Clinical Aspects, Severity, Management and Outcome of Febrile Illnesses in the Democratic Republic Congo (DRC).
Conditions: Febrile Illness
Sponsor: Institute of Tropical Medicine, Belgium

  
 
 
IMT RESEARCH STUDY 
Clinical aspects, severity, management and 
outcome 
febrile illnesses in the DRC 
FIKI² ("Febrile Illness in Kinshasa and Kimpese") 
Amendment 3 
Protocol v4.0 
4 july 2022 
Clinicaltrials.gov NCT04760678 
English translation for clinical 
trials.gov – unofficial translation 
– official protocol in French 
Promoter : 
Ins琀椀tute of Tropical Medicine 
Na琀椀onalestraat 155 
B-2000 Antwerpen - Belgium 
Inves琀椀gator 
coordinator : 
Prof. Dr. Emmanuel Bo琀�eau 
p. 1 of 36 

  
 
 
ClinicalTrials.Gov : 
Study title: 
NCT04760678 
Clinical aspects, severity, management and disease outcomes 
in the DRC 
Study acronym 
Version : 
FIKI² (Febrile Illness in Kinshasa en Kimpese) 
Amendment 3 Protocol v4.0 
Date : 
July 4, 2022 
Financial backer : 
DGD (Directie-Generaal Ontwikkelingssamenwerking en Humanitaire) 
hulp) 
Promoter : 
Institute of Tropical Medicine, Antwerp, Belgium 
Study sponsor 
Institute of Tropical Medicine 
Name 
Function 
Contact 
Email: ebottieau@itg.be 
Tel: +32(0)32476450 
Prof. Dr. Emmanuel 
Bottieau 
Coordinating investigator 
Email : kvercauteren@itg.be 
Tel : +32(0)32476332 
Email : jjacobs@itg.be 
Tel : +32(0)32476630 
Email: sdeclercq@itg.be 
Tel: +32(0)33455572 
Prof. Koen 
Vercauteren 
Co-investigator 
Co-investigator 
Prof. Jan Jacobs 
Research Scientist 
clinic 
Dr. Steven Declercq 
Achilleas Tsoumanis 
Harry Van Loen 
Email: atsoumanis@itg.be 
Tel: +32(0)32476617 
Study statistician 
Email: hvanloen@itg.be 
Tel: +32(0)32476616 
Data manager 
Project 
management/Monitoring 
Data examiner 
Email: edeviron@itg.be 
Tel: +32(0)32470778 
Emeline de Viron 
Hanne Landuyt 
Email: hlanduyt@itg.be 
Tel: +32(0)32476495 
Email: yvanherrewege@itg.be 
Tel: +32(0)33455557 
Head of Research Unit 
Clinic 
Yven Van Herrewege 
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Amendment 3 Protocol v4.0, dated July 4, 2022 

  
 
 
Site survey team 
Kinshasa General Hospital (Mama Yemo) 
Name 
Function 
Contact 
Email : elongi2002@yahoo.fr 
Tel: +243 818104366 
Prof. Jean Pierre 
Elongi 
Local principal investigator 
Institut National de Recherche Biomédicale (INRB) Clinical Research Center (CRC) 
Name 
Function 
Contact 
Email: 
Prof. Dieudonné 
Mumba 
Co-Principal Investigator 
local 
mumbadieudonne@yahoo.fr 
Tel: +243 818118511 
General Reference Hospital (HGR) of the Evangelical Medical Institute (IME), Kimpese 
Name 
Function 
Contact 
Email: docmbalap@yahoo.fr 
Tel: +243 813735641 
Prof. Charles Mbala 
Local principal investigator 
Kimpese Health Research Center (CRSK) 
Name 
Function 
Contact 
Email: 
Prof. Delphin Mavinga Co-Principal Investigator 
dmavingaphanzu@yahoo.fr 
Phanzu 
local 
Tel: +243 819481410 
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Amendment 3 Protocol v4.0, dated July 4, 2022 

  
 
 
DECLARATION OF CONFORMITY 
By signing this protocol, the researcher(s) acknowledge(s) and agree(s) 
to 
This protocol contains the information required to carry out this research study. In 
By signing this document, the investigator undertakes to conduct the study in compliance with the 
protocol 
applicable ethical guidelines such as the Declaration of Helsinki, the European General 
Data Protection (GDPR), the ESF/ALLEA Code of Conduct for Research Integrity, 
and in accordance with international scientific standards, as well as all requirements 
applicable regulations. The Investigator will also make every reasonable effort to complete 
the study on time. 
Once the final protocol has been published and signed by the researcher(s) and authorized signatories, it 
cannot be modified informally. Amendments to the protocol have the same status 
and must go through the obligatory review and approval stages before being implemented. 
implementation. 
COORDINATING INVESTIGATOR : 
Title, Name: Prof. Dr. Emmanuel Bottieau 
Signed: 
Date: 
4/07/2022 
PRINCIPAL INVESTIGATOR : 
Title, Name: Prof. Jean Pierre Elongi 
Signed: 
Date: 
Date: 
PRINCIPAL INVESTIGATOR : 
Title, Name: Prof. Charles Mbala 
Signed: 
By signing this document, I undertake to carry out the trial in accordance with the protocol 
clinical practices and applicable ethical and regulatory requirements. I also acknowledge the 
paragraph concerning the confidentiality of the study and I authorize the Institute of Tropical Medicine, 
Antwerp, 
Belgium, to record my data on a computerized system containing all data 
relevant to the study. 
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Amendment 3 Protocol v4.0, dated July 4, 2022 

  
 
 
 
 
TABLE OF CONTENTS 
DECLARATION OF CONFORMITY 
SYNOPSIS 
4 
6 
1 . INTRODUCTION 
12 
12 
13 
15 
16 
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18 
18 
18 
19 
19 
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23 
23 
24 
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25 
25 
26 
28 
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31 
32 
34 
1 .1 BACKGROUND 
1.2 JUSTIFICATION 
2. STUDY OBJECTIVES 
2 .1 PRIMARY ENDPOINT 
2.2 SECONDARY ENDPOINTS 
3. STUDY DESIGN 
3.1 GENERAL STUDY DESIGN 
4. PARTICIPANTS, POPULATION AND ELECTIONS 
4 
4 
4 
4 
4 
.1 RECRUITMENT SITES 
.2 SELECTION AND RECRUITMENT 
.3 INCLUSION AND EXCLUSION CRITERIA 
.4 SAMPLE SIZE 
.5 WITHDRAWAL AND TERMINATION OF 
THE STUDY 
5. STUDY PROCEDURES 
5.1 STUDY/VISIT SCHEDULE 
5.2 OBTAINING INFORMED CONSENT 
5.3 LABORATORY PROCEDURES 
6. STATISTICAL METHODS 
6 
6 
6 
.1 VARIABLES OF INTEREST 
.2 STATISTICAL METHODS 
.3 SAMPLE SIZE AND POWER 
7 
8 
9 
. QUALITY CONTROL AND ASSURANCE 
. DATA MANAGEMENT 
. ETHICAL ISSUES 
9 
9 
9 
9 
9 
9 
9 
9 
.1 ETHICAL AND REGULATORY REVIEW 
.2 PROTOCOL MODIFICATIONS 
.3 INFORMED CONSENT 
.4 CONFIDENTIALITY 
.5 RISKS AND BENEFITS 
.6 ACCIDENTAL DISCOVERIES 
.7 COMPENSATION FOR PARTICIPATION 
.8 INSURANCE 
1 
1 
1 
1 
1 
0. DISSEMINATION OF RESULTS, INTELLECTUAL PROPERTY 
1. ARCHIVING 
2. REFERENCES 
3. LIST OF ABBREVIATIONS 
4. 
APPENDICES 
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SYNOPSIS 
BACKGROUND AND RATIONALE 
The current epidemiology and outcome of febrile illnesses in 
The Democratic Republic of Congo (DRC) is poorly documented. 
A better description of the causes, presentation 
and host biomarker profiles will make it possible to 
future studies targeting diagnostic and 
optimized therapies. 
The FIKI² clinical trial initiated (see protocol version 1.0), which 
prospectively describes febrile illness in 2 sites 
of DRC, has integrated a prospective sample collection 
of enrolled patients; it will enable analysis of the 
host responses (biomarker profiles, in particular the 'C- 
Reactive Protein' - CRP) and explore the pathogens involved 
(blood cultures; metagenomic sequencing for the detection of 
pathogens, with a primary focus on RNA viruses and 
arboviroses). 
The storage of these samples, well documented with 
clinical data, will enable the development and evaluation 
diagnostic and monitoring platforms, as well as 
secondary etiological or immunological investigations. 
DESIGN 
Bi-centric prospective observational cohort study 
of adults and children presenting to emergency departments or 
outpatient clinic with community-acquired febrile illness, 
with laboratory analysis and sample storage in 
a biobank. 
STUDY SITES AND 
POPULATION 
1. Pediatric Emergency Department, Hôpital Général de 
Kinshasa (HGK / Mama Yemo), DRC : urban site, children < 
15-year-old with febrile illness. 
2. Hospital outpatient and emergency services 
Reference General (HGR) of 
Institut Médical Evangélique (IME); health services 
outpatient consultation at one or more health centers 
in the IME reference area, Kimpese, DRC: rural site, 
adults and children with febrile illnesses. 
OBJECTIVES AND CRITERIA 
JUDGEMENTS 
Primary objectives 
1. Estimate the proportion of survival with resolution of 
symptoms, assessed on day 21, of febrile illnesses. 
2 . Describe the biomarker profile (CRP, blood cell count 
with differentiation) on inclusion of the 
patients with febrile illnesses. 
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Secondary objectives 
1 . Estimate the proportion of survival with non-resolving 
symptoms and death, assessed on day 21, of the 
febrile illnesses. 
2 . Estimate the proportion of survival with resolution of 
symptoms, survival with non-resolution of symptoms, 
and death, assessed on days 7 and 14, of the diseases 
feverish. 
3 
4 
5 
. 
. 
. 
Describe epidemiology, clinical aspects and severity 
and management of febrile illnesses. 
Identify factors associated with outcomes (resolution, 
unresolved, death) of febrile illnesses. 
Determine the frequency and profile of malaria (by 
rapid diagnostic test and thick drop), including 
in co-infection, in patients with severe 
feverish. 
6 
7 
. 
. 
Observe the association between CRP values and 
with patient outcome at day 21. 
Observe the association between CRP values and the rate 
of 
white blood cells with specific etiologies (malaria), 
bacteremia, dengue fever, etc.). 
Exploratory objectives 
1 . Describe the frequency, etiology and profiles of 
antibiotic resistance in bacteremia 
detected in patients with 
febrile illnesses. 
Evaluating the performance and potential contribution 
of a test 
rapid immunochromatographic test for dengue fever 
under field conditions in the DRC. 
2 . Evaluate metagenomic sequencing for the 
detection of fever-causing pathogens 
(with a primary focus on RNA viruses). 
Strategic objectives 
1 . Create at ITM and in research institutions 
a biobank of clinical samples with a strong 
to enable the development and 
the future evaluation of diagnostic platforms and 
monitoring, as well as other secondary research. 
Strengthen clinical research capacity and 
laboratory training at participating sites 
and direct support. 
2 
3 
. 
. Establish etiologies, epidemiology and 
inflammatory/immunological characteristics and 
the basic outcome of febrile illnesses in preparation 
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Amendment 3 Protocol v4.0, dated July 4, 2022 

  
 
 
subsequent studies focused on optimizing 
diagnostics (biomarkers, platforms, etc.) or 
therapeutic interventions. 
Primary endpoints 
● Proportion of survival with symptom resolution 
evaluated on day 21, febrile illnesses. 
CRP and white blood cell values with 
differentiation on day 0 (inclusion). 
● 
Secondary endpoints 
● Clinical status on a 3-category ordinal scale 
(resolution, non-resolution, death) evaluated on days 7, 14 
and 21. 
● Diagnostic hypotheses at inclusion and diagnoses 
final. 
● 
- 
Disease severity on days 0, 7, 14 and 21. 
in adults : 
o 
o 
Simplified ambulatory severity index (SAHI) 
Quick Sepsis Related Organ Failure Assessment 
(qSOFA) 
- 
in children under 5 years of age: 
o criteria of the algorithm for managing 
childhood illnesses (ALMANACH) 
● 
● 
Initial and secondary hospitalization (assessed on day 21). 
Length of hospital stay (initial hospitalization and/or 
secondary) (assessed on day 21). 
● 
● 
Number of secondary visits (assessed on day 21). 
Number and types of laboratory and radiology tests 
prescribed / actually carried out. 
● 
● 
Number and types of medications and supportive care 
prescribed / actually administered. 
Detection of malaria by rapid test (HRP II antigen), 
pLDH antigen) and by thick drop (type and parasitaemia). 
Exploratory judgment criteria 
● Frequency of bacteremia, with etiological distribution 
and antibiotic resistance profiles of germs 
identified. 
● 
● 
Detection of dengue by rapid test (NS1 antigen), 
IgG and IgM antibodies). 
Detection and profiling of disease-causing pathogens 
by metagenomic sequencing. 
INCLUSION CRITERIA AND 
EXCLUSION 
Inclusion criteria: 
● Ongoing fever detected on presentation, or 
documented at home or in another health center 
within 24 hours of presentation, defined by: temperature 
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Amendment 3 Protocol v4.0, dated July 4, 2022 

  
 
 
axillary or tympanic > 37.5°C, or oral temperature or 
rectal > 38°C. 
● 
● 
Possibility of contact between the patient (or a relative) and 
and the study team on days 7, 14 and 21. 
Informed consent to participate signed by patient 
(adult) or a legally acceptable representative (child) 
or patients whose condition does not allow them to sign the 
informed consent), with the children's assent to 
From the age of 12, wherever possible. 
Exclusion criteria 
● 
● 
Children under two months old. 
Hospitalization > 48 hours in the last 14 days. 
SCREENING AND RECRUITMENT 
Sample size (participants recruited in the 
study laboratory, following amendment 1 of the protocol) 
- 
- 
500 children at HGK / Mama Yemo, Kinshasa 
500 children at and 500 adults at HGR-IME/centres de 
health, Kimpese 
Patients will be enrolled in the study at the time of 
hospital presentation. The 1,500 participants will include 
uniformly throughout the study period. 
FOLLOW
-UP 
Each patient will be monitored for 21 days. Patients will be 
evaluated at inclusion, and during fixed time points (D7, 
J14 and J21) for ambulatory patients, and daily 
for patients hospitalized at study sites. 
A blood sample (venous +/- capillary) is taken at the time of 
inclusion. Venous blood volume of maximum 14.5 ml 
(in children aged <14 years) or 30.5 ml (≥14 years) is taken. 
to meet the clinical and monitoring needs of 
for additional laboratory analyses related to 
and for sample storage. 
For children < 14 years of age, the maximum volume to ensure 
safety during blood sampling will be calculated on the basis of 
age, weight and capillary haemoglobin level. If the 
total quantity required is not possible, priority will be established 
as follows: 
1 
2 
3 
4 
. 
. 
. 
. 
clinical necessity and monitoring 
primary study analyses 
secondary and exploratory study analyses 
sample storage 
LABORATORY ANALYSES 
The following samples will be collected (according to 
consent and site capacity) 
1. Fingertip capillary blood test (participants) 
of < 14 years) 
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a. Hemoglobin (quantity determination 
in children under 14 years of age): 
QuikRead Go®, bowls 
CRP/hemoglobin or HemoCue® Hb 301 
b. CRP (only on capillary blood if 
simultaneously with hemoglobin by 
Quikread Go®) 
c. Preparation of up to 5 bloodstains 
dried: for storage + secondary research 
For participants ≥ 14 years of age, blood sampling 
The capillary blood sample is taken after the 
venous blood sample. 
2. Venous blood sampling: volume-dependent 
that can be collected, the samples 
in order of priority 
a. 4 ml (2 ml if < 14.5 ml can be taken from 
children < 14 years) EDTA tube 
▪ 
▪ 
According to clinical need: blood glucose 
Malaria TDR: SD Bioline pan-LDH & pf-. 
HRP2 
▪ 
▪ 
▪ 
Thick drop 
Dengue TDR: SD Bioline Dengue Duo 
White blood cells and differentiation : 
HemoCue® WBC/DIFF 
▪ 
CRP 
(if not yet performed on blood 
capillary) : QuikRead Go ®, cuvettes 
CRP/Hemoglobin (with hemoglobin); 
CRP TDR 
Centrifuge the remaining volume to 
aliquoting (in 2-5 aliquots of 330 µL plasma) 
and stored in the biobank at -80°. The 
aliquots will be used in part for 
metagenomic analyses, the remaining volume (the 
will be stored for research purposes 
secondary. 
b. Blood culture bottle (1-4 ml in the case of 
children < 14 years, 20 ml in patients ≥ 14 
years): according to 
routine 
c. 2 ml serum tube: biochemical analysis according to 
clinical necessity 
d. 2 ml additional EDTA: storage and 
secondary research 
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e. 2.5 ml PAXgene: storage in the biobank at -. 
0°C, for secondary research 
8 
Blood cultures are taken and analyzed in accordance with the 
monitoring protocol (protocol approved in DRC and 
Belgium; IRB IMT 613/08, UZA CE 8/20/96). 
Study analyses (except metagenomic analyses) will be 
carried out at t h e  IME laboratory and the study 
l a b o r a t o r y  
of HGRK. Samples for metagenomic analysis 
will be transported to the ITM laboratory in Antwerp. 
Samples for secondary research will be 
stored in the biobank on research sites, or at ITM, 
Antwerp. 
ANALYSIS METHODS 
Description of the proportion of patients meeting the criteria for 
3 
2 
categories (resolution, non-resolution, death) on days 7, 14 and 
1, overall and by basic characteristics (age, gender...), 
geographical location, syndromic category, characteristics 
biomarker results) in terms of medians 
and interquartile ranges for continuous characteristics, 
and in terms of numbers and percentages for 
categorical characteristics. Estimates will be given with 
a Wilson 95% confidence interval. Models of 
univariate and multivariate logistic regression will be used to 
analyze the association between basic characteristics and condition 
on day 21. Biomarker data will be 
described mainly using descriptive statistics of the 
as described above. The prevalence of 
malaria in the study population will be calculated as 
proportion with a Wilson 95% confidence interval. 
The association of CRP and white blood cell values with 
patient results at day 21, or with specific etiologies, 
will be examined using the Mann-Whitney test. The evaluation 
diagnostic performance will be calculated by calculating the 
standard diagnostic performance measures, such as the 
sensitivity and specificity. 
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1. INTRODUCTION 
1.1 
Background 
Of the 54.8 million deaths worldwide in 2013, 8.3 million (15.2%) were caused by 
products in sub-Saharan Africa. Over 60% of deaths in Africa are linked to disease. 
infectious diseases, and more than 50% of all infectious disease-related deaths worldwide occur in the 
workplace. 
Africa. Compared with global mortality data, the incidence of death from disease 
is 3.5 times higher in sub-Saharan Africa (1). This burden of disease 
is all the more intolerable given that many of the infectious diseases that kill 
are both preventable and curable. This also means that the main causes of death in 
Africa are avoidable, and any effort to prevent and cure them can lead to a 
significant reduction in mortality. 
The aetiology and burden of syndromes such as diarrhoea and pneumonia have been the subject of 
much debate. 
particularly in children (2,3). However, many pathogens can 
cause fever with focal signs in various organ systems, leading to 
different syndromic presentations, or cause an "undifferentiated fever" without signs of 
focalization (4,5). Febrile syndromes are generally divided into localized infections 
(respiratory, intestinal, cutaneous, genitourinary, etc.) and undifferentiated infections (without 
associated symptoms or focal signs). Fever (undifferentiated or not) is one of the most common 
main reasons for seeking healthcare in Africa 
sub-Saharan Africa, but it represents a huge challenge, given the wide range of differential and 
the wide etiological spectrum (6,7). Among hospitalized patients with a febrile illness 
undifferentiated, for example, case-fatality rates reach 12% (8). 
The burden of specific infections, such as malaria (9) and certain bacterial infections 
septicemia, is relatively well quantified (1), but viral infections, infections 
fungal diseases, bacterial zoonoses and parasitic diseases that have not been adequately 
may be underestimated. New emerging diseases such as fevers 
hemorrhagic viruses, or COVID-19 risk developing into major health threats. 
and pose important and dynamic challenges for disease diagnostic capacity. 
feverish. 
With the decline in malaria transmission and increased use of diagnostic tests 
(RDT), appropriate management of febrile patients with a malaria RDT 
is a growing clinical challenge for which there is no clear recommendation (9). 
In some contexts, the deployment of RDTs for malaria has ultimately contributed to 
transform the problem of antimalarial drug abuse into that of antibiotic abuse (10, 11). The 
antibiotic resistance has become a major issue for global health, and 
the rational use of antibiotics in the care of sick people is seen as an important 
key strategy for combating this problem (alongside veterinary control). 
Clinical research is a key element in the development of evidence-based strategies for 
to improve the prevention, diagnosis and management of febrile illnesses. The WHO has therefore 
stated that "multicenter studies on the causes of fever are needed to identify 
the main conditions that can be treated" and "provide evidence for the adaptation of 
clinical algorithms for fever management by countries" (11); and that studies on "the importance 
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Amendment 3 Protocol v4.0, dated July 4, 2022 

  
 
 
 
 
of febrile illnesses for populations and patients are necessary" to "produce 
evidence-based recommendations for the management of febrile patients, improve 
health status, reduce the prevalence of serious illness and death, and maintain the effectiveness of 
drugs" (11). 
However, research capacity in Africa is severely limited, as illustrated by the 
the fact that only 5003 (2.4%) of the 208491 clinical trials registered on clinicaltrials.gov in 2019 are 
on the African continent (www.clinicaltrials.gov), which contrasts sharply with the needs 
in research linked to the burden of morbidity. 
1.2 Justification 
As in most sub-Saharan African countries, very little data is available in this area. 
Democratic Republic of Congo (DRC) on the epidemiology, clinical presentation, causes and 
the outcome of febrile illnesses; research capacity is generally very low. 
The CREDO Project, an extension of the agreement between Antwerp's Institute of Tropical Medicine 
(ITM) and 
the Directorate General for Development Cooperation and Humanitarian Aid (DGD) aims to 
improve research capacity on (re)emerging epidemics in the DRC. The study 
is part of this general objective and is planned for 2 clinical sites (see below). 
below), who have little experience of clinical research. In collaboration with 2 partners 
local: Centre de Recherche Clinique (CRC) of the Institut National de Recherche Biomédicale (INRB), and 
the Kimpese Health Research Centre (CRSK). In this way, the first foundations to build 
these hospitals into research centers will be established, and, even more important 
Congolese research teams will be trained through ongoing supervision in the field. 
In addition, the study provides the epidemiological and clinical basis for future 
research into optimizing diagnostic and therapeutic strategies. 
The initial protocol (and its title) were inspired by experience gained during the FISSA study. 
("Febrile Illness in sub-Saharan Africa"). This is a multi-center cohort study designed to 
document the clinical aspects, severity, management and outcomes of febrile illnesses in 
health centers in West, Central and East Africa. This study was developed by 
African Coalition for Epidemic Research, Response and Training (ALERRT) : 
a consortium of 21 African and European institutions. The consortium has secured funding 
of the European Union's 5-year EDCTP program. The study protocol has been approved 
by the ITM IRB. 
The use of a fairly similar protocol in the DRC will have the advantage of enabling studies 
with common inclusion and evaluation criteria, and thus complete the picture. 
with scientific information from a Central African country, which is relatively under-represented in the 
the FISSA study. 
The purely descriptive and observational clinical component of the initial study is in this 
This phase is complemented by laboratory procedures, with the additional aim of providing 
better understanding of the causes of febrile illness and optimization of strategies 
diagnosis and monitoring : 
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1. Analysis of inflammatory biomarkers: C-reactive protein (CRP), white blood cells and 
their differentiation by different types of point-of-care (POC) equipment: analyzers 
and RDTs. An evaluation of the performance of a few TDRs for CRP will be carried out. 
as an additional comparative analysis. The objectives and methodology of 
this laboratory-only analysis is not described in a specific sub-protocol 
(Appendix 1). This new component will enable the future evaluation of strategies 
Diagnostics based on the use of RDTs for these inflammatory biomarkers, integrated 
into clinical routine. 
2. Etiological research into febrile illnesses 
a. Blood culture monitoring. Although pilot projects exist on both sites 
already, as part of a project to improve monitoring of resistance to 
antibiotics and clinical management (protocol approved in the DRC and the 
Belgium; IRB IMT 613/08, UZA CE 8/20/96), the systematic application on 
FIKI² study populations will make it possible to evaluate bacteremia as an etiology of 
febrile illnesses, and to evaluate the biomarkers analyzed as factors of 
prediction of blood culture positivity. 
b. Systematization of febrile illnesses using RDTs targeting specific 
specific pathogens: malaria and dengue fever. Unlike malaria, RDTs for 
dengue are poorly validated under field conditions in sub-Saharan Africa. A 
systematic use of dengue RDTs, and reference diagnoses (in 
metagenomic analyses, or other secondary research) 
will enable us to assess their respective roles in the diagnostic approach to diseases 
feverish in this context. 
c. Evaluation of a new platform for the detection of pathogens causing 
febrile diseases (primary focus on RNA viruses such as arboviruses) by 
metagenomic sequencing (performed at ITM in Belgium or on site after 
implementation). 
To identify the etiology of an infection, clinicians rely on tests that 
specific laboratory diagnostics that focus on one or a small 
number of pathogens (by PCR, antigen detection, etc.). The narrow scope of 
these targeted tests by their very nature neglects untested pathogens or agents 
previously unknown emerging pathogens. Metagenomic sequencing, which is 
proposed here will provide an interesting alternative to targeted testing with a potential 
for the diagnosis and monitoring of febrile illnesses (12). This 
technique enables the sequencing of all the genomic material present in a given 
patient sample without prior knowledge of the target. The sequences 
recovered can be compared with databases from 
sequences of well-coded infectious pathogens and new pathogens 
viruses can also be identified in this way. Given that 
RNA have a marked epidemic potential (13), we will first focus on 
detection of RNA viruses. Our aim here is to demonstrate that sequencing 
metagenomics can be a useful tool for epidemiological and environmental mapping. 
regional surveillance of pathogens linked to acute fevers (observational). 
Well-documented sample storage with clinical data will enable the 
development and evaluation of future diagnostic and monitoring platforms, and 
3. 
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Amendment 3 Protocol v4.0, dated July 4, 2022 

  
 
 
 
 
other secondary post-hoc research. Future analyses may include profiling 
of peripheral blood to search for early biomarkers 
to distinguish bacterial from viral infections. 
Separate ethical approvals will be requested from all ethics committees 
with the submission of a detailed study protocol. 
These objectives will make it possible to integrate a laboratory component consisting in the 
development and 
strengthening the clinical research capacity of the 2 sites, with a focus on the ITM biobank 
and in partner research institutions, and the evaluation of POC analyses. 
2. STUDY OBJECTIVES 
Primary objectives 
1. Estimate the proportion of survival with symptom resolution, assessed at day 21, of 
febrile illnesses. 
2. Describe the biomarker profile (CRP, white blood cell count with differentiation) in the case 
of 
inclusion of patients with febrile illnesses 
Secondary objectives 
1. 
2. 
3. 
4. 
5. 
6. 
7. 
Estimate the proportion of survival with non-resolution of symptoms and death, assessed 
on day 21, febrile illnesses 
Estimate the proportion of survival with symptom resolution, survival with non 
resolution of symptoms, and death, assessed on days 7 and 14, of febrile illnesses 
Describe the epidemiology, clinical aspects, severity and management of diseases 
feverish. 
Iden琀椀fy factors associated with disease outcomes (resolution, non-resolution, death) 
feverish. 
Determine the frequency and profile of malaria (by rapid diagnostic test and gout) 
including co-infection, in patients with febrile illnesses. 
Observe the association of CRP values and white blood cell counts with the outcome of the 
patients on day 21 
Observe the association of CRP and white blood cell values with etiologies 
specific (malaria, bacteremia, dengue fever, etc.) 
Exploratory objectives 
1. Describe the frequency, etiology and antibiotic resistance profiles of bacteremias 
detected according to the surveillance criteria in patients with 
febrile illnesses 
2 . 
. 
Assessing t h e  performance and potential contribution of a rapid immunochromatographic test 
targeting dengue under field conditions in the DRC 
Evaluating metagenomic sequencing for the detection of pathogens causing 
febrile diseases (with a primary focus on RNA viruses) 
3 
Strategic objectives 
1. Set up a biobank at the ITM and partner research institutions 
of well-documented clinical samples to enable the development and 
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Amendment 3 Protocol v4.0, dated July 4, 2022 

  
 
 
 
 
future evaluation of diagnostic and monitoring platforms, as well as other 
secondary research. 
2 . 
. 
Strengthen the clinical and laboratory research capacity of participating sites by 
formal training and direct support 
Establish etiologies, epidemiology, inflammatory/immunological characteristics 
and basic outcome of febrile illnesses in preparation for subsequent focused studies 
on diagnostic optimizations (biomarkers, platforms, etc.) or interventions 
therapeutics 
3 
2.1 Primary judging criteria 
● 
● 
Proportion of survival with symptom resolution assessed at day 21, for febrile illnesses. 
CRP and WBC values with differentiation on day 0 (inclusion) 
The word " resolution" refers to the following situations: (i) the signs and symptoms have 
completely disappeared and treatment has been completed; or (ii) certain signs or symptoms 
and / or treatment is still underway, but there has been a marked improvement in these 
signs and symptoms suggest that the person will recover quickly without needing 
exploration or treatment other than those already administered. 
Any other combination suggests that the situation is "unresolved". 
2.2 Secondary endpoints 
● Clinical status according to a 3-category ordinal scale (resolution, non-resolution, death) 
evaluated on days 7, 14 and 21. 
● 
● 
Diagnostic hypotheses at inclusion and final diagnoses. 
Disease severity on days 0, 7, 14 and 21. 
- 
o 
o 
- 
in adults : 
Simplified ambulatory severity index (SAI) 
Quick Sepsis Related Organ Failure Assessment (qSOFA) 
in children under 5 years of age: 
o criteria of the algorithm for the management of childhood illnesses (ALMANACH) 
● 
● 
● 
● 
● 
● 
Initial and secondary hospitalization (assessed on day 21). 
Length of hospital stay (initial and/or secondary hospitalization) assessed on day 21). 
Number of secondary visits (assessed on day 21). 
Number and types of laboratory and radiology tests prescribed/actually performed. 
Number and types of medications and supportive care prescribed/actually administered. 
Detection of malaria by rapid test (HRP II antigen, pLDH antigen) and thick drop test 
(type and parasitemia) 
2 .3 Exploratory judgment criteria 
● Frequency of bacteremia, with etiological distribution and drug resistance profiles 
antibiotics for identified germs 
● 
● 
Detection of dengue by rapid test (NS1 antigen, IgM) 
Detection and profiling of pathogens causing febrile illnesses by sequencing 
metagenomics 
3. STUDY DESIGN 
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3.1 General study design 
This is a bi-centric, prospective, observational cohort study of adults and children in the 
presenting to the emergency department or outpatient clinic with a community-acquired febrile illness 
(with 
or without focal signs) at 2 clinical sites in the DRC. The study included blood sampling 
at inclusion for study analysis and storage. 
The study will describe the epidemiology, causes, clinical aspects and biomarker profile of the 
disease. 
the severity, management and outcome of febrile illnesses, using the data from 
collected during routine diagnostic and therapeutic procedures, and study analyses. 
Each patient will be monitored for 21 days. Follow-up will include: 
● 
● 
Daily visits for hospitalized patients, 
Telephone calls (or a visit to the study center or home visit) on days 7, 14 and 21 
for outpatients and discharged patients. 
4. PARTICIPANTS, POPULATION AND ELECTIONS 
4.1 Recruitment sites 
Kinshasa General Hospital (also known as Mama Yemo Hospital) is a 2,000-bed hospital, 
centrally located in the Commune de Gombe, serving an urban population. The 
pediatrics has 170 beds. Participants in the FIKI² study will be recruited from the following departments 
pediatric emergencies, which currently see around 5 febrile children a day. 
The Institut Médical Evangélique (IME) de Kimpese is the General Reference Hospital (HGR) of 
Kimpese, a 400-bed hospital in the province of Kongo Central, west of DRC. The department 
The pediatrics department has 64 beds, while the internal medicine department has 78 beds. 
Some of the participants in the FIKI² study will be recruited there from emergency departments and the 
outpatient consultations. The monthly number of outpatient consultations and admissions to the HGR is 
of around 2257 and 167 respectively. Approximately 10% of patients seen at the outpatient clinic were 
present with fever. 
The population served by this health facility is estimated at 187,796 (Zone de Santé de 
Kimpese). There are several health centers in this area. Recruitment will take place 
also, if necessary, in one or more of these health centers (selected on the basis of their 
proximity, attendance, clinical standards), by the study team based at the IME and following 
exactly the same study protocol. 
4.2 Selection and recruitment 
The frequency of febrile illnesses can vary from season to season. In order to balance the 
seasonal recruitment, the FIKI² study will be carried out in two phases 
staggering inclusions evenly over the duration of the study, to take account of these 
seasonal variations. Recruitment will be carried out in such a way as to avoid, as far as possible, any 
influence from members of 
the study team on the choice of participants to be included. Recruitment will be stratified by age and 
site, with the number of patients (adults and children) to be recruited defined for the 2 sites. 
This number will be evenly distributed over the duration of the study, with a recruitment target of 
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weekly. Participants will be recruited consecutively from the beginning of the week. 
until the target is reached. If the planned number of patients has not been reached by the end of a 
week, the supplement will be paid the following week. 
4.3 Inclusion and exclusion criteria 
Inclusion criteria 
● Fever in progress and objectified at presentation, or documented at home or in another 
loca琀椀on 
health center within 24 hours of presentation, defined by: 
o Axillary or tympanic temperature > 37.5°C 
OR 
o Oral or rectal temperature > 38°C 
AND 
● 
● 
Possibility of contact between the patient (or his or her designated relative) and the team 
responsible for 
study on days 7, 14 and 21. 
Informed consent signed by patient (adults) or legal representative 
acceptable (children or patients whose condition does not allow them to sign informed 
consent), 
with the consent of children aged 12 and over, wherever possible 
Exclusion criteria 
● 
● 
Children under two months old. 
Hospitalization > 48 h in the last 14 days (to exclude nosocomial fevers). 
4.4 Sample size 
The sample size is of convenience and was adapted to the number of febrile patients. 
and feasibility forecasts. The sample size refers to the 
participants recruited in the laboratory phase of the study (following amendment 1 of the protocol). We 
plans to include 500 children in the HGK / Mama Yemo site, and 500 children + 500 adults in the 
HGR-IME and health centers in Kimpese. Sample size calculations can be found in 
section 6.3. 
4.5 Withdrawal and termination of 
the study 
Participants may be withdrawn from the study if: 
● 
● 
The participant or legal representative withdraws consent 
The investigator believes that continued participation would have a negative effect on the 
health of the 
participant 
Data from participants withdrawn from the study will be used until the time of the last visit. 
study. 
In the absence of any news from a participant, the interviewer will do everything within his or her 
power to contact the participant. 
(limited to a maximum of 3 calls to the available telephone numbers and/or visits spread over a 
number of days) 
within the visit period (window of ± 2 days) to try to re-establish a connection with the 
participant for follow-up. A patient included in the study who, however, is no longer available for the 
follow-up visits, will be considered as a loss of follow-up/abandonment. 
If the study is prematurely interrupted or suspended due to force majeure, the sponsor will 
promptly inform the investigators/institutions and the regulatory authority of any interruption or 
18 
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of the suspension and the reason(s) for the interruption or suspension. CEs will also be 
informed without delay and will receive from the sponsor or investigator/institution the reason(s) for 
interruption or suspension, as specified in the applicable regulatory requirements. 
5. STUDY PROCEDURES 
5.1 Study/visit schedule 
Inclusion visit 
Participants will be included in the study as soon as the consent form has been signed. 
The study investigator or his or her representative will fill in the data collection form (box 
report form (CRF), the patient's demographic information, history, symptoms and 
signs (type and severity), co-morbidities, initial diagnosis, medications and examinations 
and his immediate decision (hospitalization or discharge). 
The study team will work with patients to arrange the following contacts on days 7, 14 and 
21. 
- 
For patients with whom telephone contact is considered possible, 
the study team records the patient's telephone number and the number of the 
telephone number of one or more relatives authorized by the patient to provide information on 
health status up to day 21. Patients will also receive 
the member of staff responsible for the study. 
- 
- 
Patients for whom telephone contact is not an option will receive 
offer to return to the study center on days 7, 14 and 21. Home visits by 
community health workers participating in the study will also be considered if necessary and 
feasible. 
If the participant gives permission for the laboratory part of the study, a blood sample will be 
taken. 
(venous +/- capillary blood) is taken during the inclusion visit. This blood test 
is integrated with the routine sampling required for patient management. 
Immediate test results (CRP, white blood cell count with differentiation), 
blood cultures, thick drop, malaria RDT, dengue RDT) will be collected in the 
and some of them (hemocultures) communicated directly to the clinicians at 
study. Samples stored for further analysis (metagenomic evaluation) or 
of secondary research will be registered in the ITM biobank and the institutions 
research partners, depending on the logistical capacities being developed and 
well-established memorandums of understanding (based on existing collaborations). 
Follow-up visits 
After inclusion, each patient will be followed up for 21 days. Follow-up will include: 
● Telephone calls (or visits to the study center or home visits by health agents) 
community) on days 7 (D5-9), 14 (D12-16) and 21 (D19-23) for patients followed in 
outpatients, discharged patients, or patients hospitalized in facilities other than the hospital. 
inclusion site. 
● Daily bedside visits for hospitalized patients 
Every daily hospital visit (inpatients) and every visit or phone call 
whether planned or not, the study staff will collect the patient's symptoms and signs in the CRF. 
(type and severity), comorbidities, drugs and tests prescribed, medications received, 
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Amendment 3 Protocol v4.0, dated July 4, 2022 

  
 
 
examinations performed, results of examinations, secondary visits and/or hospitalizations and the 
status (resolved, unresolved, dead). 
After day 21, the investigator will establish a "final diagnosis", based on examination of the entire 
file. This final diagnosis will be syndromic and/or specific, according to standard definitions. 
There are no laboratory procedures for follow-up visits. 
Figure 1: Schedule of visits and clinical data collected*. 
Day 7 
Day 14 
Day 21 
Day 0 
Phone call or visit 
Inclusion 
Phone call or visit 
Phone call or visit 
Inclusion criteria 
Signed consent 
Data collected 
Blood sampling 
Data collec琀椀on from 
pt management. 
Data collec琀椀on from 
care of the 
pa琀椀ent. 
Data collec琀椀on from 
care of the 
pa琀椀ent. 
Vital status (member contact) 
essen琀椀al home) 
Physician-reported data : 
Patient-reported data 
Patient-reported data 
Pa琀椀ent-reported data 
Anonymous identifier 
demographic variables 
clinical variables 
(comorbidities) 
anamnesis of the episode 
feverish 
Symptoms on admission 
Diagnostic hypotheses 
Outcome (hospitaliza琀椀on, 
discharge, 
death...) 
Evolu琀椀on of symptoms 
Clinical events 
(consulta琀椀on, hospitalization, 
Evolu琀椀on of symptoms 
Clinical events 
(consultation, hospitalization, 
Evolution of symptoms 
Clinical events 
(consulta琀椀on, hospitalization, 
etc.) 
etc.) 
etc.) 
Prescribed tests 
Prescribed tests 
Prescribed tests 
Treatments received 
Treatments received 
Status: resolved, unresolved, 
deceased 
Treatments received 
Status: resolved, unresolved, 
deceased 
Status: resolved, unresolved, 
deceased 
* For hospitalized patients, the same variables will be collected daily 
5.2 Obtaining informed consent 
Adults who agree to participate in the study, children's representatives who agree to the 
their child's participation and the representatives of adults whose condition does not allow them to 
sign the form. 
informed consent (impaired consciousness, insufficient level of comprehension) is impaired and who 
agree to the patient's participation will be invited to sign the consent form after a 
oral explanation by a qualified professional. If a participant (or parent or representative) is 
unable to read or write, the signature of a witness to the informed consent discussion 
will be obtained. 
For the witness, preference will be given to persons indicated by the patient or representative, or 
if not possible, it will be carefully checked that the proposed independent witness is worthy of 
and freely accepted by the patient. 
If the child's condition so permits, children a g e d  12 and over, the age at which the 
to give informed consent, will be asked to sign a consent form. 
consent (in addition to parental consent). 
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After the informed consent process, the form will be given to the patient or his/her representative. 
A patient with a disturbance of consciousness whose legal representative has given consent, 
will receive information about the study and will be asked to give formal consent when his or her 
condition is diagnosed. 
will allow. 
Informed consent and assent forms will be available in French, Lingala and English. 
(for Kinshasa) and Kikongo (for Kimpese). 
The informed consent procedure will describe the (legitimate) purpose of the study, the procedures to 
be followed, the 
risks and benefits of participation, and data management. Participants (or parents 
or representatives) will be informed that participation in the study is entirely voluntary and that the 
participant may withdraw from the study at any time without any negative consequences. 
It will be explained to participants that the study and its laboratory procedures include a 
sample storage for secondary research (e.g. transcriptomics): 
transcriptional profiling of peripheral blood to identify early biomarkers for 
distinguish bacteria from viral infections), and that these investigations can also include 
genetic analysis *. Specific consent will be required for these analyses. 
*Note for the metagenomics protocol: unlike procedures with the intention 
host sequences, we do not consider the metagenomic protocol to be 
a "genetic analysis", as it aims to sequence, analyze and report RNA sequences 
rather than human sequences. However, since the technique is based on 
on the sequencing of all the RNA material present in the patient's sample, a small amount of material 
RNA of human origin will inevitably (incidentally) be sequenced in addition to the material 
pathogenic RNA. We will use laboratory and bioinformatics procedures to 
depleting human RNA sequences and enriching pathogenic sequences. Since this 
subtraction is not 100% efficient, some human sequences may remain in the 
the final data set used for pathogen identification. Some 
publishers can request free access to the raw data set. 
For participants whose samples will be used for secondary research, which may include 
influence clinical management (e.g. important endemic infections such as 
trypanosomiasis, which is a severe disease that can remain asymptomatic for a long time), he will be 
explained that the medical team would be informed in the event of detection, so as to warn them. 
They will 
will then be advised to go to a nearby referral center, where a diagnostic evaluation will be carried out. 
(and possibly treatment) will be offered free of charge. 
Similarly, patients will be informed that during metagenomic analyses and the 
secondary research, certain chronic infection diagnoses could be made in the same way. 
examples of HIV, or hepatitis B or C will be provided in the information form. 
so that the participant fully understands what it's all about and what the implications are. 
The consent process and documents will enable participants to give their 
consent only for the clinical and purely observational part, or to add a 
consent for (i) study laboratory analyses, (ii) sample storage for 
minimum of 10 years in the biobank for secondary research (iii) genetic analyses in 
secondary research. If participants give their permission for the storage of 
21 
Amendment 3 Protocol v4.0, dated July 4, 2022 

  
 
 
 
 
samples, it will be explained that this consent for storage can be withdrawn at any time. 
without having to provide any particular justification, and that at that time the patient's samples will 
be 
destroyed. 
5.3 Laboratory procedures 
A blood sample (venous +/- capillary) is taken at inclusion. A volume of blood 
of a maximum of 14.5 ml (in children < 14 years) or 30.5 ml (≥14 years) is taken for 
to meet routine clinical and monitoring needs, study laboratory analyses and 
sample storage. 
For children < 14 years, the maximum volume to ensure safe sampling will be 
calculated on the basis of age, weight and capillary haemoglobin. If less than the 
required is possible, priority will be given as follows: 
1. 
2. 
3. 
4. 
clinical necessity and monitoring 
primary study analyses 
secondary and exploratory study analyses 
sample storage 
The following samples will be collected (subject to consent and site capacity) 
1. Fingertip capillary blood test (participants < 14 years of age) 
a. Hemoglobin (determination of maximum blood volume in children < 14 
years): QuikRead Go®, CRP/hemoglobin cells or HemoCue® Hb 301 
b. CRP (only on capillary blood if simultaneously with hemoglobin by 
QuikRead Go ®) 
c. Preparation of a maximum of 5 dried bloodstains: for storage + for 
secondary research 
For participants ≥ 14 years of age, capillary blood is taken after the blood test. 
venous. 
2. Venous blood sampling: depending on the maximum volume that can be withdrawn, the 
The following samples will be taken in order of priority 
a. 4 ml (2 ml if < 14.5 ml can be collected from children < 14 years) EDTA tube 
▪ 
▪ 
▪ 
▪ 
▪ 
▪ 
According to clinical need: blood glucose 
Malaria TDR: SD Bioline pan-LDH & pf-HRP2 
Thick drop 
Dengue TDR: SD Bioline Dengue Duo 
White blood cells and differentiation: HemoCue ® WBC/DIFF 
CRP (if not yet performed on capillary blood) : QuickRead Go ®, cuvettes 
CRP/Hemoglobin (with Hemoglobin) or RDT 
Centrifuge the remaining volume for aliquoting (into 2-5 aliquots of 350 µL of 
plasma) and stored in the biobank at -80°. The aliquots will be partly 
used for metagenomic analyses, the remaining volume (if any) will be 
stored for secondary searches. 
b. Blood culture bottle (1-4 ml in children < 14 years, 20 ml in adults) 
patients ≥ 14 years of age): according to routine monitoring criteria 
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c. 2 ml serum tube: biochemical analysis according to clinical need 
d. 2 ml additional EDTA: storage and secondary research 
e. 2.5 ml PAXgene tube: storage in the biobank at -80°C, for research purposes. 
secondary (e.g. transcriptional profiling) 
Blood cultures will be taken and analyzed in accordance with the monitoring protocol. 
(protocol approved in DRC and Belgium; IRB IMT 613/08, UZA CE 8/20/96). 
Study analyses (except metagenomic analyses) will be performed at the 
IME and the HGK study laboratory. Samples for metagenomic analysis 
will be transported to the ITM laboratory in Antwerp (as agreed in a 'Material 
Transfer Agreement' - MTA). Local empowerment of partner institutions is planned as part of a 
second time. 
Samples for secondary research will be stored in the biobank at the following sites 
or at the ITM, Antwerp. All relevant sample data will be 
recorded in the biobank database. Samples obtained and stored in the 
biobank belong to the research site. 
The sponsor guarantees that an MTA will be drawn up and signed for each sample transfer in 
outside the research site. The intellectual property of the results belongs to the institution which 
receives samples An agreement will be reached between the study sponsor and the institutions 
partners (INRB, CRSK or IME) prior to any secondary research, as well as 
the approval of the relevant ethics committees. No data will be shared before signature 
a data-sharing agreement. 
The study will ensure that all laboratory activities, including transport, processing and storage, are 
carried out in compliance with the applicable standards, 
analysis, reporting of results and storage of samples, will be carried out in accordance with 
to GCLP. The laboratory will perform diagnostic tests in accordance with the following procedures 
and EN-ISO 15189 standards. A laboratory analytical plan will be created 
before the start of the study, describing all aspects in accordance with the WHO GCLP. 
6. STATISTICAL METHODS 
A detailed statistical analysis plan will be drawn up before recruitment begins. The analysis plan 
will provide a full description of the variables of interest, statistical methods (including the 
management of missing data, statistical adjustment, management of confounding factors and 
interactions), subgroup definitions and sensitivity analyses. 
6.1 Variables of interest 
● 
● 
● 
● 
● 
● 
Demographic characteristics 
Basic clinical data (previous treatment, history, symptoms, clinical examination) 
Diagnostic hypothesis 
Patient outcome 
Examinations and treatments prescribed and carried out 
Outcome (survival with symptom resolution, survival without symptom resolution, 
death) 
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● 
● 
Results of inflammatory biomarker analyses: CRP, white blood cells, and 
differentiation 
Results of etiological analyses: malaria, blood cultures, and germ antibiograms 
identified, metagenomic analysis, dengue TDR 
6.2 Statistical methods 
Baseline demographic and clinical characteristics will be described in terms of median and mean 
values. 
of interquartile ranges for continuous characteristics, and in terms of numbers and 
percentages for categorical characteristics. Certain demographic and 
will be compared between clinical outcome groups: survival with resolution of clinical 
symptoms vs. survival with no symptom resolution vs. survival with symptom resolution 
vs. death. Comparisons between groups will be made using the Mann-Whitney test. 
for continuous characteristics and the Chi-square test or Fisher's exact test for continuous 
characteristics. 
categorical characteristics for the three different strata (combination of site and group) 
age) 
We describe the proportion of patients meeting the criteria of resolution, non-resolution and death, 
overall and by basic characteristics (age, gender, geographical location, category, etc.). 
syndromic, patient characteristics) using percentages and intervals of 
Wilson's 95% confidence level. Univariate and multivariate logistic regression models 
will be used to analyze the association between baseline characteristics and clinical status on the 
following days. 
21. 
We will use Kaplan-Meier estimates to describe the probability of survival and resolution. 
over time, globally and by category. In addition, death will be used as a risk 
in this model. Univariate Cox proportional hazard models and 
will be used to analyze the association between baseline and follow-up characteristics. 
and mortality. Biomarker data will be described mainly using 
descriptive statistics in the same way as described above. Prevalence of malaria 
in the study population will be calculated as a proportion with a confidence interval of 
Wilson at 95%. The association of CRP and white blood cell values with patient outcome 
at day 21, or with specific etiologies, will be examined using the Mann-Whitney test. 
Diagnostic performance will be assessed by calculating the following performance measures 
standard diagnostic parameters, such as sensitivity and specificity. 
6.2.1 Subgroup analyses 
As this is a stratified study, all analyses will be presented by stratum (combination of strata). 
site and age group). 
6.2.2 Multiplicity and missing data 
An analysis of all available cases will be the main analysis for all objectives. The data 
data points, with the exception of clinical status, will be reported, and in 
In the case of limited missing data, multiple imputation will be used to recover the data. 
A subsequent analysis with the full set of imputed data will be carried out and compared with 
primary analysis. No adjustment for multiplicity is required in the analyses. 
6.3 Sample size and power 
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Amendment 3 Protocol v4.0, dated July 4, 2022 

  
 
 
 
 
Taking into account the main and strategic aims of this study (to describe the presentations 
and issues on the 2 sites, initiate the research capacity of the teams, provide the bases 
epidemiological studies), the expected sample size is based on the 
budget, reality and logistical feasibility. Size refers to the number of participants 
recruited in the laboratory phase of the study (following amendment 1 of the protocol). We plan to 
to include 500 children at HGK / Mama Yemo, and 500 at HGR-IME + health centers in Kimpese. 
children + 500 adults for a one-year study period. This number is sufficient to provide 
data relevant to the study's objectives, and will also enable teams to manage 
study requirements. With an expected drop-out rate of 20%, we hope to have an accuracy of at least 
less ± 4.9% in each stratum (combination of site and age group) in the estimation of the 
proportion of the primary result. 
7. QUALITY CONTROL AND ASSURANCE 
The study will be conducted and monitored in accordance with this protocol and the applicable 
regulations. 
for clinical research, in particular ICH-GCP and WHO-GCLP, and the procedures manual 
(MOP). 
The investigator 
coordinates 
nator and the local principal investigators will coordinate all 
study follow-up activities, including : 
● 
● 
Develop and monitor workflows, schedules, milestones and performance measurement tools. 
progress. 
Hold regular team meetings to discuss project progress, 
activities and to resolve any problems that may arise. These can be face-to-face meetings 
or remotely, depending on distance. 
● 
● 
Ensure data quality and query resolution. 
Visit each clinical site regularly to : 
o 
o 
o 
check compliance with study protocol and procedures 
verify that informed consents are valid 
verify the source data by comparing it with 
data stored in the database 
o 
o 
verify that confidentiality is fully respected. Monitoring standards 
require full verification of the presence of informed consent and the 
compliance with eligibility criteria. 
Verify/monitor the quality of laboratory tests carried out according to 
GCLP practices and SOPs 
The PI and the research staff at the site concerned will devote the necessary time and resources to this 
project. 
to these monitoring activities. The investigator will also ensure that the insurance monitors 
of the sponsor (ITM CTU), the CRC (INRB) and the CRSK have access to all the documents and 
facilities related to the above-mentioned study and has the necessary space and resources 
to monitor and verify source data. The promoter will contact 
investigators concerned as soon as it is informed of an ongoing study center inspection by a 
regulatory authority or funder. Similarly, the investigator will inform the sponsor of 
any ongoing inspections. Monitoring of the study will be carried out by the CTU monitor and will be 
done by 
according to the monitoring plan. 
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Amendment 3 Protocol v4.0, dated July 4, 2022 

  
 
 
 
 
8. DATA MANAGEMENT 
Data management procedures will comply with the 
ITM. 
At each participating clinical site and each coordinating center, the investigator will keep a record of all 
the data collected. 
hard copy of the main study file (IF: Investigator File), containing all the elements of the study 
(protocol, CRF, MOP) and regulatory requirements (ethics committee approvals), 
task delegation descriptions), CVs, memorandums of understanding). 
Data management will be organized and carried out by the ITM/CTU data manager in 
collaboration with the INRB/CRC team and the CRSK. Full details of the treatment of 
study data will be described in the "data management plan", which will comply with SOPs 
and regulatory requirements. The 
study data will take into account the following essential elements. 
Data collection 
Only data defined by the study protocol will be collected. Data collected 
will be registered via electronic forms (eCRF) in the study database at 
using a REDCap platform. 
REDCap is a data capture and management software for clinical studies. REDCap complies with 
ICH quality standards and good clinical practice, as well as the recommendations and 
requirements such as CFR 21, Part 11 (e.g. electronic signature, audit trail, etc.). 
electronic). The software includes, among other things, an integrated system for tracking and managing 
queries, as well as data import and export functions in a variety of formats. 
shapes. 
The eCRF and database have been designed in accordance with the specifications drawn up in the 
FIKI² study. The specifications include the forms, the list of included variables 
in forms, programmed checks on eCRF to validate data at the time of 
data entry and automated queries to verify data and quickly identify 
or inconsistent. A complete validation of the eCRF system will be carried out, by 
in particular the input controls. It is only after final approval of the test phase and the 
validation report that data entry can begin. 
Data capture and further processing will be carried out at all sites by our own staff. 
under the responsibility of the site's principal investigator. Participants will be identified by 
study-specific participant code on the CRF and in the database (pseudonymization). The 
name and any other identifying details will be kept only on the recruitment site and will not be disclosed 
to third parties. 
will NOT be included in any electronic study data file. 
An ITM server hosts the REDcap platform and eCRF data. ITM will have access at all times to 
access to a database export for follow-up activities. 
Confidentiality and security of participant data 
Study participants' personal information will be treated confidentially. 
Information such as the participant's name or any other data likely to make it possible to 
identification will not be recorded in the study database, nor in the database of the 
no other paper documents or electronic files used for the study. Each patient will receive a 
unique study code that will be used for registration in the study database. The 
26 
Amendment 3 Protocol v4.0, dated July 4, 2022 

  
 
 
 
 
the name and contact details of each participant and the link to the unique study code will be retained 
and access to them will be restricted to clinical site personnel. 
Access to paper documents and electronic files required for data management will be provided by 
strictly limited to ITM-authorized study personnel. Study computers and files 
eCRF will only be accessible with a personal username and password. A list of 
of authorized users of eCRFs (and the database) will be kept at ITM and updated as required. 
study. 
A data backup will be performed at server, computer and/or database level. 
data. 
Data quality and verification 
Best practices in CRF design, such as the use of codes and checkboxes, 
and drop-down menus, will improve data quality. A system will be 
set up for data entry, verification, query resolution, backup and, last but not least, 
database locking. In addition to automatic verification checks 
will also be carried out to identify out-of-spec data, data with a high 
and inconsistent data. The central data manager, the coordinating investigator, 
the INRB team and the sites, will hold regular teleconferences on data management 
throughout the study. 
Record-keeping 
Electronic documents for data management purposes, including txt and pdf formats, the 
Excel files, REDCap databases and their extractions, eCRF-generated files (in 
study descriptions), programs and e-mails will be stored in a secure 
and organized in a specific access-controlled data management subfolder on the server 
of ITM. Paper documents used for data management, such as mail printouts, 
data lists and data management documentation will be stored at the ITM in special 
specific files. ITM will ensure that access to these paper and electronic files is controlled. 
(e.g. in closed cabinets or rooms). Details on shelf life 
paper documents and electronic files are available in the section 
Archiving (see below). 
Data sharing 
Study data relating to participants will be available for sharing within a given timeframe. 
reasonable after the study and in accordance with the research data sharing principles of 
ITM. 
The notion of "data sharing for secondary research" will also be indicated in the 
informed consent form. 
The study's anonymized data will be supplemented by metadata and the 
documentation, such as the study protocol, annotated CRFs and other information such as 
codes that together form a dataset, but will not include any direct identifiers. The 
researchers can ask ITM to access anonymized data for specific research purposes. 
or secondary analyses via a controlled access procedure. 
Training 
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Amendment 3 Protocol v4.0, dated July 4, 2022 

  
 
 
 
 
For the study to be optimally implemented, all the teams involved must have a good understanding of 
the subject. 
knowledge of the study documents and tools, as well as good clinical practice, and 
principles of research ethics. Specific training will be given to all members 
of t h e  study team involved in patient care, data collection and data entry 
data verification and data monitoring. All those involved in the study will be 
trained before inclusion begins. They will also receive ongoing training throughout the year. 
throughout the study. 
9. ETHICAL ISSUES 
9.1 Ethical and regulatory review 
This clinical trial will be submitted to the Institutional Review Board for formal review and approval. 
of the ITM, the CE of the University Hospital of Antwerp and the relevant ethics committees in the DRC. 
No 
specific intervention will not take place until written approval has b e e n  obtained from the study 
committees. 
ethics, compliance with local regulatory requirements and signature of the clinical study protocol. 
of each contractual party involved. Ethics committees to be kept informed of progress 
and then informed of the study's conclusions. 
The study will be carried out in compliance with current national and international regulations, 
including 
including CIOMS international ethical guidelines for biomedical research 
involving human subjects (16), the Declaration of Helsinki (17), the harmonized tripartite directive 
for ICH Good Clinical Practice E6 (ICH-E6) (14), the WHO GCLP (15) and the 
Nagoya (18). 
The study will also be included in the public registry [Clinicaltrials.gov] before recruitment begins. 
of participants. 
9.2 Protocol changes 
Once the final clinical study protocol has been issued and signed by the authorized signatories, it cannot 
be used for any other purpose. 
can be amended informally. Amendments to the protocol have the same legal status 
and must pass through the appropriate stages b e f o r e  being implemented. Any modification 
substantial must be approved by all the organizations and CEs that have approved the protocol 
before i t  is implemented, unless it is due to concerns about the safety of 
participants (in which case immediate implementation may be necessary to protect 
participants). If changes to the protocol or an amendment are requested by an EC/CA 
during the review process, they must be discussed and agreed with the promoter 
before any new submission incorporating these changes. 
9.3 Informed consent 
No participant may be admitted to the study until the investigator or designated person 
has obtained the written informed consent form. 
9.4 Privacy policy 
Individual medical information collected in this study will remain confidential. Visit 
members of t h e  study team are bound by professional secrecy. The data 
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Amendment 3 Protocol v4.0, dated July 4, 2022 

  
 
 
 
 
will be made available to doctors in charge of patients on request. 
to the investigators. Disclosure to third parties is strictly forbidden. 
The electronic files will not contain any data that could allow the identification of the customer. 
(e.g. surname, first name, full date of birth, telephone number). Each 
participating in the study will be assigned a unique identification code (ID). On all documents or 
study files, study participants will be identified by the participant identification code. 
At each clinical site, a correspondence list will establish the link between the participant ID and the 
personal data of the participant so that the results of the examinations can be used for the 
patient care. This list containing personal data and other documents containing the 
names or signatures of participants (e.g., informed consent) will be retained. 
separately from other study documents. All study documents will be stored in a 
a secure, locked area, with access limited to the site's principal investigator and staff 
authorized. 
Access to all paper documents and electronic files required for data management 
and monitoring of the study will be limited to authorized personnel, at international, regional and local 
levels. 
The study computers and the eCRF files will only be accessible via a connection with a 
user name and a personal password. A list of authorized users of eCRF and 
the database will be kept at ITM and updated throughout the study. 
9.5 Risks and benefits 
A venous +/- capillary blood sample is part of the study procedures, but is in any case 
also necessary in the clinical management of most patients. The 2 
samples will be integrated. Sampling can be uncomfortable, even painful. Visit 
risk of complications from blood sampling is very low (may include haemorrhage in elderly patients). 
people with coagulation disorders, puncture site infection or thrombosis 
superficial veins). The amount of blood to be collected for this study, both for routine 
and additional analyses, in no case exceeds the maximum of what is considered acceptable. 
as safe for the patient. In any case, routine analyses for management will always be 
priority, if sufficient volume cannot be achieved. 
The greatest risk for participants is a potential breach of confidentiality of 
data, for which all measures will be put in place to limit them as much as possible. 
Apart from access to laboratory analyses that can improve management (some research 
biomarkers), there are no advantages to participating in the study. Compensation 
is still provided for a requested trip, this compensation does not exceed the 
usual burden of effort. 
9.6 Incidental discoveries 
All results of immediate laboratory analyses in the FIKI² study (with the exception of TDR 
for dengue fever, currently being evaluated in the African context) will be communicated to the team. 
the patient. In general, these are analyses that should already be part of the 
routine management of febrile illnesses. 
As explained above, however, participation in the study can lead to chance discoveries. 
pathogens that can give rise to chronic infections (asymptomatic for long periods), 
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Amendment 3 Protocol v4.0, dated July 4, 2022 

  
 
 
requiring further action at the level of the individual patient. These discoveries can 
occur in metagenomic sequencing and secondary research. 
If an endemic infection with immediate importance for management such as 
trypanosomiasis is detected during secondary research, the patient and medical team will be 
informed, and the patient will be offered a free diagnostic evaluation 
(and possible treatment) at a nearby referral center. 
If a chronic infection (HIV, HBV, HCV, etc.) is discovered incidentally during the analyses 
metagenomics or subsequent secondary research), only the results considered to be 
will be communicated. The results of this technique are not 
considered conclusive for the time being (the metagenomic analysis currently being 
development is not yet a validated clinical laboratory test) and would require a 
additional confirmation through a standard clinical workup. The study teams 
will then be responsible for informing the participant in question and directing him or her to the care 
structure 
appropriate for the health problem detected. 
9.7 Compensation for participation 
There is no compensation for participation in the study. Patients who cannot be 
contacted by telephone and who return to the study center on days 7, 14 and 21 will receive a 
amount sufficient for a round trip using standard means of transport in addition to any 
daily allowance, in line with that given by similar studies on this site, and 
approved by local ethics committees. 
9.8 Insurance 
The coordinator of this study, the Institute of Tropical Medicine, obtained study insurance (without 
liability) to cover any injury, damage or loss suffered by study par琀椀cipants and which is 
caused directly or indirectly by their participation in the study. 
Information on insurance is not specified in the GSI of this study, due to the fear that 
of unreasonable claims for compensation. In addition, we do not anticipate any 
physical injury or mental health problems as a result of participating in this event. 
observational (non-interventional) study. 
10. DISSEMINATION OF RESULTS, INTELLECTUAL PROPERTY 
All study documents are provided by the sponsor to the investigators and their staff. 
confidentiality. None of these documents may be disclosed to any party who is not a 
not directly involved in the study, without the written authorization of the sponsor. 
Once the results are considered final, arrangements will be made for the 
communicate to the community, in the most appropriate way, with the active participation of 
health authorities. 
Oral communications and written publications must mention the name of the sponsor and the name of 
the author. 
all the partners and researchers involved. 
Each declaration and publication will be made in accordance with the CONSORT 
declaration. 
(h琀琀p://www.consort-statement.org/consort-statement) 
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Amendment 3 Protocol v4.0, dated July 4, 2022 

  
 
 
 
 
11. ARCHIVING 
The sponsor and the investigator must keep adequate and accurate records to allow for 
fully document the course of t h e  study and verify study data. Visit 
relevant (essential) documents are those documents that individually and 
to assess the progress of the trial, the quality of the data produced and compliance with 
PCM standards and applicable regulatory requirements. The investigator's file must contain 
at least all the (essential) documents listed in the "Setting up and maintaining the 
investigator's file". A copy of all source data and report forms from 
must always be kept on site. 
Once the study has been completed, the study master file (IF) on the site and in the coordination center 
remain available for internal audits and/or inspections by regulatory authorities 
for a period of 20 years, unless otherwise requested by the national authorities. The sponsor must 
be informed before the files are destroyed. 
12. REFERENCES 
1 . 
Reddy EA, Shaw AV, Crump JA. Community-acquired bloodstream infections in Africa: a 
systematic review and meta-analysis. Lancet Infect Dis. Jun 2010;10(6):417-32. 
Kotloff KL, Nataro JP, Blackwelder WC, Nasrin D, Farag TH, Panchalingam S, et al. Burden and 
2. 
aetiology of diarrhoeal disease in infants and young children in developing countries (the Global 
Enteric Multicenter Study, GEMS): a prospective, case-control study. Lancet Lond Engl. 20 Jul 
2 013;382(9888):209-22. 
Levine OS, O'Brien KL, Deloria-Knoll M, Murdoch DR, Feikin DR, DeLuca AN, et al. The 
3. 
Pneumonia Etiology Research for Child Health Project: a 21st century childhood pneumonia etiology 
study. Clin Infect Dis Off Publ Infect Dis Soc Am. Apr 2012;54 Suppl 2:S93-101. 
4. 
Bah EI, Lamah M-C, Fletcher T, Jacob ST, Brett-Major DM, Sall AA, et al. Clinical presentation 
of patients with Ebola virus disease in Conakry, Guinea. N Engl J Med. 1 Jan 2015;372(1):40-7. 
5 
6 
. 
. 
Tissot-Dupont H, Raoult D. Q Fever. Infect Dis Clin North Am. Sep 2008;22(3):505-14. 
Roth PJ, Grant DS, Ngegbai AS, Schieffelin J, McClelland RS, Jarrett OD. Factors associated with 
mortality in febrile patients in a government referral hospital in the Kenema district of Sierra Leone. 
Am J Trop Med Hyg. Jan 2015;92(1):172-7. 
7. 
Crump JA, Ramadhani HO, Morrissey AB, Saganda W, Mwako MS, Yang L-Y, et al. Invasive 
bacterial and fungal infections among hospitalized HIV-infected and HIV-uninfected adults and 
adolescents in northern Tanzania. Clin Infect Dis Off Publ Infect Dis Soc Am. 1 Feb 2011;52(3):341-8. 
8 . 
Prasad N, Murdoch DR, Reyburn H, Crump JA. Etiology of Severe Febrile Illness in Low- and 
Middle-Income Countries: A Systematic Review. PloS One. 2015;10(6):e0127962. 
Murray CJL, Rosenfeld LC, Lim SS, Andrews KG, Foreman KJ, Haring D, et al. Global malaria 
mortality between 1980 and 2010: a systematic analysis. Lancet Lond Engl. 4 Feb 2012;379(9814):413- 
9. 
3 
1 
1. 
0. 
D'Acremont V, Kahama-Maro J, Swai N, Mtasiwa D, Genton B, Lengeler C. Reduction of anti- 
malarial consumption after rapid diagnostic tests implementation in Dar es Salaam: a before-after and 
cluster randomized controlled study. Malar J. 29 Apr 2011;10(1):107. 
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Amendment 3 Protocol v4.0, dated July 4, 2022 

  
 
 
11. 
D'Acremont V, Bosman A. WHO informal consultation on fever management in peripheral 
health care settings: a global review of evidence and practice. Geneva World Health Organ. 2013;759- 
764. 
12. Greninger AL, Naccache SN, Federman S, Yu G, Mbala P, Bres V, et al. Rapid metagenomic 
identification of viral pathogens in clinical samples by real-time nanopore sequencing analysis. 
Genome Med. 2015;7:99. 
1 3 Kreuder Johnson C, Hitchens PL, Smiley Evans T, Goldstein T, Thomas K, Clements A, et al. Spillover 
and pandemic properties of zoonotic viruses with high host plasticity. Sci Rep. 2015;5:14830. 
4. ICH. Efficacy Guidelines [Accessed December 31, 2018]. 
https://www.ich.org/products/guidelines/efficacy/article/efficacy-guidelines.html 
5. WHO. TDR | Good Clinical Laboratory Practice (GCLP) [Accessed December 31, 2018]. URL: 
https://www.who.int/tdr/publications/tdr-research-publications/gclp-web/en. 
6. Council For International Organizations Of Medical Sciences. International Ethical Guidelines 
1 
URL: 
1 
1 
for Biomedical Research Involving Human Subjects. [Accessed December 31, 2018]. URL: ICH 
Harmonised Guideline. Available at: 
https://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Efficacy/E6/E6_R2__Step 
_ 
1 
4_2016_1109.pdf 
7. The World Medical Association. Declaration of Helsinki - Ethical Principles for Medical 
Involving Human Subjects [Accessed December 31, 2018]. URL: 
Research 
https://www.wma.net/policies-post/wma-declaration-of-helsinki-ethical-principles-for-medical- 
research-involving-human-subjects 
18. 
Convention on Biological Diversity. Nagoya Protocol [Accessed December 31, 2018]. URL: 
https://www.cbd.int/abs/text/default.shtml 
13. LIST OF ABBREVIATIONS 
ACVPU 
alert, confusion, voice, pain, unresponsive' 
ALMANACH 'algorithm for the management of childhood illness'. 
ALERRT 
the African Coalition for Research, Intervention and Training in the 
epidemic 
AQ 
Quality assurance 
CA 
Competent authorities 
CIOMS 
CFR 
Council for international organizations of medical sciences 
Code of Federal Regulations 
CONSORT 
CRC 
Consolidated Standards of Reporting Trials 
Clinical Research Center 
(e-)CRF 
CRP 
'(electronic -) Case Report Form' - the data collection form 
C-reactive protein 
CRSK 
CQ 
Kimpese Health Research Center 
Quality control 
DGD 
Swiss Agency for Development and Humanitarian Aid 
Data and Security Control Board 
The European & Developing Countries Clinical Trials Partnership 
'Febrile Illness in sub-Saharan Africa'. 
DSMB 
EDCTP 
FISSA 
GCP 
Good Clinical practice 
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Amendment 3 Protocol v4.0, dated July 4, 2022 

  
 
 
GCLP 
GDPR 
HGK 
HGR 
IC 
Good clinical laboratory practice 
General Data Protection Regulation 
Kinshasa General Hospital 
General Reference Hospital (Kimpese) 
Investigator Coordinator 
IC(F) 
ICH 
Informed Consent (Form)' - Formulaire de Consentement Eclairé 
The International Council for Harmonisation - 'Conférence internationale sur 
harmonization 
(I)CE 
IGSA 
IME 
Ethics Committee (independent) 
Simplified ambulatory severity index 
Evangelical Medical Institute 
ITM 
Institute of Tropical Medicine 
INRB 
IRB 
MOP 
MTA 
IF 
French National Institute for Biomedical Research 
Institutional Review Board 
Operating procedures manual 
Material Transfer Agreement 
Investigator's File' - main study file 
Principal Investigator 
IP 
WHO/WHO 
POC 
qSOFA 
GROUND 
FLOOR 
SAP 
World Health Organization/Organisation mondiale de la Santé 
Point of care 
Quick Sepsis Related Organ Failure Assessment 
Democratic Republic of Congo 
Statistical analysis plan 
SDV 
SOP 
TDR 
TMG 
EU 
Checking source data 
Standard Operational Procedure 
Rapid diagnostic tests 
Trial Management Group 
European Union 
UZA 
Universitair Ziekenhuis Antwerpen (~ University Hospital Antwerp) 
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Amendment 3 Protocol v4.0, dated July 4, 2022 

  
 
 
14. 
APPENDICES 
Appendix 1: Protocol for the evaluation of rapid diagnostic tests for 
CRP 
Background 
and 
justification 
Acute febrile illnesses are a major cause of death and disability. 
morbidity worldwide. In countries with limited resources, they are 
often treated empirically with antibiotics due to a lack of 
the absence of reliable diagnostic methods, whereas in most 
In some cases, this is not necessary. It is well established that inappropriate 
use of 
antibiotics, including in the first line of care, contributes to the 
the spread of antibacterial resistance. 
C-reactive protein (CRP) is a biomarker of inflammation, which can 
help distinguish between bacterial and non-bacterial causes 
fever. Currently, rapid diagnostic tests (RDTs) 
for CRP determination, are becoming increasingly available and can be used 
to 
could be very useful in the management of febrile illnesses 
in primary health care centers. 
Before evaluating the use of these tests in the routine practice of 
primary health centers in the DRC, we want to evaluate a selection of 2 
immunochromatographic tests (lateral flow) measuring CRP (a 
qualitative and semi-quantitative) in a controlled laboratory environment. 
Evaluation study of 2 RDTs targeting CRP in the clinical laboratory, 
as part of the FIKI² study 
Design 
Site and population The study uses existing samples (EDTA blood) from a subpopulation of 
study 
participants in the FIKI² study, and will be carried out in the site laboratory 
correspondents : 
1 . Laboratory of the pediatric emergency department of the Hôpital 
Général de 
Kinshasa (HGK) 
. Institute General Reference Hospital (HGR) Laboratory 
2 
Evangelical Medical Institute (IME) of 
Kimpese 
Objectives 
and 
criteria 
judgment 
1. Evaluation of the performance of 2 CRP RDTs in comparison with a 
reference test (QuikRead go®) on patient samples 
of fever obtained in the FIKI² study. 
Judging criteria 
● degree of correlation between RDTs and reference test (with 
reading at minimum and maximum time depending on 
mode 
and late reading) 
● 
● 
● 
● 
● 
● 
● 
proportion of invalid tests 
proportion of migration errors 
low background lightening ratio 
proportion of test line intensities by scale 
degree of inter-observer agreement 
degree of repeatability of TDRs 
presence of the hook effect for very high CRP values 
high (> 150 mg/l) 
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2. Assessment of operational features and ease of use 
2 CRP RDTs in the laboratory 
Judging criteria 
● systematic analysis of the characteristics of TDRs and their 
packaging, labelling and instructions for use as per checklist 
Inclusion criteria: 
Criteria 
inclusion and 
exclusion 
● samples from patients who have given consent 
for the laboratory part of the FIKI² study 
CRP value (Quikread Go®) according to selection and 
planned distribution (as described in the 'Selection' section) 
and sample distribution') 
● 
Exclusion criteria : 
● samples with a maximum sampling volume of 
blood (depending on age, weight and Hb, as described in 
the FIKI² study procedures) could not be obtained 
Selection and 
distribution of 
samples 
1. Assessment of correlation between RDTs and reference test, degree of 
concordance between 2 readers, proportion of invalid tests, errors in 
migration, low background brightening, and line intensity 
● Number of samples planned: 140 (70 per site, at 
adapt to recruitment speed) 
● Distribution 
■ 
■ 
■ 
■ 
■ 
2x14 samples with CRP < 5 mg/L 
2x14 samples with CRP ≥ 5 - < 10 mg/L 
2x14 samples with CRP ≥ 10 - < 40 mg/L 
2x14 samples with CRP ≥ 40 - < 80 mg/L 
2x14 samples with CRP ≥ 80 mg/L 
● 2 TDR 1x on each sample, with 2 readings 
by TDR 
2. Evaluation of the croquet effect (HGK only): 
● 
● 
● 
Number of samples: 5 
Distribution: 5 samples with CRP > 150 mg/L 
2 TDR 1x on each sample, with 1 reading 
by TDR 
3. Repeatability assessment (HGK only) 
● 
● 
Number of samples: 5 
Distribution 
■ 
■ 
■ 
■ 
■ 
1 sample with CRP < 5 mg/L 
1 sample with CRP ≥ 5 - < 10 mg/L 
1 sample with CRP ≥ 10 - < 40 mg/L 
1 sample with CRP ≥ 40 - < 80 mg/L 
1 sample with CRP ≥ 80 mg/L 
● 2 TDR 5 x on each sample, with 1 reading 
by TDR 
Procedures 
laboratories 
2 RDTs for CRP will be analyzed: 
- 
- 
Qualitative Biopanda® RDT (threshold 10 mg/l) 
Actim® semi-quantitative RDT (thresholds 10 mg/l, 40 mg/l 
and 
0 mg/l) 
8 
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Reference test for performance evaluation: PoC QuikRead go®. 
(CRP+Hb kit; range 5-200mg/l ~ hematocrit) 
Assessment of correlation between RDT and reference test, degree of 
concordance between 2 readers, proportion of invalid tests, test errors, etc. 
migration, low background brightening, and line intensity : 
TDRs will be performed on the EDTA samples of FIKI² participants. 
according to the planned distribution and the producer's recommendations, 
and read out 
minimum and maximum incubation times, and also beyond the set time 
by the manufacturer TDR. Readings will be taken and recorded by 
two independent readers at the 2 sites and 
result by Quikread Go. A test result will be considered 
as invalid when the control line is missing, mottled or 
irregular. A scale will be used to note the intensity of the line of 
test for each reading. 
A photo will be taken for each reading. In case of discrepancy for a test 
invalid, migration error or poor background illumination, the 
result of a third reading of the photo by the principal investigator will be 
taken as a reference. 
Evaluation of the hook effect, evaluation of repeatability : 
TDRs will be performed on the EDTA samples of FIKI² participants. 
according to the manufacturer's recommendations. These 
analyses will only be performed at HGK by the principal investigator 
of this assessment. 
Systematic analysis of TDR characteristics and packaging, 
labeling and instructions for use (based on a checklist of features) will be 
made and evaluated for ease of use. 
Methods 
analysis 
The kappa index (Cohen's coefficient of agreement) will be used to evaluate 
the degree of correlation between TDR (reading at minimum, maximum, and 
and reference test for the 2 different readings, and for the 
inter-observer agreement. With sample size, the distribution 
and an expected kappa value of 0.6 - 0.7, the interval size of 
confidence would be 0.184-0.22 (Actim® test) and 0.26-0.30 (Actim® test). 
Biopanda®) for correlation with the reference test. The proportion of 
invalid tests, migration errors, clarification of the background 
At a low level, the intensities of the test lines according to scale will be 
calculated 
with the 140 TDRs performed and read at maximum time as the 
denominator. 
Evaluation of the hook effect and repeatability will be carried out in the 
following way 
descriptive. 
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