ADEQUATE Advanced Diagnostics for Enhanced QUality of Antibiotic Prescription in Respiratory Tract Infections in Emergency Rooms - Paediatric
Phase: NA
Conditions: Community-acquired Acute Lower Respiratory Infection
Sponsor: PENTA Foundation

 
 
 
 
 
 
 
 
 
ADEQUATE Advanced Diagnostics 
for Enhanced QUality of Antibiotic 
prescription in respiratory Tract 
infections in Emergency rooms 
Clinical protocol WP4b - Paediatric 
JULIA ANNA BIELICKI, MARC BONTEN, MALTE KOHNS VASCONCELOS, CRISTINA PRAT AYMERICH, 
HENRI VAN WERKHOVEN 

 
 
 
 
This project has received funding from the Innovative Medicines Initiative 
2 Joint Undertaking under grant agreement No 820755. This Joint 
Undertaking receives support from the European Union9s Horizon 2020 
research and innovation programme and EFPIA and bioMérieux SA, 
Janssen Pharmaceutica NV, Accelerate Diagnostics S.L., Abbott, Bio-Rad 
Laboratories, BD Switzerland Sàrl, and The Wellcome Trust Limited. 

 
 
 
 
 

 
 
 
Randomised controlled trial of Rapid Syndromic Diagnostic Testing (RSDT) for 
enhancing the quality of antibiotic prescribing for community acquired acute 
respiratory tract infection (CA-ARTI) in Emergency Rooms in Europe.  
 
Protocol ID 
WP4b - paediatric 
Short title 
Advanced Diagnostics for Enhanced QUality of 
Antibiotic 
prescription 
in 
respiratory 
Tract 
infections in Emergency rooms - ADEQUATE 
Version 
5.0 
Date 
26 January 2023 
NCT number 
NCT04781530  
Coordinating Investigator 
Malte Kohns, St George9s, University of London 
Chief investigator 
Julia Bielicki, St George’s, University of London 
 
 
Sponsor  
Fondazione PENTA ONLUS 
Torre di Ricerca Pediatrica 
Corso Stati Uniti, 4 
35127, Padova 
Italy 
Subsidising party 
Innovative Medicines Initiative 2  
Grant agreement No 820755 
Steering Committee 
Marc Bonten, UMC Utrecht 
Cristina Prat, UMC Utrecht 
Herman Goossens, University of Antwerp  
Julia Bielicki, Fondazione PENTA 
Florence Frager, bioMérieux 
 
Trial methodologist, statisticians 
 
 
Data Safety Monitoring Board 
(DSMB) 
Henri van Werkhoven, UMC Utrecht  
Andrew Atkinson, University of Basel Children9s 
Hospital 
 
 
Mark Lyttle, Chair 
Harry Heijerman, Pulmonologist 
Michelle Clements, senior Statistician 
 
 

 
 
 
PROTOCOL SIGNATURE SHEET 
Study Title:  
Advanced Diagnostics for Enhanced QUality of Antibiotic 
prescription in respiratory Tract infections in Emergency 
rooms – ADEQUATE 
Version Clinical Protocol:  
V5.0, 18-Oct-2023 
Name 
Signature 
Date 
Sponsor: 
Laura 
Mangiarini, 
Chief 
Program 
Officer, Fondazione PENTA ONLUS  
 
 
 Chief Investigator: 
Julia Bielicki, Senior Lecturer, St 
George’s, University of London 
 
 
I have read the above-named Clinical 
Protocol and agree to conduct the 
trial as outlined and in compliance 
with country, local and internal 
institutional requirements. 
 
 

 
 
 
 
Name of Site Principal Investigator: 
Signature 
Date 
Hippokration 
Hospital 
of 
Thessaloniki, Greece 
Prof Emmanuel Roilides  
 
 
 
 
 

 
 
 
Name of Site Principal Investigator: 
Signature 
Date 
Hospital Universitario 12 de Octubre, 
Spain 
Dr Pablo Rojo Conejo 
 
 
 
 
 

 
 
 
Name of Site Principal Investigator: 
Signature 
Date 
University 
of 
Basel 
Children’s 
Hospital, Switzerland 
Dr Julia Anna Bielicki   
 
 
 
 
 

 
 
 
Name of Site Principal Investigator: 
Signature 
Date 
University Hospital of Lewisham, 
United Kingdom 
Dr Maggie Nyirenda  
 
 
 
 
 

 
 
 
Name of Site Principal Investigator: 
Signature 
Date 
University 
Children’s 
Hospital 
Tuebingen, Germany  
Dr Hanna Renk  
 
 
 
 

 
 
 
Name of Site Principal Investigator: 
Signature 
Date 
St 
George’s 
University 
Hospital, 
United Kingdom 
Dr Louisa Brock 
 
 
 
 
 

 
 
 
Name of Site Principal Investigator: 
Signature 
Date 
Ospedale Regionale Bellinzona e 
Valli, Switzerland 
Dr Federica Vanoni 
 
 
 

 
 
 
Table of contents 
 
1. LIST OF ABBREVIATIONS ..................................................................................................... 5 
2. SUMMARY .............................................................................................................................. 6 
3. General considerations Work Package 4 VALUE-Dx overview ................................... 7 
4. BACKGROUND AND RATIONALE ......................................................................................... 9 
4.1. Background........................................................................................................................ 9 
4.2. Rationale.......................................................................................................................... 10 
5. OBJECTIVES ......................................................................................................................... 12 
5.1. Co-primary objective: .................................................................................................... 12 
5.2. Secondary objectives: ................................................................................................... 12 
5.3. Exploratory objectives .................................................................................................. 12 
6. Study parameters/endpoints ........................................................................................ 12 
6.1. Main study parameter/endpoint................................................................................. 12 
Co-primary study endpoints: .............................................................................................. 12 
6.2. Secondary endpoints .................................................................................................... 12 
7. STUDY DESIGN and DURATION ........................................................................................ 13 
7.1. Study design and justification ..................................................................................... 13 
7.2. Study duration ................................................................................................................ 14 
8. STUDY POPULATION ......................................................................................................... 15 
8.1. Study population ............................................................................................................ 15 
8.2. Inclusion criteria ............................................................................................................ 15 
8.3. Exclusion criteria ........................................................................................................... 15 
8.4. Recommended study site selection criteria ............................................................. 16 
9. DIAGNOSTIC INTERVENTION ............................................................................................ 16 
9.1. Description of the test and Intended use .................................................................. 16 
9.2. Summary of findings from clinical studies ............................................................... 16 
9.3. Summary of known and potential risks and benefits .............................................. 17 
10. METHODS .......................................................................................................................... 18 
10.1. Screening and enrolment ........................................................................................... 18 
10.2. Randomisation and blinding...................................................................................... 18 
10.3. Data collection .............................................................................................................. 18 
10.4. Clinical data set ............................................................................................................ 19 

 
 
 
10.5. Baseline and follow-up data for health-economic analysis ................................ 21 
10.6. Microbiological testing ............................................................................................... 21 
10.7. Microbiology study and biobanking ......................................................................... 22 
11. Training ............................................................................................................................. 22 
11.1. Good Clinical practice training. .................................................................................. 22 
11.2. Medical device training, being part of the Site Initiation Visit ............................ 22 
11.3. Data management training ......................................................................................... 23 
12. SAFETY REPORTING ......................................................................................................... 23 
12.1. Adverse events .............................................................................................................. 23 
12.2. Reporting Adverse Events ........................................................................................... 26 
12.3. Device Deficiency .......................................................................................................... 27 
12.4. DEVIATIONS FROM THE CLINICAL STUDY PROTOCOL .............................................. 27 
12.5. Data Safety monitoring board (DSMB)...................................................................... 27 
13. STATISTICAL CONSIDERATIONS ..................................................................................... 28 
13.1. Sample size calculation ............................................................................................... 28 
13.2. Analysis populations.................................................................................................... 31 
13.3. Primary study parameter(s)........................................................................................ 32 
13.4. Secondary study parameter(s) .................................................................................. 32 
13.5. Mid-term data analysis ............................................................................................... 33 
14. ETHICAL CONSIDERATIONS ............................................................................................ 33 
14.1. Regulation statement .................................................................................................. 33 
14.2. Recruitment and consent ........................................................................................... 33 
14.3. Withdrawal of individual subjects ............................................................................ 34 
15. ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION .................................. 34 
15.1. Steering Committee...................................................................................................... 34 
15.2. Handling and storage of data and documents ....................................................... 35 
15.3. Storage of samples. Biobanking ................................................................................ 35 
15.4. Monitoring and Quality Assurance ............................................................................ 35 
15.5. Public disclosure and publication policy................................................................. 35 
16. REFERENCES ..................................................................................................................... 37 
 

 
 
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1. LIST OF ABBREVIATIONS 
AE  
 
Adverse event 
ARTI  
Acute Respiratory Tract Infections 
CA-ARTI 
Community acquired acute respiratory tract infections 
CRP 
 
C Reactive Protein 
COVID-19 
Coronavirus Infectious Disease 2019 
DALY  
disability adjusted life years 
eCRF  
electronic Case Report Form 
Dx 
 
Diagnostics 
EQ5D   
EuroQoL (Europe Quality of Life) 5 dimensions 
ER 
 
Emergency Room 
ETA 
 
Endotracheal aspirate 
GCP 
 
Good Clinical Practice 
GPDR  
General Data Protection Regulation 
ICD 
 
International Classification of Diseases 
ICF 
 
Informed Consent Form 
MDR  
Multidrug Resistant 
LAR   
Legally Accepted Representative  
LRTI   
Lower Respiratory Tract Infections 
NPS 
 
Nasopharyngeal swabs 
PCT 
 
Procalcitonin 
POCT  
Point of Care Testing 
PPAS  
Point Prevalence Audit Study 
RP2.1. plus 
Respiratory Panel version 2.1 plus (BioFire®FilmArray®) 
RSDT  
Rapid syndromic diagnostic testing 
SAE 
 
Serious adverse event 
SARS-CoV-2 Severe Acute Respiratory Syndrome Coronavirus 2 
SOP 
 
Standard Operating Procedures 
SCC 
 
Site selection committee 
SSP 
 
Site selection plan 
WP 
 
Work Package 

 
 
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2. SUMMARY  
Background and Rationale: Community-acquired acute respiratory tract infections (CA-ARTI) 
are among the most frequent infectious diseases worldwide. At the same time, uncomplicated 
acute respiratory infections (ARIs) are the most frequent cause of inappropriate antibiotic use. 
Antibiotic resistance rates are related to antibiotic use in any setting, but opportunities to 
implement a more judicious antibiotic prescribing are probably most apparent in primary care 
and emergency departments. Optimal clinical management of CA-ARTI is hampered because 
of diagnostic delays and suboptimal test sensitivity, leading to incorrect or missing etiologic 
diagnosis, and over prescription of antibiotics. Highly sensitive molecular assays have 
increased the detection of respiratory pathogens, but the impact in clinical decision-making 
needs further evaluation. Accurate and rapid identification of infected patients allows for more 
rational and effective infection control practices and public health responses which will limit 
morbidity and mortality, economic damage, and can allow low risk/non-infected and 
recovered people to return to the workforce. 
Objective: To assess the impact of rapid diagnostic testing of patients with Acute Respiratory 
Tract Infection (ARTI) at the emergency department, on (1) hospital admission rates and 
(2) antibiotic prescriptions (days of treatment). Geographical and seasonal variation will be 
assessed on a real time basis including pathogens of public health interest. The impact will be 
stratified within age groups and risk factors in order to determine the long-term clinical, public 
health and economic determinants for the integration of diagnostics in a global and 
sustainable perspective. 
Study design: Prospective, multi-centre, individually randomised, controlled trial. 
Study population: Children of any age consulting in selected participating sites with CA-ARTI 
with initial uncertainty about management regarding hospitalisation and/or antibiotic 
therapy. 
Study Intervention: The diagnostic intervention is rapid syndromic testing with 
BioFire FilmArray Respiratory Panel 2.1 plus (RP2.1plus): Nasopharyngeal swab 
Main study parameters/endpoints:  
• Days alive out of hospital (superiority endpoint), within 14 days  
• Days on Therapy (DOT) with antibiotics (superiority endpoint), within 14 days 
Nature and extent of the burden and risks associated with participation, benefit and group 
relatedness: Participation in the study involves collection of data that can be obtained from 
medical charts and follow up questionnaires and interviews. Participants randomised to the 
intervention will receive a nasopharyngeal swab at the ER. This will cause a temporary 
uncomfortable feeling. Based on the results of diagnostic testing (BioFire FilmArray) antibiotics 
may be withheld when deemed unnecessary, or a different antibiotic class may be selected 
when certain bacterial pathogens are detected. The risks and benefits of management 
decisions, complemented with adequate training, are subject to the current investigation. 
 
 
 

 
 
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3. General considerations Work 
Package 4 VALUE-Dx overview  
The purpose of VALUE-Dx consortium (www.value-dx.eu) is to facilitate and accelerate the 
rigorous assessment and implementation of (new) diagnostic technologies into healthcare 
settings, by establishing the infrastructure, methods, processes and approaches needed to 
understand, evaluate, assess, and demonstrate the multi-faceted value of diagnostics and 
overcome the associated barriers to their widespread adoption and use. VALUE-Dx focuses its 
research on community acquired acute respiratory tract infections (CA-ARTI).  
 
The objectives of VALUE-Dx are: 
• To design a health-economic framework to assess and demonstrate the value of 
diagnostics both for individual patients and for public health impact by reducing 
antibiotic use and subsequent antibiotic resistance among patients; 
• To establish a sustainable European Standardised Care Network adequately trained and 
resourced to conduct clinical trials evaluating the value of diagnostics; 
• To design and implement clinical studies to demonstrate the value of diagnostics in the 
optimal management of CA-ARTIs; 
• To explore, define and attempt to resolve the psychological, ethical and social barriers 
which prevent the more widespread adoption of diagnostics delivering healthcare to 
the population. 
 
The clinical studies, to be implemented in WP4, aim to gather evidence on:  
i) 
doses or days of antibiotics prescribed,  
ii) 
proportion of patients not receiving antibiotics;  
 
WP4 studies will be performed separately in primary care and long-term facilities (WP4a)- 
POCT with Abbott and BD and Emergency Rooms (WP4b)- Rapid syndromic diagnostic testing 
with bioMérieux.  
 
The clinical trial will align – where possible with the other WPs of the project.  
• WP1 aims to develop evidence-based clinical algorithms that integrate point of care tests. 
Initial analysis has provided results on accuracy of the selected groups of items: signs and 
symptoms, biomarkers, imaging and rapid diagnostic tests, for prediction of influenza and 
bacterial pneumonia, respectively.  
• WP2 will explore analytical performance of some tests in the trials and will collect pathogen 
and host biomarker data.  
• WP3 will provide data management.  
• Within WP4a, during the winter season from January to March 2020, a web-based point 
prevalence audit survey (PPAS) on presentation and management of CA-ARTI in primary 
care and long-term facilities was performed. The aim is to retrospectively characterise 

 
 
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patients who seek healthcare for CA-ARTI, quantify antibiotic prescriptions and to 
benchmark patterns of diagnostic in different European countries.   
• WP5 is setting-up data collection for health economic modelling, that includes a disease 
model, diagnostic models and antibiotic resistance predictions. The variables to be 
included to assess direct and indirect medical costs and quality of life need to be collected 
in the clinical trials of WP4.  
• WP4b, The ADEQUATE study is an individual randomised study that aims to assess the 
impact of rapid syndromic diagnostic testing (Biofire® bioMérieux) in patients with Acute 
Respiratory Tract Infection (ARTI) at the emergency department, on (1) hospital admission 
rates and (2) antimicrobial prescriptions (days of treatment). 
• The WP4b clinical protocol has been split into an adult (WP4b – adult) and paediatric 
population (WP4b – paediatric), to address the differences in standard of care in the two 
populations, but with common sample size calculation and analysis of the results. The 
current protocol describes the trial of WP4b for the paediatric population. 
 
 

 
 
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4. BACKGROUND AND RATIONALE 
4.1. Background 
Community-acquired acute respiratory infections (CA-ARTI), including upper and lower 
respiratory tract infections, are among the most frequent infectious diseases worldwide. Lower 
respiratory tract infections (LRTI) are among the most lethal communicable diseases and the 
fourth cause of death globally, responsible for an estimated 3 million deaths in 2016 [1]. LRTI 
disproportionately affects children younger than 5 years and is the second cause of disability 
adjusted life years (DALYs). A study published in the Lancet in 2018 estimated the global, 
regional, and national morbidity, mortality, and aetiologies of LRTI in 195 countries, between 
the years 1990–2016.  In 2016, LRTI caused 652,572 deaths (95% uncertainty interval 586,475–
720,612) in children younger than 5 years, 1,080,958 deaths (943,749–1,170,638) in adults older 
than 70 years, and 2,377,697 deaths (2,145,584–2,512,809) in people of all ages, worldwide  S. 
pneumoniae was the leading cause of LRTI  morbidity and mortality globally, contributing to 
more deaths than all other aetiologies combined in 2016 (1,189,937 deaths (690,445–
1,770,660)[2], particularly as community acquired pneumonia. Within the European region, 
geographical variations are present. Over the years, the epidemiology has changed due to 
changing populations, with increased disease burden in elderly (>70 years) in many regions, 
varying prevalence of smoking and varying patterns of vaccine usage [3, 4].  
On the other hand, uncomplicated ARTI is the most frequent cause of inappropriate antibiotic 
use [5, 6], and there is a need of more judicious antibiotic prescription to prevent exposure to 
drug-related adverse events, selection of antibiotic resistance and emergence of opportunistic 
pathogens that substitute the indigenous microbiota. At the same time, the clinical role of 
bacteria whose normal ecological niche is the airways is an unresolved issue because of 
contamination with oropharyngeal flora Antibiotic resistance rates are related to antibiotic 
use in any setting, but opportunities to decrease unnecessary treatments are probably most 
apparent in primary care and emergency departments. Not only the ecological but also, the 
economic cost of antimicrobial resistance per antibiotic consumed is considerable [7-9]. 
Management is heterogeneous in diverse geographical areas due to non-uniform guidelines, 
both for diagnostic and antimicrobial stewardship [10]. 
One of the major challenges in clinical decision making is the absence of microbiological 
evidence of the aetiological agent in CA-ARTI at the time the antibiotics must be initiated so 
rapid diagnostic testing may have an impact. The required sample is not always available, and 
with conventional testing there may be low sensitivity (40-60% cases without aetiological 
diagnosis) and an important diagnostic delay before results are available. An accurate and 
reliable distinction between bacterial and viral causes of CA-ARTI would provide an important 
opportunity for better antimicrobial stewardship. However, due to substantial overlap in 
clinical disease presentation and laboratory parameters it is currently impossible to reliably 
distinguish viral from bacterial aetiology based on available tools. 
It has been proposed that implementation of Point-of-Care tests (POCT) with biomarkers or 
microbiological tests to differentiate viral from bacterial infections may reduce inappropriate 

 
 
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antibiotic prescriptions [11]. There is encouraging evidence from randomised trials for 
biomarkers-guided antimicrobial management. In a multicenter study in adults in primary care 
the combination of selected clinical symptoms with the addition of C-reactive protein (CRP) 
measurement improved diagnostic information, but measurement of procalcitonin (PCT) did 
not add clinically relevant information [12]. In a Cochrane review of controlled trials of 
biomarkers in patients with CA-ARTI, PCT appeared to be more informative than CRP and other 
inflammatory markers, as it was associated with an earlier increase upon infection, a better 
negative predictive value, and was influenced by immunosuppressive medication [13].  
The development of highly sensitive molecular assays has increased the detection of 
respiratory pathogens in patients with CA-ARTI, and increased our understanding of the role 
of viruses in CA-ARTI [14]. However, diagnostic methods that detect a virus do not always rule-
out bacterial infection. Additional diagnostic yield has been demonstrated by using molecular 
tests but evidence is limited regarding the impact on antibiotic use or costs [15-17].  
In this context, a new molecular rapid syndromic testing platform (BioFire; Salt Lake City, UT, 
USA) might improve clinical decision making in patients with CA-ARTI. The BioFire FilmArray 
Pneumonia Panel plus (PP) is a multiplexed nucleic acid amplification test that identifies 34 
bacterial and viral targets, including antimicrobial resistance genes from sputum or 
bronchoalveolar lavage (BAL) specimens. The BioFire FilmArray Respiratory Panel 2.1 plus 
(RP2.1plus) can simultaneously detect 24 viruses and atypical pathogens from nasopharyngeal 
swabs. Both panels allow syndromic testing and results can be provided in less than one hour 
with high sensitivity and specificity [18, 19]. Several single-center studies have reported 
promising results and ongoing clinical trials are summarised in section 9.2. Additional data is 
needed to prospectively assess the impact of rapid syndromic testing in daily clinical decision 
making as well as to determine its costs and effects.  
The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [20] has had 
an unprecedented and dramatic impact on the health care system and on the world economy. 
One of the most effective tools in the management of this global pandemic is the ability to 
rapidly and accurately test patients with signs and symptoms of ARTI or with risk factors for 
exposure. Some countries have used aggressive and widespread testing paired with contact 
tracing to manage the crisis and have been apparently the most successful at reducing 
mortality rates, the strain on the health care system and the spread of the virus. Expanding 
testing locations could prevent patients from spreading infections due to lack of availability 
of testing outside hospitals and by reducing the time between sample collection and test 
result. 
4.2. Rationale.  
Optimal clinical management of CA-ARTI is hampered because of diagnostic delays and 
suboptimal test sensitivity, leading to incorrect or missing aetiological diagnosis, and over 
prescription of antibiotics. Diagnostic and antimicrobial stewardship is important to get the 
optimal patient management.  
There is a need to assess the impact of rapid syndromic diagnostic testing in patients with CA-
ARTI presenting to Emergency Rooms on clinical decision making related to: 

 
 
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• Hospitalisation yes or no; 
• Start antibiotics yes or no; 
At the same time, it must be determined whether the decisions guided by the rapid syndromic 
diagnostic testing results do not compromise patient safety. 

 
 
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5.  OBJECTIVES 
5.1. Co-primary objective:  
To assess the impact of rapid syndromic testing in patients presenting with CA-ARTI in the ER 
on: 
1. Days in hospital within 14 days after study enrolment 
2. Days with antibiotic therapy within 14 days after study enrolment 
5.2. Secondary objectives:  
1. To assess the impact of rapid syndromic testing on occurrence of adverse outcome 
within 30 days after study enrolment. 
2. To assess the impact of rapid syndromic testing on healthcare utilisation. 
3. To assess the impact of rapid syndromic testing on quality of life.  
4. To quantify the additional diagnostic yield and sensitivity of rapid syndromic testing 
because of targets not included in standard of care testing. 
5. To quantify the impact of rapid syndromic testing on antimicrobial de-escalation and 
the choice of antibiotics and prescription of antivirals. 
6. To quantify the impact of rapid syndromic testing on detection of antibiotic 
resistance.  
7. To assess the impact of rapid syndromic diagnostic testing on patient bed 
management and-or isolation measures.  
5.3. Exploratory objectives 
• To biobank a subset of well characterised clinical samples to encompass 
epidemiological descriptions of public health interest 
• Collect comprehensive data on clinical status and laboratory results for development 
and validation of clinical algorithms.  
• To describe the current routine diagnostic and antimicrobial stewardship policy in 
Europe and identify good practices  
• To estimate the impact of rapid syndromic testing on primary and secondary 
endpoints for subcategories of hospitals with similar routine diagnostic and 
antimicrobial stewardship programs.  
6. Study parameters/endpoints 
6.1. Main study parameter/endpoint 
Co-primary study endpoints: 
1. 
Days alive out of hospital (superiority endpoint), within 14 days after study enrolment 
2. 
Days on Therapy (DOT) with antibiotics (superiority endpoint), within 14 days after study 
enrolment 
6.2. Secondary endpoints  
Adverse outcome (non-inferiority safety endpoint)  
• 
Safety endpoint: 

 
 
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• 
For initially hospitalised patients: i) any readmission, ii) ICU admission => 24 
hours after hospitalisation, or iii) death, within 30 days after study enrolment 
• 
For initially non-admitted patients: any admission or death within 30 days after 
study enrolment. 
• 
Direct costs and indirect costs within 30 days after enrolment.  
• 
Change in quality of life as determined by EQ-5D-5L (or suitable alternative for age), 
days away from usual childcare routine or school and healthcare utilisation on day 1, 
14, and 30 after enrolment. 
• 
Microbiological results obtained as standard of care and with the diagnostic 
intervention  
• 
Empirical antibiotics, antibiotic type switches, de-escalation based on antimicrobial 
agent categories [21]. Prescription of antivirals during the main study. 
• 
Detection of antimicrobial resistance (carriage or infection) related to the diagnostic 
intervention results compared to standard of care and impact on antimicrobial 
stewardship guidelines and prevention of hospital acquired infections. 
• 
Impact on decisions regarding isolation measures related to test result. 
 
7.  STUDY DESIGN and DURATION 
7.1. Study design and justification 
This is a prospective, multi-centre, individually randomised controlled open-label trial. 
Approximately 520 subjects will be randomised in the trial in up to 10 investigational sites in 
the European region. Subjects will be followed at day 14 and day 30 after randomisation. 
Enrolment will be competitive across sites.  
Justification of the design. The aim is to deliver a study outcome that is valid (absence of bias) 
precise (sufficiently powered to achieve clinically relevant absence and presence of 
difference) and generalisable (recognizable population). The study needs to be feasible in 
terms of costs (determined by number of study sites and number of tests to be performed), 
with an easy patient enrollment and an achievable burden on laboratories. Therefore, 
although an individually randomised trial - in contrast to cluster randomisation - requires 
informed consent from all recruited patients fulfilling inclusion criteria prior to performing the 
test. This approach has several advantages: 1. Internal validity of the trial is guaranteed by the 
randomisation, whereas in cluster-randomised trials, patient selection criteria can be difficult 
to apply in a consistent manner, inducing a risk of selection bias; 2. Required sample size is 
lower compared to a cluster-randomised trial; 3. Recognisable population can be realised, 
although generalisability will always be lower compared to enrolling all-comers; 4. Feasibility: 
compared to a cluster-randomised trial, a lower sample size is needed, reducing costs for 
study execution; fewer hospitals, fewer patients, and a lesser burden on labs because tests are 
only performed in 50% of enrolled patients instead of 100% of all-comers. Moreover, although 
requirement of informed consent to be obtained at the ER could be considered as a drawback 
of this design, it in fact may help in enrolling the pursued patient population for this specific 

 
 
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trial. We aim for patients in which the result of the rapid syndromic testing can guide clinical 
decision making. This implies that there is also time for obtaining informed consent.  
In choosing the primary endpoints the following considerations were made:  
• 
The composite endpoint is designed to capture the relevant outcomes and will be 
combined for analysis  
• 
In the paediatric population the non-inferiority co-primary endpoint is unlikely to be 
clinically relevant  
• 
Because of the non-blinded nature of the study, outcomes should be defined 
objectively, so e.g., we cannot use cause-specific re-admission. 
• 
Reassessment when other microbiological results are available. Discrepant results 
should be handled by the treating physician based on best practice.  
• 
Different time windows have been chosen because i) impact of rapid syndromic 
testing on hospitalisation / antibiotics is immediate. ii) Shorter follow-up increases 
power for difference of 1 day. iii) Adverse outcomes may reflect late sequelae 
(reflected in the key safety secondary endpoint).  
 
Modifications were made following the decision to terminate the recruitment of adult 
patients (Value Dx WP4b ADEQUATE Protocol, adult version 3.0 18-Feb-2022) on 3rd May 2022. 
Details are provided in sections 5, 6 and 13 of the current protocol.  
 
7.2. Study duration  
The study will encompass at least 3 influenza seasons (autumn/winter months) in Europe. 
Justification. i) most CA-ARTI occur in autumn winter months. ii) Timeframes allow for training, 
initiation and patient follow up. iii) To achieve an adequate sample size iv) Fluctuations in 
microbiological epidemiology of CA-ARTI (seasonal outbreaks Mycoplasma, Bordetella, 
variability on virus lineages, pneumococcal serotypes, etc.) are better represented with more 
than one season. 
The End of Trial is defined as the date of receipt of the last data point from the last subject 
that is required for primary, secondary and/or exploratory analysis. 
 
 

 
 
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8. STUDY POPULATION 
8.1. Study population  
Children of any age presenting at the Emergency Room in selected participating sites with CA-
ARTI, after informed consent by parents or legal guardians and participant9s assent where age-
appropriate.  
8.2. Inclusion criteria  
1. 
Children of any age presenting to the Emergency Room with an acute illness (present 
for 
14 days or less) with  
Temperature ≥38.0°C measured at presentation or parental report of fever within the 
previous 72 hours 
AND at least two of the below: 
- 
Cough 
- 
Abnormal sounds on chest auscultation (crackles, reduced breath sounds, bronchial 
breathing, wheezing) 
- 
Clinical signs of dyspnea (chest indrawing, nasal flaring, grunting) 
- 
Signs of respiratory dysfunction: tachypnoea for age or decreased oxygen saturation 
(<92% in room air) 
- 
Signs of reduced general state: poor feeding, vomiting or lethargy/drowsiness 
  
2. 
At time of screening: 
- 
Patient has undergone first assessment by managing clinical team (doctor or nurse, incl. 
triage) 
- 
Hospitalisation is not yet determined, i.e., neither by clinical presentation definitely 
requiring hospitalisation (e.g., per local guideline) nor by fixed decision of managing 
clinical team; admission to a short-stay unit or surveillance unit is not considered a 
hospitalisation for this trial 
- 
Antibiotic treatment or hospitalisation is being considered 
- 
The rapid syndromic diagnostic test result can be awaited up to 4 hours before the 
decision to discharge the patient or to initiate antibiotic treatment is made 
8.3. Exclusion criteria 
1. Development of ARTI more than 48 hours after hospital admission (hospital acquired); 
2. Patients with a severe underlying medical condition dictating management decisions 
including hospitalisation and/or antibiotic treatment (e.g., cystic fibrosis, 
immunosuppression);  
3.  Hospitalisation for at least 24 hours within the last 14 days (healthcare-associated); 
4. Confirmed pregnancy and/or breastfeeding; 
5. Any clinically significant abnormality identified at the time of screening that in the 
judgment of the Investigator would preclude safe completion of the study or constrain 
endpoints assessment such as major systemic diseases or patients with short life 
expectancy;  
6. Inability to obtain informed consent; 

 
 
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7. Alternative noninfectious diagnosis that explains clinical symptoms. 
  
8.4. Recommended study site selection criteria 
• 
Does not currently use equivalent rapid testing routinely in patients with CA-ARTI at 
the ER (rapid defined as time from sample collection to result interpretation by the 
physician within 4 hours).  
• 
At least 25% of CA-ARTI patients seen at ER are not hospitalised.  
• 
At least 50 paediatric patients with CA-ARTI are seen per month at ER during influenza 
season 
• 
Microbiology lab is capable of performing molecular testing.  
• 
Highly motivated and GCP-trained local Principal Investigator. Clinical research nurse. 
• 
Chosen timelines for intervention will be considered based on number of 
consultations and site characteristics.  The possibility to obtain real data about costs 
will be explored. 
• 
Geographical balance We will prioritise EU Member States and H2020 Associated 
Countries with high and medium antibiotic use and with a range of country level 
income and antibiotic stewardship programs. (if present, the antibiotic stewardship 
program will be documented, including the list of participant roles). Variations in 
microbiological etiologies and vaccination policies will be taken into consideration. 
9. DIAGNOSTIC INTERVENTION 
9.1. Description of the test and Intended use  
A rapid syndromic diagnostic test - the results of this test should not be used as the sole basis 
for diagnosis, treatment, or other patient management decisions. 
• 
BioFire FilmArray Respiratory Panel 2.1 plus (RP2.1plus): Nasopharyngeal swab 
 
The FilmArray Respiratory Panel 2.1 plus (RP2.1 plus) is a multiplexed nucleic acid test intended 
for use with FilmArray® 2.1 or FilmArray® Torch systems for the simultaneous qualitative 
detection and identification of multiple respiratory viral and bacterial nucleic acids in 
nasopharyngeal swabs (NPS) obtained from individuals suspected of respiratory tract 
infections. The test is FDA approved. 
Virus: 
Adenovirus, 
Coronavirus 
(229 
E, 
HKU1, 
NL63, 
OC43, 
SARS-CoV-2), 
human 
Metapneumovirus, Human Rhinovirus/Enterovirus, Influenza A, including subtypes H1, H1-
2009, and H3, Influenza B, Middle East Respiratory Syndrome Coronavirus (MERS-CoV), 
Parainfluenza Virus (1, 2, 3, 4), Respiratory Syncytial Virus. Bacteria: Bordetella parapertussis 
(IS1001), Bordetella pertussis (ptxP), Chlamydia pneumoniae, Mycoplasma pneumoniae  
 
9.2. Summary of findings from clinical studies 
The diagnostic test had a high sensitivity (overall 97%) and specificity (overall 99%) in US FDA 
product registration trials (available from https://www.biofiredx.com/support/documents/  
under Instructions for Use and Manuals)  and in independent research [23]. In a performance 

 
 
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study that compared the BioFire RP2 Panel to the BioFire RP Panel or PCR and sequencing, the 
overall percent agreement between the BioFire RP2 Panel and the comparator testing was 
99.2% [24].  
Biofire respiratory panels: 
Several studies have been conducted to examine the clinical impact of the BioFire RP Panel. 
Findings from such studies included an increase in diagnostic yield, decrease time to 
diagnosis, lower probabilities of hospital admission, reduced time in isolation, reduced 
number of supplementary tests such as chest radiographs, reduced hospital length of stay, 
decreased duration of antimicrobials use, and increase in appropriate antiviral use [25, 26].  
As a rapid syndromic diagnostic testing it expedites turnaround time for results, leading to 
higher rates of early discharge and early discontinuation of antibiotics [27].  In a large 
retrospective study in paediatric patients with acute viral respiratory tract infections its use 
was associated with less exposure to antibiotics and chest X-rays and more timely 
administration of antivirals [28].  
 
9.3. Summary of known and potential risks and benefits 
Based on the BioFire FilmArray, antibiotics may be withheld when deemed unnecessary, or a 
different antibiotic class may be selected when certain bacterial pathogens are detected. 
These management decisions are made by the patient9s treating physician. The risks and 
benefits of these management decisions are subject to the current investigation.  
A risk assessment and monitoring plan will be implemented but based on known intended use 
of the test (see below). Special effort will be invested on adequate training and monitoring of 
the sites (both laboratory and clinicians) for an optimal use of the test, and to support in the 
clinical and therapeutic decisions based on test results.  
Safety and warning precautions of the product are summarised in the Instructions for Use as 
well as the Summary of Results and Limitations, with focus on the following: 
• Asymptomatic carriage of viruses does occur, or the virus could be a co-pathogen 
together with a bacterial pathogen, or a recent viral infection could have predisposed 
to a secondary bacterial pneumonia. 
• Negative results in the setting of a respiratory illness may be due to infection with 
pathogens that are not detected by this test and pathogens below the limit of detection 
• There is a risk of false positive results due to non-specific amplification and/or cross-
reactivity with organisms found in the respiratory tract and they are summarised in the 
Instructions for use. Erroneous results due to cross-reactivity with organisms that were 
not evaluated or new variant sequences that emerge is also possible. 
 
Expected benefits of the study are, together with the main objective of reducing 
hospitalisation rates and antimicrobial use without adverse outcome, the opportunity for 
improved training, harmonised improvement of existing guidelines (local and European), 
opportunity to grow the different existing networks, opportunity to improve surveillance 

 
 
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because of underreporting or not available diagnostic tests. Assessing the diagnostic test in 
children is very important given the high rate of antimicrobial prescription when presenting 
with respiratory symptoms and the scientific research may directly benefit this target 
population. The potential benefit is much bigger than the risk of withholding a treatment when 
it should be given, which is a remote situation (<1%). 
 
10. METHODS  
10.1. Screening and enrolment 
Patients in the emergency department and/or short-stay unit will be screened for eligibility 
by local coordinators, clinicians, nurses in collaboration with members of the research team.  
Study staff will seek informed consent from all patients meeting the eligibility criteria at the 
time of screening. The health status of patients might rapidly deteriorate between screening 
and randomisation. Therefore, all eligibility criteria need to be re-evaluated and confirmed 
prior to the decision to randomise the patient. 
Screening failures are defined as patients, who were found eligible per screening but have 
either not given informed consent, or have deteriorated between screening and 
randomisation, and therefore no longer fulfil eligibility criteria. Screening failures are 
recorded anonymously on a screening log detailing the reason for screening failure. They are 
not randomised. Of note, no diagnostic procedures will be performed for the purpose of 
checking eligibility criteria specifically, i.e., any procedures indicated for the standard of care 
patient management will be performed but none will be added in order to check eligibility 
criteria.  
10.2. Randomisation and blinding 
Due to the nature of the intervention, blinding is not possible. After all eligibility criteria have 
been verified and informed consent has been obtained, randomisation will be performed using 
the built-in randomisation module of the eCRF system. Allocation will be concealed until the 
moment of randomisation. To this end, block randomisation will be used with variable blocks 
of size 2, 4 and 6. Randomisation will be stratified by centre. After the decision to randomise 
the subject is made, subjects will not be excluded from the trial. If the allocated intervention 
is not applied for any reason, this will be recorded and follow-up for the participant will be 
completed.  
10.3. Data collection 
A secured electronic case record form (eCRF) will be specifically designed for this study. 
• 
Main study (Follow up until 30 days) – Standard of care clinical and microbiological data 
will be collected but no study specific biological samples will be obtained with the 
exception of the diagnostic test in the respective allocation group. The clinical data set 
will summarise the illness episode and outcome, microbiological testing and 
antimicrobial use including the total hospitalisation, or in case of discharge, the time 
window defined on the primary endpoints. Follow-up information including data for 

 
 
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health economic analysis will be collected on day 14 (visit window: day 12 – 16) and on 
day 
30 
(visit 
window: 
day 
28 
– 
32) 
after 
randomisation. 
Parents/legal 
guardians/participants (where age-appropriate) will be asked to consent to being 
contacted by study staff for the follow-up visits to minimise loss to follow-up. In case 
of failure to successfully contact parents/legal guardians/participant (where age-
appropriate) 
at 
the 
end 
of 
trial 
participation, 
the 
participant9s 
general 
practitioner/family doctor will be contacted to complete information on the primary 
endpoints.   
• Microbiology study Clinical data will be collected as defined above. At participating 
study sites, additional biological samples as specified in section 10.7 will be collected, 
provided separate informed consent is given. Day 30 follow-up will include a face-to-
face visit for participants in the microbiology study. 
10.4. Clinical data set 
Inclusion. Check for inclusion and exclusion criteria. 
Randomisation. Informed Consent Form. Baseline registration and investigations.  
Signs and symptoms  
at ER and management plan. 
Participant background. 
Vaccination 
Co-morbidities  
and chronic medication 
Standard of care haematology and biochemistry 
EQ-5D-5Y questionnaire 
Management: Clinical decision after randomisation and initial results 
Investigations: respiratory, urine, faeces, blood, SARS-CoV-2, radiology (only when standard of 
care) 
Day 14:  symptoms, EQ-5D-5Y questionnaire 
Day 30: symptoms, EQ-5D-5Y questionnaire 
Outcomes and safety:  
Antibiotic treatment 
Antiviral and antifungal agents 
Hospital course 
Device deficiency 
(Severe) Adverse Event 
Deviation 
End of study 
 
Day 14 and 30 follow-up  
Days away from normal daily routines, healthcare utilization, antibiotic prescriptions, any 
hospital admission, ICU admission or death.  
 
 
 
 

 
 
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Table 1. Visit Schedule 
 
Procedures 
Screening 
Enrollment Hospitalisation 
Periode 
Dischargee 
Online questionnaire, 
phone call or face-to-
face visit 
Pre- study 
procedures 
Day 1 
Day 1 
> Day 1 
 
Day 14 
Day 30 
Informed 
consent 
X 
  
  
  
  
  
Eligibility 
criteria 
X 
  
  
  
  
  
Demographicsa  X 
  
  
  
  
  
Baseline 
assessment b 
X 
  
  
  
  
  
Vaccination 
status 
X 
  
  
  
  
  
Comorbidities 
and 
chronic 
medications 
X 
  
  
  
  
  
Hospital 
coursec 
  
X 
Xe 
Xe 
  
  
Microbiological 
testingd 
  
X 
  
  
  
X 
Concomitant 
therapy review 
X 
X 
Xe 
Xe 
X 
X 
Symptoms 
review 
and 
hospitalizations 
X 
X 
Xe 
Xe 
X 
X 
Quality of life 
questionnaire 
EQ5D 
  
X 
  
Xe 
X 
X 
 
 
a        Age in months or years date of birth, sex, weight, date of admission, partial postal code 
b        Symptoms, signs at clinical examination, duration of complaints, patient attending ER spontaneously or referred by general 
practitioner 

 
 
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c        ICU admission, hospital length of stay, ICU length of stay, non-invasive ventilation, vasopressor use, extracorporeal support 
d        Assessments only performed if patient is hospitalized 
e        Only applicable when informed consent is obtained for the additional study 
 
10.5. Baseline and follow-up data for health-economic analysis 
Follow up Assessments: Timepoints: Day 1, Day 14 and Day 30 
Individual patient data will be clustered in several main categories, and will reflect patient 
status prior to consultation (including quality of life assessment), diagnostics (including 
microbiological diagnostic tests and thoracic imaging), medicines (antibiotics and any other 
prescribed medication at the ER/admission including several parameters, antibiotic 
prescription in the previous 14 days) and patient follow-up (quality of life, adherence to 
treatment, duration of complaints –diary- and productivity costs including productivity loss 
and disability and designed differently for adults and children). 
Quality of life- (see eCRF completion guidelines on guidance) The EQ-5D (https://euroqol.org) 
contains 5-dimensions (<5D=) related to everyday living: mobility, self-care, usual activities, 
pain/discomfort, and anxiety/depression. Patients rate the problems they have with each of 
these dimensions on a 5-level scale (<5-L=) from 1 (no problems) to 5 (extreme problems). The 
second part of the EQ-5D-5L asks patients to grade their current global health status from 0 
(worst health you can imagine) to 100 (best health you can image). The questionnaire form, 
takes ± 5-10 min to administer. It needs to be completed by patient or person who has known 
the patient for > 5 years. (Order of preference: caregiver > spouse > children 18+ > sibling > 
other acquaintance). For patient questionnaires, patients will receive a link per email to 
complete the questionnaires online, which will directly be incorporated into the eCRF and be 
regarded as source data once digitally completed by the patients. If this is not possible or if 
patients are seen face to face, questionnaires will be completed per paper (source data) and 
entered in the eCRF by study staff at the site. In this case paper versions will be stored as 
source data. Quality of life data for the paediatric trial population will be captured using age-
appropriate tools, including proxy questionnaires for completion by parents or guardians for 
younger children. 
10.6. Microbiological testing 
Results from standard of care testing. Microbiological tests according to standard of care from 
site.  
Known previous colonization with multidrug resistant microorganism. 
Known previous SARS-CoV-2 infection if required hospitalisation and data are available 
• Type of samples collected: blood, ETA, BAL, pleural fluid, urine  
• Type of tests: cultures, quality markers for sputum (<10 epithelial cells and >25 
leukocytes per field magnification x 100), antigen detection, antibody detection, 
molecular testing 
• Test results: list of microorganisms. Selected susceptibility patterns 
Susceptibility testing will be grouped based on antimicrobial resistant phenotypes definitions: 
methicillin 
resistant 
Staphylococcus 
aureus 
–MRSA-, 
carbapenem 
resistant 
Enterobacteriaceae, Extended Spectrum Beta Lactamases Enterobacteriaceae –ESBL-, 
carbapenem-nonsusceptible Pseudomonas aeruginosa, multidrug resistant P. aeruginosa.   

 
 
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(Based on https://www.cdc.gov/nhsn/PDFs/pscManual/11pscAURcurrent.pdf) 
The data collected will allow also reporting pathogens of public health interest. 
Within VALUE-Dx WP3 there is current work on inter-operability with lab devices, with the 
objective to achieve direct connectivity with these systems to have antimicrobial resistance 
information from lab results easily available. 
 
Study samples. For diagnostic intervention: nasopharyngeal swab. Specific procedures for 
collection and processing will be provided. Data to be recorded: 
• Test result (including data on genomic copies provided by the test) 
• Time at which Test Result was Generated 
• Time at which Test Result was received by care team and how it is communicated 
• Time at which antibiotics were prescribed 
• Results from standard of care diagnostic tests (e.g., cultures) with comparable targets 
and assessment of discrepant results 
10.7. Microbiology study and biobanking 
In a subset of study sites and participants (up to 150 participants), additional oropharyngeal 
samples will be obtained from participants. One sample will be obtained on the day of 
randomisation and one sample on day 30 (visit window: day 28 – 32) after randomisation. 
Specific procedures for collection and processing will be provided. The aim of the microbiology 
study is to use suitable methods, including metagenomic sequencing, to characterise changes 
in microbiological colonisation and antimicrobial resistance patterns dependent on treatment 
with antibiotics. Inclusion in the microbiology study will require separate informed consent 
and participation in the main study will not depend on consent for the microbiology study. 
Biological samples obtained for the study (including leftovers from the specimens obtained 
for the intervention and for the microbiology study) can be stored at all sites and shipped to 
the University of Antwerp for inclusion in a biobank, subject to the condition that separate 
informed consent for biobanking is given. Participation in the study will not depend on consent 
for biobanking. 
11.  
Training 
11.1. Good Clinical practice training.  
The Principal Investigator and all site personnel involved in the study  need to provide a valid 
ICH-GCP certificate to timely identify and enrol eligible patients, collect informed consent 
forms, collect source data, enter data into clinical database. See the ADEQUATE monitoring 
plan for more details. Guidelines will preferably follow ISO20916:2019 because of the specific 
differences in device related events and deficiencies reporting. 
11.2. Medical Device training, being part of the Site Initiation Visit. 
Training of the hospital staff involved in the study will be implemented, with an emphasis on 
sample collection, sample processing, correctly identifying causative organisms and 
performance of susceptibility testing in coordination with bioMérieux.  Where required, 

 
 
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guidance will be provided to improve procedures and align them with standardised uniform 
manners following the principles of Good Clinical Laboratory Practice (GCLP). Quality Control 
of the device will be performed as described in the ADEQUATE monitoring plan. 
11.3. Data management training 
All sites will be trained to use the data management system (Research Online) as part of the 
site initiation visit. To assure high quality the Data Management Department of the Julius 
Centre (JC) of UMCU works according to a Quality Management System. All work is carried out 
in accordance with UMCU written Standard Operating Procedures (SOP) and Work instructions.  
12. SAFETY REPORTING  
12.1. Adverse events 
Definitions 
Adverse Event (AE) 
For this study, an adverse event is defined as any untoward medical occurrence, inappropriate 
patient management decision, unintended disease or injury, or untoward clinical signs in 
subjects, users, or other persons, with any connection to study related activities, whether or 
not related to the IVD medical device. 
 
Adverse device effect (ADE) 
An adverse event related to the use of the BioFire®.  
 
Serious Adverse Event (SAE) 
All SAEs, regardless of relationship to the study device or procedure will be documented in the 
source. It is the Investigator9s responsibility to determine the <seriousness= of an AE using the 
protocol defined terms, listed below. A SAE is an AE that results in one or more of the following 
for this study: 
• Resulted in death: An AE that resulted in the patient9s death. 
• Life-threatening illness or injury: The patient was at imminent risk of dying at the time 
of the adverse event. 
• Permanent impairment: An AE that resulted in permanent impairment of a body 
function, including chronic diseases or permanent damage to a body structure. 
• Required in-patient or prolonged hospitalisation. 
• Resulted in medical or surgical intervention to prevent life threatening illness or injury 
or permanent impairment to a body structure or body function. 
• Led to fetal distress, fetal death or congenital abnormality or birth defects, including 
physical or mental impairment. 
 
Notes: 

 
 
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1. Hospitalisation on Day-1 should not be reported as an AE or SAE if this is a direct 
consequence of the initial referral to the hospital.  
2. SAEs resulting in death should be reported using the primary cause of death as the 
event term. The only exception is <Sudden Death= when the cause is unknown. 
3. Planned hospitalisation for a pre-existing condition is not considered a SAE. 
 
Serious adverse device effect (SADE) 
Adverse device effect that has resulted in any of the consequences characteristic of a serious 
adverse event. 
Categories of adverse events 
ADVERSE 
EVENTS 
Non-device-related 
Device-related 
 
 
Applies to: 
- Inaccurate test result leads to indirect harm to 
the subject 
- Device causes direct harm to user or another 
person 
Non-serious 
Adverse event 
Adverse device effect 
Serious  
Serious 
adverse 
event  
Serious adverse device effect  
 
SEVERITY OF ADVERSE EVENTS 
It is the Investigator9s responsibility to assess the severity of an AE. A change in severity may 
constitute a new reportable AE. 
Also, the following guideline should be used to determine the severity of each adverse event: 
• MILD: Awareness of signs or symptoms, but does not interfere with the patient9s usual 
activity, or is a transient event that resolves without treatment and with no sequelae. 
• MODERATE: A sign or symptom, which interferes with the patient9s usual activity. 
• SEVERE: Incapacity with inability to do work or usual activities. 
 
RELATIONSHIP OF ADVERSE EVENTS 
It is the Investigator9s responsibility to assess the relationship between all AEs to the study 
device and procedure. The following guidelines should be used in determining the relationship 
of an AE to a device, procedure, or other causality: 
 
Not related 
 
Relationship to the procedures or device can be excluded when: 
• The event is not a known side effect of the product category 
the device belongs to or of similar device and procedures; 

 
 
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• The event has no temporal relationship with the use of the 
device or the procedures; 
• The event involves a body-site or an organ not expected to 
be affected by the device or the procedure or the disease 
under investigation; 
• The event can be attributed to another cause (e.g., an 
underlying or concurrent illness/clinical condition, an effect 
of another device, drug, treatment, or other risk factors); 
• Harms to the subject are not clearly due to use error; or 
• To establish the non-relatedness, not all the criteria listed 
above might be met at the same time, depending on the type 
of device/procedure and the event. 
Unlikely 
The relationship with the use of the device seems not relevant 
and/or the event can be reasonably explained by another cause, but 
additional information may be obtained. 
Possible* 
The relationship with the use of the device is weak but cannot be 
ruled out completely. Alternative causes are also possible (e.g., an 
underlying or concurrent illness/condition and/or an effect of 
another device, drug, or treatment). Cases where relatedness cannot 
be assessed, or no information has been obtained should also be 
classified as possible. 
Probable* 
 
 
 
 
The relationship with the use of the device seems relevant and/or 
the event cannot reasonably be explained by another cause, but 
additional information may be obtained.  
Causal 
Relationship* 
 
The event is associated with the device or with procedures beyond 
reasonable doubt when: 
• The event is a known side effect of the product category the 
device belongs to or of similar device and procedure 
• The event has a temporal relationship with the device 
uses/application or procedures 
• The event involves a body-site or organ that 
o The device or procedures are applied to 
o The device or procedures have an effect on 
• The event follows a known response pattern to the medical 
device (if the response pattern is previously known) 
• Other possible causes (e.g., an underlying or concurrent 
illness/clinical condition and/or an effect of another device, 
drug, or treatment) have been adequately ruled out 
• Harm to the subject is due to error in use 
• To establish the relatedness, not all the criteria listed above 
might be met at the same time, depending on the type of 
device/procedure and the event. 
*Denotes <related= to the study procedure or device and should be reported (AE) as part of the 
study. 

 
 
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12.2. Reporting Adverse Events 
The principal investigator shall: 
a) record all AEs and device deficiencies, regardless of relationship to the study device or study 
procedure in the source, together with an assessment as to whether the device or study 
procedure were a cause of the event,  
b) report to the Sponsor, without unjustified delay (max. 24 hours after becoming aware of the 
event), all SAEs and SADEs, regardless of relationship to the study device or procedure, from 
the time of signing the informed consent through study completion (day-30 follow-up visit, 
including window, for the main study and the microbiology study) and device deficiencies that 
could have led to a SADE; this information shall be promptly followed by detailed written 
reports, as specified in the Safety Management Plan, 
c) report to the Sponsor, without unjustified delay (max. 7 calendar days after becoming aware 
of the event), all AEs, that deemed to have a possible, probable, or causal relationship to the 
study procedure, or device, from the time of signing the informed consent through study 
completion (day-30 follow-up visit, including window, for the main study and the microbiology 
study); this information shall be promptly followed by detailed written reports, as specified in 
the Safety Management Plan, 
d) ensure reporting of SAEs or SADEs to the ethics committee, or if applicable a competent 
authority, per the timelines of the national regulations, from the time of signing the informed 
consent through study completion (day-30 follow-up visit, including window, for the main 
study and the microbiology study) and device deficiencies that could have led to a SADE, when 
required by the national regulations or Safety Management Plan or by the ethics committee,  
e) ensure annually reporting to the ethics committee, or per the timelines of the national 
regulations, AEs that deemed to have a possible, probable, or causal relationship to the study 
procedure or device, from the time of signing the informed consent through study completion 
(day-30 follow-up visit, including window, for the main study and the microbiology study), 
when required by the national regulations or Safety Management Plan and 
f) ensure to report to regulatory authorities, SADEs and device deficiencies that could have led 
to serious adverse device effect, as required by national regulations, and supply the Sponsor, 
upon Sponsor9s request, with any additional information related to the safety reporting of a 
particular event. Sponsor remains responsible for adequate reporting to regulatory 
authorities. 
The study site will report applicable safety events to the Sponsor by entering the event into 
the AE form in the eCRF, which will trigger an automated email to the Sponsor and 
manufacturer. Refer to the ADEQUATE Safety Management Plan for more extensive guidance 
on reporting.  
 

 
 
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12.3. Device Deficiency 
Device Deficiency Definition 
A device deficiency is defined as inadequacy of the BioFire® and/or kits with respect to its 
identity, quality, durability, reliability, usability, safety or performance. 
Device deficiencies include, but are not limited to, malfunctions, use errors, and inadequacy 
in the information supplied by the manufacturer including labelling. 
A device deficiency may or may not be associated with an AE/SAE. 
 
Reporting Device Deficiency 
All device deficiencies related to devices in the procedure shall be documented throughout 
the study. The study site must report device deficiencies related to the BioFire and/or kits, 
within 24 hours after becoming aware of the event through the Device Deficiency form in the 
eCRF.  
Manufacturer representatives will organise collection of the device for evaluation, as needed. 
 
12.4. DEVIATIONS FROM THE CLINICAL STUDY PROTOCOL 
A protocol deviation is any non-compliance with the study protocol, Good Clinical Practice. A 
deviation  can be identified from a number of sources. Potential sources include, but are not 
limited to, a member of the Investigator9s staff, a Sponsor representative during monitoring 
visits, or a member of the data management or statistical groups when entering or analyzing 
data. Regardless of the source, it is crucial to document the deviation in the protocol deviation 
eCRF. The Investigator will report protocol deviations to the IRB/EC as required by the IRB/EC 
procedures.  
Any deviation from the protocol or procedures should be recorded in the source documents. 
Standard of care assessments not completed at a site should not be considered protocol 
deviations. 
Steps to be taken to assure the accuracy and reliability of data include the selection of 
qualified investigators and appropriate sites, the review of protocol procedures with the 
Investigator and associated personnel prior to the study, and periodic monitoring visits by the 
Sponsor. The Sponsor will review eCRFs for accuracy and completeness during on-site and/or 
remote monitoring visits; any discrepancies will be resolved with the Investigator or designees, 
as appropriate. 
 
12.5.  Data Safety monitoring board (DSMB) 
A Data Safety Monitoring Board has been established with the aim to safeguard the interests 
of trial participants, assess the safety of the interventions during the trial, and monitor the 
overall conduct of the trial.  A specific charter document describes the roles and 

 
 
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responsibilities of the independent DSMB for the ADEQUATE trial, including the timing of 
meetings, methods of providing information to and from the DSMB, frequency and format of 
meetings, statistical issues and relationships with other committees. 
13. STATISTICAL CONSIDERATIONS 
13.1. Sample size calculation – joint paediatric and adult trial (protocol version 
2.0, 23-Nov-2020 (paediatric)/protocol version 3.0, 18-Feb-2022 (adult)) 
Co-primary endpoints used in the study:  
1. 
Days on antibiotic treatment within 14 days (superiority endpoint) 
2. 
Days alive out of the hospital within 14 days (superiority endpoint) 
3. 
Clinical failure within 30 days (non-inferiority safety endpoint) 
Required sample sizes are presented for endpoint 2 and 3. Endpoint 1 is non-limiting due to 
having an expected smaller standard deviation compared to endpoint 2 and having the same 
clinical relevant effect size. 
The intervention is considered successful if superiority is demonstrated for either or both 
endpoints 1 and 2 AND non-inferiority is demonstrated for endpoint 3. We use a hierarchical 
nested design: superiority primary endpoints are tested first. Only if superiority to at least one 
of the two superiority endpoints is confirmed, the non-inferiority safety endpoint is taken into 
consideration. To maintain an overall alpha < 0.05, the two superiority endpoints will be tested 
using a two-sided alpha of 0.025, while the non-inferiority endpoint will be tested using a one-
sided alpha of 0.05. The minimal power for the superiority endpoints is set to 0.9 and for the 
non-inferiority endpoint to 0.95. This is to maintain overall power of the trial, given the 
hierarchical testing. For 8Clinical failure9 we considered different non-inferiority margins. For 
8Days on antibiotic treatment9 and 8Days alive out of the hospital9 we defined 1 day as a 
clinically relevant effect size. 
The distribution of endpoint 2 is yet unknown for the ARI population. We supported our sample 
size calculation using data of patients presenting with CAP to Dutch ERs and hospitalised to a 
non-ICU ward (ClinicalTrials.gov: NCT02604628). The standard deviation (SD) of 8Days alive out 
of the hospital within 14 days9 in this population was 4 days. The theoretical maximum SD with 
a 14-day follow-up period is 7 (which would be the case if 50% had zero days and 50% had 14 
days alive out of the hospital). However, we expect that in the target population the variability 
for this endpoint will be less than in the CAP population and in children less than in adults. We 
varied the SD to assess the impact on the sample size. We used a standard correction factor 
of 1.15 for non-normality of the data. (Table 2) Hence for the superiority endpoints a sample 
size of 257 per arm for children would be adequate. 
Table 2: Sample sizes for Days alive out of the hospital using different assumptions 
SD 
Relevant effect size 
Alpha 
Beta 
Correction 
Sample size per 
arm 

 
 
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29 
 
3 
1 
0.025 
0.1 
1.15 
257 
4 
1 
0.025 
0.1 
1.15 
457 
5 
1 
0.025 
0.1 
1.15 
714 
6 
1 
0.025 
0.1 
1.15 
1,028 
7 
1 
0.025 
0.1 
1.15 
1,399 
For clinical failure, if we assume an incidence of 5% it would be 412, using a non-inferiority 
margin of 5% (Table 3). A high power for clinical failure is considered important because both 
the superiority and the non-inferiority hypothesis need to be confirmed in order to declare 
the intervention a success so a total of 412 participants per study arm would be required to 
reach 95% power 
 
 

 
 
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Table 3: Sample sizes for clinical failure using different assumptions. 
Incidence * 
Non-inferiority 
margin 
Alpha** 
Beta 
Sample size per arm 
0.05 
0.05 
0.05 
0.05 
412 
* same for intervention and control arm; ** one-sided alpha 
To account for potential drop-outs we originally set an aim of a total of 900 randomised 
children in the study.  
 
13.2. Review of sample size calculation for the current version of the protocol  
The sample size calculation was redone after the decision to terminate the recruitment of 
adult patients (Value Dx WP4b ADEQUATE Protocol, adult version 3.0 18-Feb-2022) on 3rd May 
2022. We first considered the three co-primary endpoints listed in 13.1. 
 
Co-primary endpoint EP3: Clinical failure within 30 days (non-inferiority safety endpoint) 
The non-inferiority safety endpoint, defined as any inpatient admission or death within 30 
days after study enrolment for initially non-admitted patients (expected to be >90% of 
paediatric participants) and any readmission, secondary ICU admission or death within 30 
days after study enrolment for initially admitted patients, is unlikely to be relevant or 
appropriate for the paediatric population considered alone. 
 
Rationale: Mortality in the study population is extremely low, and secondary admission rates 
among children initially managed in the community as well as readmission and secondary 
ICU admission rates among primarily admitted children are in fact likely to be in the range of 
2-2.5%. Secondary admissions are highly unlikely to be related to the initial illness episode 
due to the relative conservative approach in current paediatric emergency medicine practice 
erring on the side of primary admission if there is any doubt about appropriate safety 
netting being possible for the family. Based on this, EP3 will be considered a key secondary 
endpoint for the statistical analysis plan and will no longer be used to inform the target 
sample size.  
 
Co-primary endpoints EP1 and EP2: Days on antibiotic treatment and Days alive out of 
hospital, both within 14 days (superiority endpoints) 
The original assumptions for EP1 and EP2 remain unchanged. In particular, a reduction of one 
day in antibiotic treatment or increase of one day in days alive out of hospital appear to be 
relevant for a clinically relevant reduction in antibiotic prescribing and a reduction in hospital 
costs, respectively. In children, the co-primary superiority endpoints are likely to be 
dominated by EP1, as ambulatory exposure to antibiotics is likely to be common in the absence 
of hospital admission, whereas many admitted children would be expected to be treated with 
antibiotics as well. The superiority comparison on EP1 will be considered the primary outcome 
comparison for the trial.  

 
 
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The sample size estimation was revisited for co-primary endpoint EP1. From a recent 
publication on variations in antibiotic prescribing in febrile children presenting to European 
EDs, the standard deviation for days on antibiotic treatment was estimated as 3.7 days. Based 
on this, recruitment of 170 children per arm (total of 340 children) will be sufficient to detect 
a difference of one day in EP1 (power 80%, alpha 0.05; table 4).  
Table 4: Sample sizes for Days on antibiotic treatment (paediatric) using different assumptions. 
SD 
Delta 
Alpha 
Beta 
Correction 
Sample size per arm 
2.5 
1 
0.025 
0.2 
1 
99 
3.0 
1 
0.025 
0.2 
1 
142 
3.5 
1 
0.025 
0.2 
1 
193 
3.7 
1 
0.025 
0.2 
1 
215 
4.0 
1 
0.025 
0.2 
1 
252 
4.5 
1 
0.025 
0.2 
1 
318 
5.0 
1 
0.025 
0.2 
1 
393 
 
To account for uncertainty about the variability in EP1 (and EP2) in the paediatric study 
population, we propose to adopt a highly conservative approach aiming to recruit 252 children 
per arm (total of 504 children), resulting in adequate power to detect a difference in one day 
in EP1 or EP2. To adjust for loss to follow-up of up to 10%, we will aim for a total of 554 
randomised children.  
 
13.3. Analysis populations 
A statistical analysis plan will be prepared after protocol approval and once site selection 
process has been initiated. 
The main analyses will be performed for the total population 
 
Analyses will be stratified by: 
• Country/geographical location 
• age groups (i.e., <5y/5-17) 
• clinical syndrome: clinical pneumonia/LRTI, influenza-like illness, 
laryngitis/laryngotracheitis (croup), dominant obstructive airway disease/bronchiolitis  
• risk factors: patients with and without known risk factors for severe disease; the latter 
include chronic pulmonary, cardiovascular or metabolic disease or 
immunocompromised patients. 
• severity at presentation:  patients with and without primary hospital admission. 

 
 
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• Vaccination status: Completed primary vaccinations according to local schedule y/n, 
pneumococcal vaccine receipt according to local schedule y/n, receipt of influenza 
vaccine for current season y/n, SARS-CoV-2 vaccine receipt y/n.  
 
The primary analysis will follow the intention-to-treat principle in which groups are compared 
based on the allocated regimen. In the per protocol analysis, we will exclude participants not 
receiving the test according to the randomised regimen (e.g., randomised to rapid syndromic 
testing but not receiving the test). 
13.4. Primary study parameter(s) 
Descriptive statistics will be produced and tabular summaries will be presented, stratified 
according to the allocated group (rapid syndromic testing vs. control). Categorical data will be 
summarised by the number and percentage of subjects in each category. Appropriate summary 
statistics will be used for continuous variables depending on the distributional assumptions. 
These include measures of central tendency (mean or median) and dispersion (standard 
deviation or inter-quartile range).  
8Days on antibiotic treatment9 and 8Days alive out of the hospital9 will be analysed using a linear 
regression analysis, including as covariates stratification by age groups and predefined risk 
factors. Upfront we may expect that the assumption of normally distributed residuals is 
violated, in which case we will determine the confidence interval by bootstrapping. 
13.5. Secondary study parameter(s) 
Clinical failure will be analysed using Cox proportional hazards regression adjusted for age 
groups and predefined risk factors. Risk differences and 90% CI will be inferred by comparing 
the cumulative incidence of failure at day 30 from the Cox model and bootstrapping for the 
confidence interval. A 90% CI will be used to be compatible with a one-sided alpha of 0.05. 
Economically relevant parameters will be gathered within the trial context from individual 
participants but also the following data from the participant sites/countries will be extracted 
when possible: 
• Patient numbers relative to population and hospital catchment area  
• Estimations on the number of emergency department visits associated with acute 
respiratory infections based on a list of ICD codes. 
• For the long-term economic model, the probability of susceptibility to received 
antibiotics and the resulting illness duration, additional outpatient visits and a second 
antibiotic course will be estimated based on surveillance data on every site.  When 
needed, assumptions regarding the incidence and hospitalisation rates will be derived 
from literature and expert opinion  
 
Types of costs: Direct medical costs, direct and indirect non-medical costs.  
Hospital costs will be determined using bottom up calculation based on representative sites 
that have the information available on electronic databases that allow unit cost prices 
assignment to the variables recorded. These descriptive data will include health care 
utilization for the entire hospitalisation, for ICU stay (unit cost price per hospitalisation day, 

 
 
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33 
 
unit cost price for ICU hospitalisation day). Unit cost price for recorded diagnostic tests and 
thoracic imaging (referred to the procedure of testing as coded/reimbursed by official 
nomenclature).  
When not available, resource use will be measured as volume of hospital admission days, tests 
performed, etc.    
Analysis of the antibiotic domain based on antibiotic type switches and de-escalation. For the 
health economic analysis, a decision-tree deterministic approach with specification of the 
base case and alternative scenarios, together with threshold analyses to determine efficient 
ranges for the values of some parameters. Analysis of the antibiotic domain based on 
antibiotic type switches and de-escalation. 
13.6. Mid-term data analysis 
A data snapshot will be taken after the first season, in terms of number of recruited cases per 
category, impact of seasonal outbreaks or emerging pathogens. Sample size assumptions may 
also need to be validated.  
 
14. ETHICAL CONSIDERATIONS 
14.1. Regulation statement 
The study will be performed in accordance with all applicable laws and regulations including 
the International Conference on Harmonisation (ICH) Guideline for Good Clinical Practice (GCP) 
the ethical principles that have their origins in the Declaration of Helsinki (current official 
version: Fortaleza, 2013; https://www.wma.net/policies-post/wma-declaration-of-helsinki-
ethical-principles-for-medical-researchinvolving-human-subjects/), the updated version of 
the General Data Protection Regulation (EU) 2016/679 (GDPR), ISO 20916 and other applicable 
privacy laws. 
14.2. Recruitment and consent 
The investigator or authorised delegate must obtain written informed consent before any 
clinical study related procedure/activity takes place. Parents or guardians and participants 
will be approached for the study by the local principal investigator or appropriately trained 
delegate, in co-ordination with the attending clinical team.  Written versions of the Participant 
Information Sheet and Informed Consent will detail the exact nature of the study; the 
implications and constraints of the protocol; and any risks involved in taking part.  It will be 
stated clearly in the information letter that participation in this study is completely voluntary 
and that withdrawal is possible at any time and without any consequences. Consent will be 
sought from one or both parents in accordance with local regulations. Where the participant 
has the capacity to contribute to the decision about participation, they will be involved in the 
informed consent process in an age- and capacity-appropriate way. This will include obtaining 
assent to participation where appropriate. The standard consent form will request consent 
from subjects for sample storage of specific samples to collaborating institutions for 
investigations that cannot be performed locally. Staff will explain the details of the study to 

 
 
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the participant or parent/guardian/consultee and allow them time to discuss and ask 
questions. The consenting party will be asked to sign and date an informed consent form.  
Obtaining a subject's consent to participate in medical research may be complicated by COVID-
19 measures. In the case of the patient being in isolation and unable to sign, he/she will give 
his/her oral consent, the researcher will sign directly, and the patient will sign as soon as 
possible after completion of the isolation period. Deviations in consent procedures might 
occur per participating country, following national law and locally accepted procedures with 
regards to challenging COVID-measures. 
Participant Information Sheets will be available in the common local language. Written 
Informed Consent will be confirmed by the dated signatures of the participant and by the 
person who presented and obtained the informed consent. A copy of the signed Informed 
Consent will be given to the participants. The original signed form will be retained at the study 
site. Following the code of conduct for pediatric patients, in all medical research involving 
child subjects, the burden associated with participation should be minimised; where non-
therapeutic research is concerned, the law stipulates that it must be negligible.  
14.3. Withdrawal of individual subjects 
Patients are free to withdraw consent at any time without providing a reason. Patients who 
wish to withdraw consent for the study will have anonymised data collected up to the point of 
that withdrawal of consent included in the analyses. The patient will not contribute further 
data to the study. Data up to the time of withdrawal will be included in the analyses unless the 
patient appeals to the 8right to be forgotten9 according to the national GDPR regulations. The 
investigator can decide to withdraw a subject from the study for urgent medical reasons. 
Enrolled patients meeting one or more of the exclusion criteria prior to randomisation, will be 
withdrawn by the investigator. Patients withdrawn prior to randomisation will be replaced (i.e., 
they will not count towards the sample size). Patients withdrawn after randomisation will not 
be replaced. 
The participants cannot be enrolled at the same time into any interventional clinical study 
unless it is a COVID-19 study that does not interfere with this study. Subjects may be co-
enrolled in another observational study if the local study coordinators have been informed 
and have given their approval, to ensure the other study would not interfere with the results 
of this study or compromise patient welfare. 
15. ADMINISTRATIVE ASPECTS, 
MONITORING AND PUBLICATION 
15.1. Steering Committee 
The missions of the steering committee are to define the objectives of the research, to propose 
protocol modifications during the research, to organise the research, to determine the 
methodology, to coordinate information and to monitor the conduct of research. The steering 

 
 
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committee will decide during ongoing research what to do in unexpected situation. The 
steering committee will meet regularly until the end of inclusions. 
15.2. Handling and storage of data and documents 
Data will be recorded in a secured electronic case record form (eCRF) specifically designed for 
this study and validated for authenticity, accuracy, reliability and consistent intended 
performance. An eCRF should be completed for each participant.  
All information obtained during the study (except the ICF data) will be entered digitally in 
conformity with the applicable laws and regulations. All data (except the ICF data) will be 
coded using a unique study number. Data will be handled in accordance with local privacy 
regulations and the European General Data Protection Regulation (GDPR, 2016/679, effective 
as of May 25th, 2018). Penta as Sponsor of the study will have access to all data collected from 
the start of the study  
At the end of the study all data will be transferred to Penta Foundation and will be stored for 
25 years. 
15.3. Storage of samples. Biobanking 
Biological samples will be obtained from a subset of participants as detailed in section 10.7. 
Detailed instructions for sample storage will be provided separately. Samples will be stored at 
local facilities in a pseudonymised way until shipment to the central trial laboratory.  
15.4. Monitoring and Quality Assurance 
Remote/centralised and/or on-site study monitoring will be carried out by the Sponsor. A 
monitoring plan will be scheduled and will define the monitoring frequency and procedures. 
Monitoring will start before recruitment begins, throughout the trial (data monitoring 
including but not limited to recruitment rates, consent procedures, access/storage of patient 
identifiers, sample handling for performing the diagnostic intervention test, data entry, data 
queries, unusual data patterns), between recruiting season and at the end of the trial (source 
document verification, collect trial supplies, close-out meetings/visits). We will use a risk 
adapted approach within the protocol design to enable safety reporting requirements to 
reflect the amount of safety data available and the level of risk for this non-interventional 
study. We refer to the monitoring plan for details. 
This study does not involve any experimental treatment, but the diagnostic intervention may 
interfere with the standard of care and it involves data collection. The need for additional 
insurance for study patients may differ per country local regulations.  
15.5. Public disclosure and publication policy 
All information disclosed or provided by the Sponsor (or any company/institution acting on 
their behalf) or produced during the study, including, but not limited to, the protocol, the eCRF, 
and the results obtained during the course of the study, is confidential prior to the publication 
of results and in accordance to consortium agreement and open access regulations. 
The detailed procedures for the review of publications are set out in the clinical trial 
agreement entered into with the Sponsor in connection with this study. These procedures are 
in place to ensure coordination of study data publication and adequate review of data for 

 
 
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publication against the validated study database for accuracy. UMCU will adhere to all 
applicable local laws governing transparency in clinical trials including the trial posting on 
clinicaltrials.gov and all other applicable registrations. 
 
 

 
 
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Esposito, S. and N. Principi, Defining the aetiology of paediatric community-acquired pneumonia: 
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Schuetz, P., et al., Procalcitonin to initiate or discontinue antibiotics in acute respiratory tract 
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Ruuskanen, O., et al., Viral pneumonia. Lancet, 2011. 377(9773): p. 1264-75. 
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Oosterheert, J.J., et al., Impact of rapid detection of viral and atypical bacterial pathogens by real-
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Rogers, B.B., et al., Impact of a rapid respiratory panel test on patient outcomes. Arch Pathol Lab 
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Vos, L.M., et al., More targeted use of oseltamivir and in-hospital isolation facilities after 
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respiratory viruses in immunocompromised adult patients. J Clin Virol, 2019. 116: p. 11-17. 
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Poritz, M.A., et al., FilmArray, an automated nested multiplex PCR system for multi-pathogen 
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Chan, M., et al., Comparison of the Biofire FilmArray Respiratory Panel, Seegene AnyplexII RV16, 
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Zhu, N., et al., A Novel Coronavirus from Patients with Pneumonia in China, 2019. N Engl J Med, 
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]; Available from: https://www.cdc.gov/nhsn. 
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Timsit, J.F., et al., Appropriate endpoints for evaluation of new antibiotic therapies for severe 
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Leber, A.L., et al., Multicenter Evaluation of BioFire FilmArray Respiratory Panel 2 for Detection of 
Viruses and Bacteria in Nasopharyngeal Swab Samples. J Clin Microbiol, 2018. 56(6). 
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Shu, Y.L., et al., Dual seasonal patterns for influenza, China. Emerg Infect Dis, 2010. 16(4): p. 725-6. 
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Brendish, N.J., et al., Routine molecular point-of-care testing for respiratory viruses in adults 
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controlled trial. Lancet Respir Med, 2017. 5(5): p. 401-411. 
26. 
Shengchen, D., et al., Evaluation of a molecular point-of-care testing for viral and atypical 
pathogens on intravenous antibiotic duration in hospitalized adults with lower respiratory tract 
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27. 
Brendish, N.J., et al., Impact of turnaround time on outcome with point-of-care testing for 
respiratory viruses: a post hoc analysis from a randomised controlled trial. Eur Respir J, 2018. 52(2). 
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Lee, B.R., et al., Impact of multiplex molecular assay turn-around-time on antibiotic utilization and 
clinical management of hospitalized children with acute respiratory tract infections. J Clin Virol, 
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29. 
Webber, D.M., et al., Evaluation of the BioFire(R) FilmArray(R) Pneumonia Panel for Detection of 
Viral and Bacterial Pathogens in Lower Respiratory Tract Specimens in the Setting of a Tertiary Care 
Academic Medical Center. J Clin Microbiol, 2020. 
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Edin, A., H. Eilers, and A. Allard, Evaluation of the Biofire Filmarray Pneumonia panel plus for lower 
respiratory tract infections. Infect Dis (Lond), 2020: p. 1-10. 
31. 
Hughes, A.E.O., et al., Comparable Detection of Viral Pathogens in Lower Respiratory Tract 
Specimens With the BioFire Respiratory Panel 2 and BioFire Pneumonia Panel. J Clin Microbiol, 
2020. 
32. 
Yoo, I.Y., et al., Evaluation of the BioFire(R) FilmArray(R) Pneumonia Panel for rapid detection of 
respiratory bacterial pathogens and antibiotic resistance genes in sputum and endotracheal aspirate 
specimens. Int J Infect Dis, 2020. 
33. 
Lee, S.H., et al., Performance of a multiplex PCR pneumonia panel for the identification of 
respiratory pathogens and the main determinants of resistance from the lower respiratory tract 
specimens of adult patients in intensive care units. J Microbiol Immunol Infect, 2019. 52(6): p. 920-
928. 
34. 
Yugueros-Marcos, J., et al., New molecular semi-quantification tool provides reliable 
microbiological evidence for pulmonary infection. Intensive Care Med, 2018. 44(12): p. 2302-2304. 
35. 
Hansell, D.M., et al., Fleischner Society: glossary of terms for thoracic imaging. Radiology, 2008. 
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17. Appendix 1: Summary of Changes 
 
Version Number  
Version Date 
Current Approved Protocol  
V2.0  
13-Nov 2020 
Amended Protocol 
V3.0 
11-Nov-2022 
Amended Protocol 
V4.0 
26-Jan-2023 
Reviewed Amended Protocol 
V5.0 
18-Oct-2023 
 
Section number, title 
and page number  
Changes  
Rationale for change  
Cover page 
Addition of <Julia Anna Bielicki= and <Malte Kohns 
Vasconcelos= to protocol contributors 
Developed new 
protocol 
Study overview 
Moved study acronym to end of study title 
Clarity 
Study overview 
Amended protocol version/date 
For new protocol 
 
 
 
Study overview 
Change of Coordinating Investigator from  
<Cristina Prat Aymerich= 
 
To 
 
<Malte Kohns= 
Change in sponsor 
Study overview 
Change of Chief Investigator from  
<Marc Bonten, UMC Utrecht= 
 
To 
 
<Julia Bielicki, St George9s, University of London= 
Change in sponsor 
Study overview 
Change of sponsor from  
<University Medical Center Utrecht (UMC Utrecht) 
Heidelberglaan 100  
3584 CX Utrecht The Netherlands= 
 
To  
 
<Fondazione PENTA ONLUS 
Torre di Ricerca Pediatrica 
Corso Stati Uniti, 4 
35127, Padova 
Italy= 
Change in sponsor 
Study overview 
Members of Steering Committee 
Change of format 
Study overview 
Added <Andrew Atkinson, UKBB= 
Additional statistician 
worked on amendment 
Study overview 
Added members of DSMB 
Update to members 
Protocol signature sheet:  Change of sponsor signatory from 
<Prof. Dr. K.G.M. Moons= 
 
To 
Change in sponsor 

 
 
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40 
 
 
<Laura Mangiarini=  
Protocol signature sheet:  Change of text from  
<Principal Coordinating Investigator Prof. Dr. M.J.M Bonten 
Microbiologist, UMC Utrecht= 
 
To 
 
<Principal Investigator Julia Bielicki, Senior Lecturer St 
George9s, University of London= 
Change in chief 
investigator 
Protocol signature sheet:  Removal of Penta Coordinating Investigator 
Change in sponsor – no 
longer relevant 
Protocol signature sheet:  Addition of individual pages for signatures 
Signature page for each 
site provided 
Protocol signature sheet:  Added additional sites and investigators 
Updated sites list 
Table of Contents:  
Amended <Value-Dx= to <VALUE-Dx=  
For consistency across 
project  
Table of Contents:  
Amended page numbers 
Due to pages shifting  
Table of Contents: 
Change of text from:  
<Study site selection criteria=  
 
To 
 
<Recommended study site selection criteria= 
More appropriate title 
of the paragraph that 
follows  
Table of Contents: 
Change of text from:  
<Laboratory training=  
 
To 
 
<Medical device training, being part of the Site Initiation Visit= 
More appropriate title 
of the paragraph that 
follows  
2. Summary  
Addition of <acute respiratory infections= 
Clarity 
2. Summary  
Amended <antimicrobial= to <antibiotic= 
Grammar correction 
2. Summary  
Removal of <and (3)  the non-inferiority in terms of 
clinical outcome= 
No longer a primary 
endpoint 
2. Summary  
Change of text from:  
<Individually randomised controlled trial. Hierarchical nested 
analysis design.= 
 
To 
 
<Prospective, multi-centre, individually randomised, controlled 
trial.= 
Clearer and more 
specific definition. 
2. Summary  
Following text removed: 
<Adverse outcome (non-inferiority safety endpoint)  
For initially non-admitted patients: any admission or death 
within 30 days  
For initially hospitalised patients: i) any readmission, ii) ICU 
admission >= 24 hours after hospitalisation, or iii) death, all 
within 30 days= 
This endpoint is no 
longer a primary 
endpoint – now a 
secondary endpoint 

 
 
VALUE Dx WP4B ADEQUATE Protocol, version 5.0 18-Oct-2023 
 
 
 
41 
 
2. Summary  
Following text removed: 
<and who do not produce sputum= 
Not applicable to all of 
the paediatric 
population.   
3. General 
considerations:  
Amended <Value-Dx= to <VALUE-Dx=  
For consistency across 
project  
3. General 
considerations:  
Removal of <and (3)  the non-inferiority in terms of 
clinical outcome= 
No longer a primary 
endpoint 
4.1 Background:  
Amended <effects= to <affects=  
Spelling correction 
4.1 Background:  
Amended <prescribing= to <prescription= 
Grammar correction 
4.1 Background:  
Amended <ecologic= to <ecological= 
Grammar correction 
4.1 Background:  
Addition of <The Biofire= 
Full commercial name 
5.1 Co-primary objective  
Removal of <occurrence of adverse outcome within 30 days 
after study enrolment= 
Not applicable to 
paediatric study 
5.2 Secondary objectives:  Insertion of <to assess the impact of rapid syndromic testing 
on occurrence of adverse outcome within 30 days after study 
enrolment.= 
This was a primary 
endpoint but is now a 
secondary endpoint 
6. Study parameters/ 
endpoints.  
Moved <Adverse outcome (non-inferiority safety endpoint)  
• Safety endpoint: 
• For initially hospitalised patients: i) any readmission, ii) ICU 
admission => 24 hours after hospitalisation, or iii) death, 
within 30 days after study enrolment 
• For initially non-admitted patients: any admission or death 
within 30 days after study enrolment.= from 6.1 Main study 
parameter/endpoint to 6.2 Secondary endpoints  
In paediatric population 
it is unlikely this 
endpoint will be 
clinically relevant 
6.1 Co-primary study 
endpoints:  
Following text added after each of the two co-primary 
study endpoints: 
<after study enrolment= 
Additional information 
added regarding the 
study endpoints.  
6.2 Secondary endpoints:  Following text added: 
<Change in= (second bullet point) 
  
<during the main study= (fourth bullet point) 
Additional information 
regarding the 
secondary endpoints. 
 
7.1 Study design and 
justification:  
Change of text from:  
<The study design will be an individually randomised 
controlled open-label trial= 
 
To 
 
<This is a prospective, multi-centre, individually randomised 
controlled open-label trial. Approximately 520 subjects will be 
randomised in the trial in up to 10 investigational sites in the 
European region. Subjects will be followed at day 14 and day 
30 after randomisation. 
 
Enrolment will be competitive across sites.=  
Clearer and more 
specific definition. Test 
includes change to 
sample size and 
number of 
investigational sites 
from previous protocol 
due to removal of adult 
sites. Clarification of 
the enrolment type. 
7.1 Study design and 
justification:  
Change of text from: 
<lab both in adult and paediatric sites= 
 
To 
 
<laboratories= 
Removed reference to 
adult sites 

 
 
VALUE Dx WP4B ADEQUATE Protocol, version 5.0 18-Oct-2023 
 
 
 
42 
 
7.1 Study design and 
justification:  
Change of text from: 
<It needs to be ensured that the non-inferiority endpoint is not 
positively impacted by direct effects of the intervention such 
as decisions to initiate or withhold antibiotic treatment or 
early discharge from the ER or hospital. E.g., if we would 
include hospitalisation days in the non-inferiority endpoint, we 
run the risk of 8compensating9 the putative adverse effects.= 
 
To 
 
<In the paediatric population the non-inferiority co-primary 
endpoint is unlikely to be clinically relevant= 
Change in primary/ 
secondary endpoints 
7.1 Study design and 
justification:  
Removal of text: 
<Hierarchical nested design: Superiority primary endpoints are 
tested first. Only if superiority to at least one of the two 
superiority endpoints is confirmed, is the non-inferiority safety 
endpoint taken into consideration= 
Non-inferiority safety 
endpoint no longer a 
primary endpoint 
7.1 Study design and 
justification:  
Removal of text: 
<or cause-specific death= 
Removal of death as 
primary objective 
7.1 Study design and 
justification:  
Addition of text: 
<reflected in the key safety secondary endpoint= 
Composite endpoint 
summarises the most 
relevant adverse events 
including death  
7.1 Study design and 
justification:  
Addition of text: 
<Modifications were made following the decision to terminate 
the recruitment of adult patients (Value Dx WP4b ADEQUATE 
Protocol, adult version 3.0 18-Feb-2022) 3rd May 2022. 
Details are provided in sections 5, 6 and 13 of the current 
protocol=. 
Closure of adult arm of 
trial 
7.2 Study duration:  
Change of text from  
<2 influenza seasons= 
 
To  
 
<3 influenza seasons= 
Study has been 
extended 
7.2 Study duration:  
Following text added: 
<(autumn/winter months)= 
Clarification of the 
meaning of <influenza 
seasons=. 
7.2 Study duration:  
Removal of text: 
<, however timelines might be extended related to the COVID 
19 situation= 
In line with changes 
made to the whole 
paragraph. 
7.2 Study duration: 
Addition of text: 
"The End of Trial is defined as the date of receipt of the last 
data point from the last subject that is required for primary, 
secondary and/or exploratory analysis.= 
Clearer definition of the 
study duration.  
8.1 Study population  
 
Change of text from:  
<consulting= 
 
To 
 
<presenting at the Emergency Room=  
Clearer description of 
the study population. 

 
 
VALUE Dx WP4B ADEQUATE Protocol, version 5.0 18-Oct-2023 
 
 
 
43 
 
8.2 Inclusion criteria 
 
Change of text from: 
<Temperature ≥38.0°C measured at presentation or reported 
within the previous 24 hours= 
 
To 
 
<Temperature ≥38.0°C measured at presentation or parental 
report of fever within the previous 72 hours= 
The key function of 
the fever 
requirement in the 
inclusion criteria is to 
separate children 
with infection from 
those with non-
infection-related 
respiratory symptoms 
that may otherwise 
present in similar 
ways (e.g. asthma). 
As fever is only 
present during a 
short period of time 
especially in younger 
children who mostly 
have viral causes of 
their infection, we 
extend the possible 
interval between 
fever and enrolment.  
8.3. Exclusion criteria 
 
Change of text from: 
<Less than 14 days since the last episode of respiratory tract 
infection= 
 
To 
 
<Hospitalised for at least 24 hours within the last 14 days 
(healthcare-associated)= 
Clarity 
8.4 Recommended study 
site selection criteria 
(title) 
 
Following text added: 
<Recommended= 
More specific definition 
of the paragraph title. 
8.4 Recommended study 
site selection criteria 
(first bullet point) 
 
Change of text from:  
<Does not currently use equivalent rapid testing routinely in 
patients with CA-ARTI (rapid defined as time from sample 
collection to result interpretation by the physician within 4 
hours). Specific SARS-CoV-2 molecular testing will be 
considered individually.= 
 
To 
 
<Does not currently use equivalent rapid testing routinely in 
patients with CA-ARTI at the ER (rapid defined as time from 
sample collection to result interpretation by the physician 
within 4 hours).= 
Clarification of the 
place where the rapid 
testing routinely is 
performed. 
 
Removal of sentence 
regarding SARS-CoV-2 
molecular testing. 
10.1 Screening and 
enrolment 
 
Following text added: 
<and confirmed= 
Clarification of the 
actions to take before 

 
 
VALUE Dx WP4B ADEQUATE Protocol, version 5.0 18-Oct-2023 
 
 
 
44 
 
enrolling new 
participants   
10.2. Randomisation and 
blinding 
 
Following text added: 
<the subject= 
Clarity 
10.2. Randomisation and 
blinding 
 
Change of text from:  
<patients= 
 
To 
 
<subjects= 
Consistency  
10.3 Data collection 
 
Following text removed: 
<We will collect detailed demographic and clinical data from 
patient history and examination, patient and  
caretaker questionnaires and electronic chart reviews. 
Extension of the required data collection will be  
adjusted according to stratification and time points.= 
Text in the paragraph 
that follows the 
sentences removed 
already explains data 
collection in detail  
10.3 Data collection 
 
Change of text from:  
<Main study (Routine clinical and microbiological data 
collection and follow up until 30 days) – Clinical data will be 
collected but no biological samples will be obtained for 
research purposes with exception of the diagnostic test in the 
respective allocation group, if this sample is not taken as per 
standard of care.= 
 
To 
 
Main study (Follow up until 30 days) – Standard of care – 
clinical and microbiological data will be collected but no 
biological samples will be obtained with the exception of the 
diagnostic test in the respective allocation group. 
Clarification of the 
description of the main 
study 
10.4 Clinical data set 
 
Change of text. 
The whole paragraph has been reviewed. 
 
Table 1 Visit Schedule added.  
Clearer explanation of 
Clinical data set 
10.5 Baseline and follow-
up data for health-
economic analysis 
 
 
Following text added: 
<(see eCRF completion guidelines on guidance)= 
Reference to a more 
detailed document  
10.5 Baseline and follow-
up data for health-
economic analysis 
 
Change of text: 
<To= 
 
To 
 
<It needs to= 
Clarity 
10.5 Baseline and follow-
up data for health-
economic analysis 
 
Following text moved to this paragraph: 
<For patient questionnaires, patients will receive a link per 
email to complete the questionnaires online, which will 
directly be incorporated into the eCRF and be regarded as 
source data once digitally completed by the patients. If this is 
not possible or if patients are seen face to face, questionnaires 
will be completed per paper (source data) and entered in the 
Text moved to a more 
appropriate section of 
the protocol 

 
 
VALUE Dx WP4B ADEQUATE Protocol, version 5.0 18-Oct-2023 
 
 
 
45 
 
eCRF by study staff at the site. In this case paper versions will 
be stored as source data.= 
10.6 Microbiological 
testing.  
Change of text from:  
<is= 
 
To 
 
<are= 
Grammar correction 
10.7 Microbiology study 
and biobanking 
 
Following text removed: 
<at 3 study sites= 
Harmonisation with the 
rest of the protocol 
11.1 Good Clinical 
practice training.  
 
Change of text from:  
<Site personnel will be trained to obtain a Good Clinical 
practice (GCP) certification (if not already done) to timely 
identify and enrol eligible patients,= 
 
To 
 
<The Principal Investigator and all site personnel involved in 
the study need to provide a valid ICH-GCP certificate to timely 
identify and enrol eligible patients,= 
Clearer explanation of 
one of the 
responsibilities of the 
Principal Investigator in 
relation to GCP training  
11.1 Good Clinical 
practice training.  
 
Following text added: 
<See the ADEQUATE monitoring plan for more details.= 
Reference to a more 
detailed document 
11.1 Good Clinical 
practice training.  
 
Following text removed: 
<Specific training for the follow up questionnaires will be 
implemented if needed.=   
Removal of sentence 
regarding training for 
follow up 
questionnaires 
11.2 Medical Device 
training, being part of the 
Site Initiation Visit. 
 
Change of text from:  
<Laboratory training= 
 
To 
 
<Medical Device training, being part of the Site Initiation 
Visit.= 
More appropriate title 
of the paragraph that 
follows  
11.2 Medical Device 
training, being part of the 
Site Initiation Visit. 
 
Change of text from:  
<laboratory= 
 
To 
 
<the hospital= 
Better explanation of 
who is receiving 
training 
11.2 Medical Device 
training, being part of the 
Site Initiation Visit. 
 
Following text removed: 
<During site selection, lab external accreditation and 
participation in External Quality Assessments will be 
recorded.= 
Removal of sentence 
regarding lab 
accreditation 
11.2 Medical Device 
training, being part of the 
Site Initiation Visit. 
 
Following text added: 
<Quality Control of the device will be performed as described 
in the ADEQUATE monitoring plan.= 
Reference to a more 
detailed document 
11.3 Data management 
training.  
Change of text from:  
<our=  
 
Clarity 

 
 
VALUE Dx WP4B ADEQUATE Protocol, version 5.0 18-Oct-2023 
 
 
 
46 
 
To  
 
<UMCU= 
12.1 Adverse events 
 
Change of text from:  
<2. Progression of the disease under study should not be 
reported as an SAE.  
3. <Death= should not be reported as an AE. The cause of 
death should be reported as an AE. The only exception is 
<Sudden Death= when the cause is unknown.= 
 
To 
 
<2. SAEs resulting in death should be reported using the 
primary cause of death as the event term. The only exception 
is <Sudden Death= when the cause is unknown.= 
Clearer explanation of 
what constitutes a 
Serious Adverse Event 
(SAE) 
12.2 Reporting Adverse 
Events 
 
Following text removed: 
<disease under investigation= 
Paragraph aligned with 
paragraph 12.1 
12.2 Reporting Adverse 
Events 
 
Following text added: 
<Sponsor remains responsible for adequate reporting to 
regulatory authorities.= 
Clarification of one of 
the responsibilities of 
the Sponsor in relation 
to reporting Adverse 
Events 
12.2 Reporting Adverse 
Events 
 
Change of text from: <AEs and all SAEs= to <safety events= 
Clarity 
12.2 Reporting Adverse 
Events 
 
Following text added: 
<Refer to the ADEQUATE Safety Management Plan for more 
extensive guidance on reporting.= 
Reference to a more 
detailed document 
12.2 Reporting Adverse 
Events 
 
Following text removed: 
<Additional information, including the Investigator9s 
assessment, may be added to the eCRF later. Any necessary 
medical management of the event will be recorded in the 
patient9s medical record/source document.   
If the Sponsor requires supporting documentation or other 
information, the Sponsor will contact the study  
site.  
Data related to AEs and SAEs will be collected until event 
resolution, until the event is considered stable, or until all 
attempts to determine the resolution of the event are 
exhausted.  
All AEs and SAEs that are unresolved at study completion (or 
early termination) will be recorded as  
ongoing at study end.  
 
In addition, the following information should be recorded:  
• Onset date  
• Resolution date or date of death  
• Action taken  
• Event status (ongoing at study end or resolved)  
• Relationship of AE to the BioFire device used in the study  
• Relationship of AE to the sampling procedure  
• Indication of seriousness  
Removal of text 
regarding AEs and SAEs 

 
 
VALUE Dx WP4B ADEQUATE Protocol, version 5.0 18-Oct-2023 
 
 
 
47 
 
• Was AE anticipated or not (only for serious, device-related 
AEs)= 
12.4 Deviations from the 
clinical study protocol 
 
Change of text from:  
<A protocol deviation is any noncompliance with the study 
protocol, Good Clinical Practice, or protocol-specific 
requirements. A deviation (any activity conducted outside the 
parameters established by the study protocol) can be 
identified from a number of sources.= 
 
To 
 
<A protocol deviation is any non-compliance with the study 
protocol, Good Clinical Practice. A deviation can be identified 
from a number of sources.= 
Removal of text 
regarding protocol 
deviation 
13.1 Sample size 
calculation:  
Addition to section title - <joint paediatric and adult trial 
(protocol version 2.0, 23-Nov-2020 (paediatric)/protocol 
version 3.0, 18-Feb-2022 (adult))= 
Kept in to show initial 
sample size 
calculations, title 
amended for clarity  
13.1 Sample size 
calculation:  
Change of text from  
<we will aim for=  
 
To 
 
<we originally set an aim of= 
Change in sample size 
13.2 Sample size 
calculation.  
Addition of text 
<Review of sample size calculation for protocol version 3.0, 11-
Nov-2022: paediatric study 
The sample size calculation was redone after the decision to 
terminate the recruitment of adult patients (Value Dx WP4b 
ADEQUATE Protocol, adult version 3.0 18-Feb-2022) on 3rd 
May 2022. We first considered the three co-primary endpoints 
listed in 13.1.  
Co-primary endpoint EP3: Clinical failure within 30 days (non-
inferiority safety endpoint)  
The non-inferiority safety endpoint, defined as any inpatient 
admission or death within 30 days after study enrolment for 
initially non-admitted patients (expected to be >90% of 
paediatric participants) and any readmission, secondary ICU 
admission or death within 30 days after study enrolment for 
initially admitted patients, is unlikely to be relevant or 
appropriate for the paediatric population considered alone.  
Rationale: Mortality in the study population is extremely low, 
and secondary admission rates among children initially 
managed in the community as well as readmission and 
secondary ICU admission rates among primarily admitted 
children are in fact likely to be in the range of 2-2.5%. 
Secondary admissions are highly unlikely to be related to the 
initial illness episode due to the relative conservative 
approach in current paediatric emergency medicine practice 
erring on the side of primary admission if there is any doubt 
about appropriate safety netting being possible for the family. 
Based on this, EP3 will be considered a key secondary 
endpoint for the statistical analysis plan and will no longer be 
used to inform the target sample size.   
Change in sample size 

 
 
VALUE Dx WP4B ADEQUATE Protocol, version 5.0 18-Oct-2023 
 
 
 
48 
 
Co-primary endpoints EP1 and EP2: Days on antibiotic 
treatment and Days alive out of hospital, both within 14 days 
(superiority endpoints)  
The original assumptions for EP1 and EP2 remain unchanged. 
In particular, a reduction of one day in antibiotic treatment or 
increase of one day in days alive out of hospital appear to be 
relevant for a clinically relevant reduction in antibiotic 
prescribing and a reduction in hospital costs, respectively. In 
children, the co-primary superiority endpoints are likely to be 
dominated by EP1, as ambulatory exposure to antibiotics is 
likely to be common in the absence of hospital admission, 
whereas many admitted children would be expected to be 
treated with antibiotics as well. The superiority comparison on 
EP1 will be considered the primary outcome comparison for 
the trial. The sample size estimation was revisited for co-
primary endpoint EP1. From a recent publication on variations 
in antibiotic prescribing in febrile children presenting to 
European EDs, the standard deviation for days on antibiotic 
treatment was estimated as 3.7 days. Based on this, 
recruitment of 170 children per arm (total of 340 children) will 
be sufficient to detect a difference of one day in EP1 (power 
80%, alpha 0.05; table 4). 
 To account for uncertainty about the variability in EP1 (and 
EP2) in the paediatric study population, we propose to adopt a 
highly conservative approach aiming to recruit 251 children 
per arm (total of 502 children), resulting in adequate power to 
detect a difference in one day in EP1 or EP2.  
To adjust for loss to follow-up of up to 10%, we will aim for a 
total of 554 randomised children.= 
 
Addition of table 4 – <sample sizes using different 
assumptions= 
13.2 Sample size 
calculation.  
Change of text from  
<Review of sample size calculation for protocol version 3.0, 11-
Nov-2022: paediatric study=  
 
To 
 
<Review of sample size calculation for the current version of 
the protocol= 
More appropriate title 
of the paragraph that 
follows 
13.3 Analysis 
populations.  
Change of text from: 
<Analyses will be stratified by: 
• 
age groups (i.e. <5y/5-17) 
• 
clinical syndrome: exacerbation of chronic pulmonary 
disease, influenza-like illness, laryngitis/laryngotracheitis 
(croup), acute bronchitis/bronchiolitis (depending on age) 
• 
risk factors. Patient groups at risk for developing severe 
disease are well known, such as the elderly, patients with 
chronic pulmonary, cardiovascular or metabolic disease 
or immunocompromised patients. Clinical predictors of 
severity are also well known. Still, within risk groups may 
exist the uncertainty whether to hospitalise and/or to 
treat and the impact of testing may be assessed.   
• 
Vaccination status. Influenza. Pneumococcal.= 
 
Addition of variables 
for stratification 

 
 
VALUE Dx WP4B ADEQUATE Protocol, version 5.0 18-Oct-2023 
 
 
 
49 
 
To 
 
<Analyses will be stratified by: 
• 
Country/geographical location 
• 
age groups (i.e., <5y/5-17) 
• 
clinical syndrome: clinical pneumonia/LRTI, influenza-like 
illness, laryngitis/laryngotracheitis (croup), dominant 
obstructive airway disease/bronchiolitis  
• 
risk factors: patients with and without known risk factors 
for severe disease; the latter include chronic pulmonary, 
cardiovascular or metabolic disease or 
immunocompromised patients. 
• 
severity at presentation:  patients with and without 
primary hospital admission. 
• 
Vaccination status: Completed primary vaccinations 
according to local schedule y/n, pneumococcal vaccine 
receipt according to local schedule y/n, receipt of 
influenza vaccine for current season y/n, SARS-CoV-2 
vaccine receipt y/n.= 
13.4 Primary study 
parameter(s)/13.5 
Secondary study 
parameter(s).  
Text below moved from primary to secondary study 
parameters 
<Clinical failure will be analysed using Cox proportional 
hazards regression adjusted for age groups and predefined 
risk factors. Risk differences and 90% CI will be inferred by 
comparing the cumulative incidence of failure at day 30 from 
the Cox model and bootstrapping for the confidence interval. 
A 90% CI will be used to be compatible with a one-sided alpha 
of 0.05.= 
Change in primary/ 
secondary endpoint 
13.6 Mid-term data 
analysis 
 
 
 
 
Change of text from:  
<A data snapshot will be taken at the end of month six, after 
the first season,= 
 
To 
 
<A data snapshot will be taken after the first season,= 
Clarification of time 
when mid-term data 
analysis will be 
performed  
14.1 Regulation 
statement 
 
Following text added: 
<ISO 20916= 
Additional standards 
added to the list of 
regulations to abide to 
14.2 Recruitment and 
consent 
 
Following text added: 
The investigator or authorised delegate must obtain written 
informed consent before any clinical study related 
procedure/activity takes place.= 
Clarification of 
obligation to receiving 
written IC before 
performing any study 
related procedure 
14.2 Recruitment and 
consent 
 
Following text removed: 
<The investigator or delegate will be trained in consent 
procedures that protect the rights of the patient and adhere 
to the ethical principles within the Declaration of Helsinki.=  
Removal of sentence 
regarding training in 
consent procedures 
14.2 Recruitment and 
consent 
 
Following text removed: 
<and verbal= 
Sentence aligned with 
first sentence of this 
paragraph 
14.2 Recruitment and 
consent 
 
Following text removed: 
<Therefore, it is proposed to obtain ICF from LAR or in 
presence of an impartial witness to confirm ICF is verbally 
Sentence no longer 
applicable in view of 

 
 
VALUE Dx WP4B ADEQUATE Protocol, version 5.0 18-Oct-2023 
 
 
 
50 
 
given where it is not possible to obtain written consent 
directly.= 
the first sentence of 
this paragraph 
14.2 Recruitment and 
consent 
 
Following text added: 
<Deviations in consent procedures might occur per 
participating country, following national law and locally 
accepted procedures with regards to challenging COVID-
measures.= 
Clarification of 
procedure for receiving 
consent during the 
COVID-19 pandemic 
14.3 Withdrawal of 
individual subjects 
 
Following text added: 
<unless it is a COVID-19 study that does not interfere with this 
study.= 
Clarification of co-
enrolment of 
participants into 
another interventional 
study during the 
COVID-19 pandemic   
15.1 Steering Committee 
 
Change of text from:  
<every 6 months= 
 
To 
 
<regularly= 
Clarification of the 
frequency of the 
steering committee9s 
meeting  
15.2 Handling and 
storage of data and 
documents 
 
Following text removed: 
<For patient questionnaires, patients will receive a link per 
email to complete the questionnaires online, which will 
directly be incorporated into the eCRF and be regarded as 
source data once digitally completed by the patients. If this is 
not possible or if patients are seen face to face, questionnaires 
will be completed per paper (source data) and entered in the 
eCRF by study staff at the site. In this case paper versions will  
be stored as source data.= 
Text moved to a more 
appropriate section of 
the protocol 
15.2 Handling and 
storage of data and 
documents 
 
Addition of text:  
<Penta as Sponsor of the study will have access to all data 
collected from the start of the study. At the end of the study 
the Data will be transferred to Penta Foundation and will be 
stored for 25 years.= 
Change in sponsor 
Throughout 
General administrative corrections throughout: 
 
Addition of <the=  
 
Amendment of <E.g.= to <E.g.9= 
Amendment of <I.e.= to <I.e.9= 
Grammar corrections 
Throughout, Footer 
Amendment of protocol version and date 
For new protocol 
PI and site names 
Changed the St George9s Hospital PI from 8Dr Louise Hill9 
to 8Dr Louisa Brock9 
Change in PI 
PI and site names 
Removed Padua and Greek site that are not taking part 
in the study 
Change in site list 
Throughout, Footer 
Amendment of protocol version to V5.0 and date 18-
Oct-2023 
For new protocol 
Study summary table  
Added the NCT number 
Information missing  
 
 

 
 
VALUE Dx WP4B ADEQUATE Protocol, version 5.0 18-Oct-2023 
 
 
 
51 
 
 
 

 
 
 
Copyright 2019 VALUE-Dx 
www.imi.europa.eu 
www.value-dx.eu