
methods (including management of missing data, statistical adjustment, management of 
confounding factors and interactions), subgroup definitions and sensitivity analyses. 
 
7.1 PRIMARY ANALYSIS 
A conditional logistic regression model will be fitted with the logarithm of anti-malarial 
antibody levels to Circum Sporozoite Protein (CSP) as the independent variable. The p-value 
for the test of no association between anti-malarial antibody levels and case/control will be 
presented. 

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7.2 SECONDARY ANALYSIS 
▪ Compare anti-malarial antibody levels against other antigens between severe and 
non-severe COVID-19 cases:  
o A model similar to that for the primary objective is fitted with the 
respective anti-malarial antibody levels as the independent variable. 
▪ Comparing the cellular response against malaria between severe and non-severe 
COVID-19 cases:  
o A conditional logistic regression model will be fitted with cellular response 
against malaria as the independent variable. 
▪ Compare the proportions of acute malaria infection between severe and non-
severe cases of COVID-19 using the Thick Gout (TG) and Rapid Diagnostic Test 
(RDT). 
o A conditional logistic regression model will be fitted with the proportions 
of acute malaria infection as the independent variable. 
▪ Describe the clinical characteristics and clinical status of severe and non-severe 
cases of COVID-19 with and without malaria (former exposure or active infection):  
o Medians with quartiles or numbers and proportions will be presented. 
▪ Evaluate the modulatory impact of existing antimalarial immunity on the strength 
of humoral and cellular immune responses to SARS-COV-2 in severe and non-
severe cases of COVID-19  
o A conditional logistic regression model will be fitted with the SARS-COV-2 
specific cellular immune response as the independent variable. 
 
7.3 SUBGROUP ANALYSIS 
No subgroup analysis will be performed. 
 
7.4 MULTIPLICITY AND MISSING DATA 
As this is a study with a single primary objective, no multiplicity adjustment is necessary. The 
management of missing data will be specified in the statistical analysis plan. 

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8 ETHICAL CONSIDERATIONS 
8.1  ETHICAL (AND REGULATORY) REVIEW 
The investigators would be committed to conducting the study in accordance with the 
principles of the Declaration of Helsinki. Prior to carrying out the study, official approval 
would be obtained from the ethics committee; the study protocol would be submitted for 
approval to the ITM's institutional review, to the ethics committee of Belgium's Antwerp 
University Hospital, and to the national ethics committee of the DRC's Ministry of Public 
Health. Authorization will also be sought from the relevant health zones before the study 
begins. 
8.2 PROTOCOL CHANGES  
Once the final study protocol has been issued and signed by the authorized signatories, it 
cannot be modified informally. Amendments to the protocol have the same legal status and 
must go through the appropriate steps before being implemented. Any substantial 
modification must be approved by all the bodies and the Ethics Committee (EC) that 
approved the original protocol, before being implemented, unless it is due to participant 
safety concerns (in which case immediate implementation may be necessary for participant 
protection). If changes to the protocol or an amendment are requested by a local EC during 
the review process, they must be discussed and agreed with the sponsor prior to any 
resubmission incorporating these changes. 
8.3 OBTAINING INFORMED CONSENT 
Before starting the study, the investigator assigned to the site (CTCO or ZS) will obtain 
informed consent from the participants; this will consist of asking for their opinion and 
explaining the purpose, objectives, rights of the participants and responsibilities of the 
investigation team. Consent will ensure that each participant understands all procedures. 
Participants will receive clear explanations of all possible techniques that will be used by their 
samples.  
The informed consent form will be translated into the local language (Lingala) at the language 
service of the University of Kinshasa. The informed consent procedure will be conducted in 
the patients' mother tongue or preferred language by a qualified person, indicated by the 
principal investigator. 
Patients will have sufficient time to ask questions and make a free decision. If the person is 
willing to participate, he or she will be asked to sign the consent form. If the participant 
cannot read and/or write, an impartial witness should be present during the informed 

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consent discussion. After the written informed consent form has been read and explained to 
the participant, and after he/she has orally consented to participate in the study and 
provided his/her fingerprint, the witness should complete the participant's name and add 
the date of the fingerprint, then personally sign and date the consent form. By signing the 
consent form, the witness attests that the information contained in the consent form and all 
other written information has been accurately explained to the participant, that the 
participant has apparently understood it, and that the participant has freely given consent.    
The person obtaining informed consent will be asked to sign and date a written confirmation 
that he/she has been sufficiently informed about all relevant aspects of this study and 
confirms that he/she has freely and voluntarily consented to participate in the study. 
For patients who are unable to give consent due to their clinical condition, consent will be 
obtained from a legally acceptable representative. As soon as the patient is able to give 
consent, the consent procedure will be repeated and the patient will be given the 
opportunity to confirm/refuse participation. 
 
8.4 PRIVACY 
Individual medical information collected in this study will remain confidential. Members of 
the study team are bound by professional secrecy. Individual data will be made available to 
physicians in charge of patients upon request to the investigators. Disclosure of this 
information to third parties is strictly forbidden. 
Electronic files will not contain any data that could identify the participant (e.g. surname, first 
name, full date of birth, telephone number). Each study participant will be assigned a unique 
identification code (ID). On all study documents or files, study participants will be identified 
by the participant's ID code. 
A correspondence list will establish the link between the participant ID and the participant's 
personal data so that examination results can be used for patient care. This list containing 
personal data and other documents containing participants' names or signatures (e.g. 
informed consent) will be kept separate from other study documents. All study documents 
will be kept in a secure, locked location, with access limited to the site principal investigator 
and authorized personnel. 
Access to all paper documents and electronic files required for data management and study 
follow-up will be restricted to authorized personnel at international, regional and local levels. 
Study computers and CRF files will only be accessible via a login with a personal username 

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and password. A list of authorized CRF and database users will be kept at ITM and updated 
throughout the study. 
8.5 RISKS AND BENEFITS  
The direct benefit of the study is that each participant will receive a free malaria test with an 
additional examination, namely a blood sample. This will be the only additional examination 
compared to basic care. In addition, as compensation for their time, the controls will receive 
masks and soap to limit the spread of the virus in their environment. 
Participation in the study will also benefit the community; it will contribute to the acquisition 
of knowledge about COVID-19, and the information it will generate will enable researchers 
or the response team to improve management. The only additional procedure specific to the 
study will be follow-up visits for controls: telephone contacts, or for patients for whom this 
is not possible: a physical visit either at the study site or at home.  
The greatest risk for participants is a potential breach of confidentiality, for which all 
measures will be in place to limit this as much as possible (by de-identification). The sting 
may also cause a little pain and swelling at the sampling site, but this is temporary. In the 
event of significant risk, as assessed by the clinician, the sample will not be taken and the 
patient will be excluded (see inclusion/exclusion criteria). 
8.6 SAMPLE STORAGE 
The samples collected can be stored for a maximum of 20 years, so that they can be used for 
further research into COVID-19 immunization in line with future scientific discoveries. These 
samples will be stored and registered at INRB's bio-banking facilities in Kinshasa. Subjects will 
be asked to indicate on the informed consent form whether or not they agree to long-term 
storage.   
The decision whether or not to use the samples for future research will be made by the study 
investigators in consultation with, and only after receiving approval from, the Institutional 
Review Board (IRB) and ethics committees.  
8.7 INSURANCE 
The coordinator of this study, ITM, has obtained study (no-fault) insurance to cover any 
injury, damage or loss to study participants caused directly or indirectly by their participation 
in the study. Information about the insurance is not specified in the Informed Consent Form 
for this study due to the expressed concern about unreasonable claims for compensation. 
Furthermore, we do not anticipate any specific physical injuries or mental health problems 
as a result of participation in this observational study. 

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8.8 MONITORING AND QUALITY CONTROL 
Detailed SOPs will be developed to cover all laboratory processes, from sample collection to 
shipping, analysis and storage. Immunological and parasitological tests will be carried out by 
qualified laboratory technicians. The INRB and ITM PIs will be responsible for ensuring that 
all study-specific analyses are carried out in accordance with Good Clinical Laboratory 
Practice (GC(L)P) guidelines, study-specific Standard Operating Procedures (SOPs), and the 
internal/external quality control systems of the respective tests. During visits by the ITM 
research team, compliance with the protocol and GC(L)P will be monitored, including data 
management and documentation. 
9 DISSEMINATION OF RESULTS AND INTELLECTUAL PROPERTY  
All study documents are provided by the principal investigators to the investigators and their 
designated personnel in strict confidence. None of these documents may be disclosed to any 
party not directly involved in the study, without the written authorization of the principal 
investigators. 
Once the results are considered final, arrangements will be made to communicate them to 
the Kinshasa community in the most appropriate way, with the active participation of the 
health authorities. Oral communications and written publications will mention the name of 
the sponsor and the names of all partners and researchers involved.  
 

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10 CALENDAR 
N° 
Tasks 
Managers 
Mars
21 
April
21 
Ma
y 21 
Jun
e 21 
July 21 
Augu
st 21 
Sept 
21 
Oct
21 
Nov
21 
Dec
21 
Janv
22 
Feb 
22 
Marc
h 22 
1 
Drafting the 
protocol 
- Principal 
investigator 
- Scientific 
Committee 
 
 
 
 
 
 
 
 
 
 
 
 
 
2 
Submission and 
approval Ethics 
Committee 
- 
Investigator 
- 
Developer 
 
 
 
 
 
 
 
 
 
 
 
 
 
3 
Preliminary steps : 
- 
Contact 
with 
political 
and 
administrat
ive 
authorities 
- 
Investigator 
- 
Supervisor 
- 
Scientific 
Committee 
 
 
 
 
 
 
 
 
 
 
 
 
 

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- 
Survey tool 
finalized 
- 
Interviewer 
training 
4 
Collecting data 
Investigators 
 
 
 
 
 
 
 
 
 
 
 
 
 
5 
Laboratory 
analysis of samples 
Survey team 
 
 
 
 
 
 
 
 
 
 
 
 
 
6 
Data capture and 
analysis 
- Project 
management/Monit
oring 
 
- Data management 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
7 
Summary of 
results 
Scientific Committee 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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