Personalised Risk Assessment in Febrile Illness to Optimise Real-life Management Across the European Union (PERFORM)
Conditions: Fever, Infection, Inflammation
Sponsor: Imperial College London

PERFORM – Clinical protocol - v 1.0 
 
1st June 2016 
 
This document is part of a project that has received funding from the European Union’s 
Horizon 2020 research and innovation programme, under grant agreement nr. 668303  
 
 
                    
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CLINICAL PROTOCOL  
Version 1.0   1st June 2016 
 
Project full title:  Personalised Risk assessment in Febrile illness to Optimise 
Real-life Management across the European Union (PERFORM) 
 
Project acronym: PERFORM   
 
Project type of funding scheme: Large scale integrating collaborative project 
 
H2020 call covered: H2020-PHC-2014-2015 (Project ID 668303) 
 
Chief Investigator and Global Coordinator: Prof. Michael Levin 
 
Clinical Coordinator (WP1): Dr. F. Martinon-Torres 
 
Sponsor: Imperial College London 
 
 
 
 
Information contained in this document is the property of PERFORM consortium and is confidential. 
This information will not be disclosed to third parties without the prior written authorisation of 
Michael Levin or his authorised deputy. No part of this document may be reproduced, stored in a 
retrieval system or transmitted in any form or by any means - electronic, mechanical, recording or 
otherwise - without the prior written permission of Michael Levin or his authorised deputy. 
PERFORM 
Personalised management of 
febrile illness 

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CONTENTS 
 
1. 
LIST OF ABREVIATIONS ................................................................................................................... 4 
2. 
GENERAL INFORMATION ................................................................................................................ 5 
2.1. 
STUDY ORGANIGRAM ............................................................................................................... 5 
2.2. 
PARTICIPATING SITES AND PRINCIPAL INVESTIGATORS ............................................................. 5 
2.3. 
CLINICAL NETWORK CONFIGURATION ...................................................................................... 8 
3. OBJECTIVES OF PERFORM .................................................................................................................. 9 
4. 
ACTIONS IN PERFORM ................................................................................................................. 10 
5. 
SUMMARY OF STUDIES INCLUDED IN THIS PROTOCOL ................................................................ 11 
6. 
DETAILED STUDY PROTOCOLS ...................................................................................................... 12 
a. 
Biomarkers of Viral and Bacterial Infection (BIVABI) – For biomarker discovery .................... 12 
b. 
Protocol for the Management and Outcome of Fever in children in Europe (MOFICHE) ....... 15 
c. 
Overview of Biomarker Validation Studies (BIVA): .................................................................. 19 
1. 
Biomarker Validation in Emergency Department (BIVA-ED) ................................................... 21 
2. 
Protocol for Biomarker Validation in Paediatric Intensive Care (BIVA-PIC) ............................. 26 
3. 
Protocol for Biomarker Validation in High Risk patients (BIVA-HR) ......................................... 29 
4. 
Protocol for Biomarker Validation in Inflammatory patients (BIVA-INF) ................................. 32 
7. 
IMPLEMENTATION ........................................................................................................................ 36 
8. 
REGULATORY AND ADMINISTRATIVE ISSUES ............................................................................... 37 
a. 
ETHICAL APPROVAL ................................................................................................................. 37 
b.  
ENROLLING AND CONSENT ..................................................................................................... 37 
a) 
DEFERRED CONSENT ............................................................................................................... 37 
b) 
WAIVER OF INFORMED CONSENT FOR MOFICHE STUDY ........................................................ 38 
c) 
INFORMED CONSENT FOR FUTURE RESEARCH ....................................................................... 39 
c. 
SAMPLE TYPES ......................................................................................................................... 39 
d. 
BLOOD SAMPLING VOLUME RESTRICTIONS ............................................................................ 40 
e. 
CONFIDENTIALITY .................................................................................................................... 40 
f. 
INDEMNITY .............................................................................................................................. 41 
g. 
SPONSOR ................................................................................................................................. 41 
h. 
FUNDING ................................................................................................................................. 41 
i. 
DATA PROTECTION, HANDLING AND RETENTION ................................................................... 41 
j. 
AUDITS .................................................................................................................................... 41 
10. STUDY MANAGEMENT ................................................................................................................... 42 
11.  PUBLICATION POLICY .................................................................................................................... 42 
12. REFERENCES ................................................................................................................................... 42 
APPENDIX 1A – MOFICHE and common information for biomarker studies CRF ................................ 45 

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APPENDIX 1B – MOFICHE QUESTIONNAIRE: DESCRIPTION OF THE SETTING ...................................... 50 
APPENDIX 2- BIVA-PIC CRF ................................................................................................................... 53 
APPENDIX 3– BIVA-HR CRF ................................................................................................................... 58 
APPENDIX 4– BIVA-INF CRF .................................................................................................................. 60 
  
 
 
 
 
 

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1. LIST OF ABREVIATIONS 
 
ATC 
Anatomical Therapeutic Chemical  
BIVA 
Biomarker Validation studies 
BIVABI 
Biomarkers of Viral and Bacterial Infection  
BIVA-ED 
Biomarker Validation in Emergency Department  
BIVA-HR 
Biomarker Validation in High Risk patients 
BIVA-INF 
Biomarker Validation in Inflammatory patients 
BIVA-PIC 
Biomarker Validation in Paediatric Intensive Care 
CRF 
Case Report Form 
CRP 
C-reactive protein 
ED 
Emergency Department 
eQTL 
expression Quantitative Trait Locus  
EUCLIDS 
EU Childhood Life-threatening Infectious Disease Study 
FBC  
Full blood count  
GCP 
Good Clinical Practice 
GENDRES 
Project 
Genetic influence and Vitamin D levels on the individual susceptibility and 
prognostic to Influenza Virus, Respiratory Syncytial Virus and other 
Respiratory viral infections 
GP 
General Practitioner  
ICED 
Infection in the Emergency Department  
ICU 
Intensive Care Unit 
IRIS 
Immunopathology of Respiratory Infection Study  
MOFICHE 
Management and Outcome of Fever in children in Europe  
NPV 
Negative Predictive Value  
PERFORM 
Personalised Risk assessment in Febrile illness to Optimise Real-life 
Management across the European Union 
PICU 
Paediatric Intensive Care Unit  
SBI 
Serious Bacterial Infection  
SOP 
Standard Operational Procedure 
TB 
Tuberculosis 
 
 

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2. GENERAL INFORMATION 
 
2.1. 
STUDY ORGANIGRAM 
 
Lead:  
 
SERGAS (SPAIN) 
 
F. Martinón-Torres 
 
Deputy Lead:  
RUMC (NL) 
 
 
Michiel Van der Flier 
Deputy BIVABI  
IMPERIAL (UK)  
 
Jethro Herberg 
Deputy MOFICHE  
EMC (NL) 
 
 
Henriette Moll 
Deputy BIVA-ED 
LIVERPOOL (UK) 
 
Enitan Carrol 
Deputy BIVA-PIC 
GRAZ (AUS) 
 
 
Werner Zenz 
Deputy BIVA-HR 
NEWCASTLE (UK) 
 
Marieke Emonts 
Deputy BIVA-INF 
AMC (NL) 
 
 
Taco Kuijpers 
 
 
2.2. 
PARTICIPATING SITES AND PRINCIPAL INVESTIGATORS 
 
Name of participating site 
Address, and local Principal Investigator 
Servizo Galego de Saude 
Hospital Clínico Universitario de Santiago de Compostela 
(CHUS)- Spain 
Genetics, Vaccines, Infections and Pediatrics Research group 
(GENVIP)  
Prof. Federico Martinón Torres  
 
Imperial 
College 
of 
Science, 
Technology and Medicine 
Section of Paediatrics, Wright-Fleming Institute, Norfolk 
Place, London W2 1PG, UK 
 
CI Prof Michael Levin 
PI Dr Jethro Herberg 
London School of Hygiene and 
Tropical Medicine 
Department of Clinical Research 
Faculty of Tropical and Infectious Disease 
London School of Hygiene and Tropical Medicine 
Keppel Street 
London WC1E 7HT 
UK 
Dr Shunmay Yeung 

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The University of Liverpool 
Department 
of 
Clinical 
Infection, 
Microbiology 
and 
Immunology 
University of Liverpool Institute of Infection and Global 
Health  
Ronald Ross Building 
8 West Derby Street 
Liverpool L69 7BE 
UK 
 
Prof Enitan Carrol 
University of Newcastle Upon Tyne 
Great North Children’s Hospital 
Paediatric Immunology, Infectious Diseases & Allergy 
Clinical Resources Building 
Queen Victoria Road 
Newcastle upon Tyne 
NE1 4LP 
UK 
 
Dr Marieke Emonts 
ERASMUS 
universitair 
medisch 
centrum Rotterdam 
Sophia’s Childrens Hospital 
Prof Henriëtte A. Moll 
Head Department general paediatrics,of Pediatrics 
ErasmusMC Sophia Children’s Hospital 
Wytemaweg 280 
3015 CN Rotterdam , The Netherlands 
Prof Henriette A Moll 
Academisch Medisch Centrum bij 
Universiteit van  Amsterdam 
Meibergdreef 9 
1105 AZ Amsterdam, The Netherlands 
Prof. Dr. T.W. Kuijpers 
National and Kapodistrian University 
of Athens 
 
Second 
Department 
of 
Paediatrics, 
National 
and 
Kapodistrian University of Athens, P. and A. Kyriakou 
Children’s Hospital, 115 27 Athens, Greece.  
Prof Maria Tsolia 
Stichting Katholieke Universiteit 
Radboudumc Nijmegen, The Netherlands. 
Prof. Ronald de Groot 
Dr. Michiel van der Flier 
Medizinische Universität Graz 
Department of General Paediatrics 
Auenbruggerplatz 34/2 
8036 Graz, Austria 
Prof Werner Zenz  
Univerzitetni 
klinični 
Center 
Ljubljana 
Department of Infectious Diseases 
University Medical Centre Ljubljana 
Japljeva 2 
1525 Ljubljana, Slovenia 
Prof. Franc Strle 
Dr. Marko Pokorn 
Rīgas Stradiņa universitāte 
Riga Stradins university, Dept.of Pediatrics 
Children clinical university hospital, Riga, Latvia 
Assist.prof. Dace Zavadska 

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Medical Research Council (Gambia) 
Medical Research Council Unit, The Gambia, Atlantic 
Boulevard, Fajara, The Gambia 
Dr Kalifa Bojang 
Dr Suzanne Anderson 
Ludwig-Maximilians-Universität 
München 
Division of Paediatric Infectious Diseases 
Dr. von Hauner Children's Hospital, KUM 
Ludwig-Maximilian-University Munich (LMU) 
Lindwurmstr. 4 
80337 Munich, Germany 
 
Dr. Ulrich von Both 
Universität Bern 
Department of Pediatrics, University of Bern, 3010 Bern, 
Switzerland 
 
Dr. Phlipp Agyeman (PI) 
Prof Luregn Schlapbach 
Prof. Christoph Aebi 
 

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2.3. 
CLINICAL NETWORK CONFIGURATION 
The PERFORM clinical network will be organised into nodes, as shown in the figure below (Fig. 1).  
  
 
Figure 1.- Clinical network in PERFORM study 
 
The PERFORM clinical network nodes will recruit prospective patients into one or more biomarker 
studies. To achieve this, clinical nodes will work with existing or new local networks of collaborating 
centres and investigators. The Principal Investigator in each node will take responsibility for local 
networks including budget and local ethics. 
 
 
 
 

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3. OBJECTIVES OF PERFORM 
This study aims to improve the diagnosis and management of febrile children across Europe and 
West Africa through the development of simple diagnostic tests to discriminate febrile illnesses in 
children, including bacterial and viral infection and inflammatory diseases. The project will:  
• 
Assess current management of febrile illness across Europe and West Africa using 
quantitative and qualitative methods (including cost-effectiveness analysis).  
• 
Identify personalized discriminators of bacterial and viral infection and inflammatory 
diseases, and diagnostic signatures that distinguish severe and mild disease, using a 
combination of clinical phenotypic markers, host genetic markers, and biomarkers derived 
using transcriptomic, proteomic, and bioinformatic approaches. 
• 
Develop simple proof-of-concept personalized tests for application at the point of care. 
• 
Model the impact of the introduction of optimised management strategies for febrile 
children, in the varied healthcare settings of Europe, thus providing the evidence necessary 
for European and international health systems to adopt new management strategies for 
febrile children. 
The PERFORM international clinical network constitutes a key factor for the PERFORM project as a 
whole and for each of the different participating countries in Europe and West Africa (Fig. 1). The 
network will provide all necessary patient cohorts for all working packages (Fig. 2) as needed 
(discovery and validation studies).  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 2.- Strategy and general description of the project 
An established Europe-wide network of researchers in infectious diseases, molecular and cellular 
biology, informatics and public health will combine their expertise to improve the diagnosis and 
treatment of febrile patients across Europe through the application and evaluation of sophisticated 
new personalized medicinal, molecular, transcriptomic and proteomic methods. 

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Clinical data and sample banks from previous EU-funded or national funded studies undertaken by 
members of the consortium will be combined with samples recruited by PERFORM to create a 
comprehensive and well-characterised biobank. 
 
4. ACTIONS IN PERFORM  
PERFORM will achieve its objectives through the following actions: 
• 
Detection of novel transcriptomic, proteomic and phenotypic clinical markers in existing patient 
cohorts with bacterial and viral infections and inflammatory diseases.  
• 
Correlation of these biomarkers with genetic polymorphisms known to influence phenotypes of 
infectious disease identified in the patients’ genetic sequence, including genetic changes 
discovered as part of the FP7-funded EUCLIDS study (www.euclids-project.eu), using expression 
Quantitative Trait Locus (eQTL) mapping and other approaches 
• 
Collection of data from different European countries on current practice in management of 
febrile children, including investigation, hospitalization and antibiotic use in emergency 
departments, wards and intensive care units (ICU) in hospitals, and analysis of current costs of 
management. 
• 
Evaluation of the performance of novel transcriptomic, proteomic and phenotypic markers that 
distinguish bacterial infection from viral infection, first in patients with “gold standard” confirmed 
bacterial or viral infections, and then patients with presumed bacterial or viral infections in 
whom existing culture based diagnostic methods have failed to assign accurate diagnosis. 
• 
Prospective validation of the most promising transcriptomic, proteomic and phenotypic markers 
in cohorts of febrile patients attending primary and secondary care settings in different European 
countries as well as in West Africa. 
• 
Evaluation and validation of the diagnostic biomarkers in diagnostically challenging groups of 
patients with immune-suppression, inflammatory diseases, hospital acquired infections and 
patients admitted to paediatric intensive care units (PICU).  
• 
Development of a management algorithm for febrile children including use of the novel 
diagnostic markers and evaluation of its performance in the settings of different European 
countries. 
• 
Cost-effectiveness analysis of the model of care for febrile patients including its effects on 
antibiotic use, reduction in hospital admissions and prevention of misdiagnosis. 
• 
Initiate product development and implementation of the novel diagnostic approaches identified 
by our program in collaboration with our industrial and SME partners. 
• 
Dissemination of the findings at government level to healthcare systems in different European 
countries for inclusion in the management of febrile children across Europe. 
 
 

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5. SUMMARY OF STUDIES INCLUDED IN THIS PROTOCOL 
This protocol will describe three components of the patient and sample groups used in 
PERFORM:  
1. Biomarker discovery using pre-existing sample collections (Biomarkers of Viral and 
Bacterial Infection (BIVABI)) 
The BIVABI study will use transcriptomic, proteomic and metabolomic approaches to 
discover biomarkers that distinguish the different phenotypes of childhood febrile illness, 
including children with infection (viral, bacterial, mycobacterial, other) and inflammation 
(including Kawasaki disease, juvenile idiopathic arthritis).  
 
2. Observational study of patient management and outcome, using large, aggregated 
datasets and not involving sample collection (Management and Outcome of Fever in 
children in Europe (MOFICHE)) 
MOFICHE is an observational study assessing the management and outcome of children 
presenting to Emergency Departments (ED) with fever across Europe. This study will use 
large departmental datasets to collect information on nearly 50,000 febrile episodes in 
12 ED’s in 9 European countries. This study will use large-scale, pseudo-anonymized 
departmental data to safeguard identity of participants and will not involve consented 
patient recruitment; nor will it use patient samples. Data included in MOFICHE will be 
based on that collected as part of routine clinical care. No tissue samples will be taken 
for research use, and informed consent will not be needed for the MOFICHE study.  
 
3. Biomarker validation using prospectively recruited patients and patient samples 
(Biomarker Validation studies (BIVA)) 
Prospective, observational studies will recruit a validation group of children with 
infectious and inflammatory conditions. Research blood samples will be taken and 
analysed in order to validate biomarker findings identified in the BIVABI discovery study. 
There are 4 related prospective studies. The BIVA-ED study will recruit the majority of 
the children. The other studies will target more challenging groups who will not be 
adequately recruited in an ED study, in order to increase numbers. The studies are:  
• 
Biomarker Validation in Emergency Department (BIVA-ED)  
• 
Biomarker Validation in Paediatric Intensive Care (BIVA-PIC) 
• 
Biomarker Validation in High Risk patients (BIVA-HR) 
• 
Biomarker Validation in Inflammatory patients (BIVA-INF) 
 
 

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6. DETAILED STUDY PROTOCOLS 
a. Biomarkers of Viral and Bacterial Infection (BIVABI) – For biomarker discovery 
 
SUMMARY 
The BIVABI study will use transcriptomic, proteomic and metabolomic approaches to discover 
biomarkers that distinguish the different phenotypes of childhood febrile illness, including children 
with infection (viral, bacterial, mycobacterial, other) and inflammation (including Kawasaki disease, 
juvenile idiopathic arthritis).  
 
PATIENT GROUPS 
 
Study 
Lead 
country 
Target diseases 
DNA 
metabolo
mics 
transcrip
tomic 
prote
omics 
EU action for diseases of 
poverty 
UK 
Tuberculosis, 
other 
infections, 
latent 
TB 
infection, HIV 
 
ü 
ü 
ü 
EU Childhood Life-
threatening Infectious 
Disease Study (EUCLIDS) 
UK 
Bacterial and viral illness 
ü 
ü 
ü 
ü 
ESPID/IKDGC Kawasaki 
Disease study Consortium 
UK 
Kawasaki disease 
ü 
ü 
ü 
ü 
ESPID Meningococcal study 
Consortium 
UK 
Meningococcal 
meningitis and sepsis 
ü 
ü 
ü 
ü 
International IKDGC 
collaborative study 
NL 
Rheumatological 
and 
inflammatory diseases 
ü 
ü 
ü 
ü 
GENDRES 
Spain 
Respiratory 
viral 
infections 
ü 
ü 
ü 
ü 
UCAN 
Canada 
Childhood Arthritis and 
Rheumatic Diseases 
ü 
ü 
ü 
ü 
VIRGO  NL 
Respiratory Virgo 
viral infections 
ü 
ü 
ü 
ü 
Immunopathology of 
Respiratory Infection Study 
(IRIS) 
UK 
All febrile illness 
ü 
ü 
ü 
ü 
Infection in the Emergency 
Department (ICED) 
UK 
All febrile illness 
ü 
ü 
ü 
ü 
Biomarkers of Acute Severe 
Infection Consortium 
(BASIC) 
UK 
All critical illness 
ü 
ü 
ü 
ü 
Swiss Pediatric Sepsis 
Study 
CH 
Bacteremia 
and 
systemic 
inflammatory 
response syndrome 
ü 
ü 
ü 
ü 
IPD study 
(pneumococcal disease) 
Malawi      Pneumonia 
and 
meningitis 
ü 
 
 
 
Nepal UK 
Pneumonia 
ü 
ü 
ü 
ü 
Oxford Vaccine Studies UK 
Paediatric 
vaccine 
recipients 
ü 
ü 
ü 
ü 
 
Table 1.- Studies which will support BIVABI study 

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Clinical data and stored samples from previous EU-funded or national funded studies undertaken by 
members of the consortium will be combined to form a discovery set that will be used to identify 
transcriptomic, proteomic, metabolomic and clinical markers that distinguish the different causes of 
febrile illness, including bacterial and viral infection and inflammatory disease. This study will use 
only those samples that have been collected as part of an ethically-approved study, in which 
written consent has already been obtained which permits their future use in ethically-approved 
studies. No new patients will be recruited for the BIVABI study.  
The retrospective collections used in BIVABI will be primarily drawn from samples collected in the 
studies summarized in Table 1.  
 
STUDY DESIGN 
The discovery group will include patients from across the spectrum of febrile disease, including 
bacterial infection, viral infection, inflammatory (non-infectious) disease, and children with febrile 
illness of unclear aetiology. The discovery group will also include control samples from children 
without infection, taken as part of ethically approved studies. We will apply phenotyping algorithms 
for patient selection and categorization, using defined clinical criteria, and application of both 
conventional microbial diagnostics and molecular diagnostic approaches to detect bacterial and viral 
genomes (Fig. 3). We will categorise patients into phenotypic groups, including bacterial infection, 
viral infection and inflammatory diseases, from which stored samples will be drawn for biomarker 
discovery. After analysis of RNA expression by microarray and RNAseq, and proteomic and 
metabolomic analysis by multiplex immunoassays and mass spectrometry respectively, 
bioinformatics analysis will be undertaken to identify small diagnostic signatures that accurately 
identify bacterial infection, viral infection and inflammatory diseases. We will identify diagnostic 
signatures that distinguish severe and mild disease.  
 
 

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Fig. 3.-  Phenotyping flow chart for febrile patients in the BIVABI study 
 
  
 
Febrile patient meeting entry criteria for study 
With available samples for biomarker discovery (RNA, proteomic, metabolomic) 
Categorization of patients based on clinical data 
BACTERIAL 
syndrome 
Toxic shock syn 
LRTI/empyema 
Celluli7s/
impe7go/Nec. 
fasc./Purpura 
fulminans 
Pyomyosi7s 
CLABSI 
UTI/Pyelonephri7s 
Appendici7s/
Peritoni7s 
Bone infec7on 
Mastoidi7s 
Scarlet fever 
Endocardi7s 
Abscess 
VIRAL 
syndrome 
Bronchioli7s 
Viral-induced 
wheeze 
Croup 
Varicella  
Herpes simplex 
Flu-like illness  
Hand-foot-mouth 
Roseola 
Paro77s/mumps 
Erythema 
infec7osum /
parvovirus 
Symptoms viral 
OR bacterial 
Meningi7s/
Encephali7s/
Neuri7s 
LRTI (no 
empyema) 
Sepsis/severe 
sepsis/sep7c 
shock 
FWS/PUO 
Gastroenteri7s 
URTI, Tonsilli7s, 
O77s media, 
Sinusi7s 
Conjunc7vi7s 
ARDS 
Adeni7s 
 
Other febrile 
syndrome 
Not bacterial or 
viral 
Inﬂamma7on 
without infec7on 
Other cause of 
fever 
 
Bacterial 
syndrome 
but no 
bacteria 
identified 
Unclear 
features  
OR 
microbiology 
does not fit 
syndrome 
Sterile-site 
pathogenic 
bacteria, 
match 
syndrome 
Viral 
syndrome 
but no 
virus 
identified 
Other 
infection, 
eg 
malaria 
Virus 
identified 
that 
matches 
syndrome 
Minor 
illness, & 
insufficient 
clinical 
information 
Matches 
criteria for 
inflammat-
ory 
syndrome 
Unclear 
features – 
infection 
or 
inflammat-
ory 
Probable 
Bacterial
* 
Bacterial 
syndrome 
low or no 
inflamm, 
markers* 
Definite 
Bacterial
* 
Probable 
Viral 
Other 
infection 
Definite 
Viral 
Trivial 
Unknown 
bacterial 
or viral* 
Viral 
syndrome 
high or no 
inflamm, 
markers* 
Inflammat-
ory 
syndrome 
Infection 
or 
inflamm-
ation 
no 
no 
CRP>60 
mg/l 
CRP≤60 mg/l 
neutrophils ≤12 x109/ml 
yes 
yes 
*DB/PB/unknown bacterial or viral: pa7ents CAN have iden7ﬁed viral co-infec7on  
yes 
Review clinical investigation results 
Bacteriology, virology, radiology, hematology, chemistry 

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b. Protocol for the Management and Outcome of Fever in children in Europe (MOFICHE) 
SUMMARY 
This is an observational study assessing the management and outcome of children presenting to 
Emergency Departments (ED) with fever across Europe. This study will use large departmental 
datasets to collect information on nearly 50,000 febrile episodes in 12 ED’s in 9 European countries. 
This study will use large-scale, pseudo-anonymized departmental data, and will not involve 
consented patient recruitment; nor will it use patient samples. Data included in MOFICHE will be 
based on that collected as part of routine clinical care. No tissue samples will be taken for research 
use, and informed consent will not be needed for the MOFICHE study.  
BACKGROUND 
The organisation of Emergency care varies between European countries. In several European 
countries acute medical care is delivered by general practitioners (who can act as gatekeepers), 
primary care paediatricians, emergency departments and out of office GP services. The number of 
children presenting to the ED varies across Europe, influenced by the number and availability of GPs 
primary care paediatricians and the preferences of parents1,2 . Differences in morbidity and mortality 
have been described 3,4.  
Fever is the most frequent reason for a child to attend the paediatric emergency department (20-
40%). The majority of febrile children suffer from self-limiting illness, with serious bacterial infections 
(SBI) ranging from 5% to 30% 5,6. Pneumonia and urinary tract infection constitute the majority of 
serious infections, and septicaemia/meningitis are currently rare 7. Current guidelines state that 
there is no indication for oral antibiotics in children with fever without a clear diagnosis of bacterial 
infection 6. Nevertheless, antibiotic prescription rates in febrile children remain high 8, 9 and there is 
marked variation between countries 10.  
Furthermore the diagnostic work-up and the number of febrile children hospitalised varies between 
settings 11. This study will investigate the nature and cause of variations in management practices 
and outcomes for febrile children in the ED in 9 European countries. 
OBJECTIVES 
• 
To evaluate the management of febrile children visiting the ED in different European countries. 
• 
To study the determinants of diversity between countries in hospital admission, antibiotic 
prescription and investigations related to clinical signs and symptoms in febrile children.  
• 
To provide data for comparative health evaluation and cost effectiveness modelling  
STUDY DESIGN 
An observational study on febrile children aged 0- 18 years visiting the emergency department of 12 
different hospitals in 9 European countries.  
STUDY POPULATION 
Inclusion criteria: 
Febrile  children aged 0- 18 years visiting the emergency care.  

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Febrile child: fever measured at ED or history of (measured) fever less than 3 consecutive days prior 
to presentation at emergency department,  
In the participating hospitals more than 50.000 febrile children visit the ED annually (1200-29.000 
per emergency setting) (Table 2).  
Table 2.- Data of febrile children which visit ED in the participating sites per year 
STUDY PROCEDURES 
Data of all febrile children will be flagged at triage and a standardised dataset will be collected. ED 
data will be extracted from the standardized clinical notes or electronic records of the individual 
febrile child. This will include data on age, gender, temperature, vital signs, triage category, clinical 
alarming signs, grade of doctor/under supervision of consultant, admission or discharge and the 
following outcome measures: investigations (blood, cerebrospinal fluid, nasopharyngeal aspirates 
Participant 
Name of institution 
City 
Country 
Children to 
ED/year 
Number 
with 
fever (%) 
Number 
admitted (%) 
1-
IMPERIAL 
Imperial 
College 
Healthcare NHS Trust 
London 
UK 
26,000 
8,666 (33%) 
2,340 (12%) 
2-LIV 
Alder Hey Children’s NHS 
Foundation Trust 
Liverpool 
UK 
60,000 
8,600 (14%) 
554 (6%) 
3- UNEW 
Great 
North 
Children’s 
Hospital 
Newcastle-
upon-Tyne 
UK 
30,000 
4,500 (15%) 
700-1000 (2-3%) 
4-EMC 
Erasmus Medical Centre 
Rotterdam 
Netherlands 
7,000 
1,400 (20%) 
1,050 (15%) 
5-AMC 
Amsterdam 
Medical 
Centre 
Amsterdam 
Netherlands 
6,000 
1,200 (20%) 
550 (9%) 
6- NKUA 
National and Kapodistrian 
University of Athens 
Athens 
Greece 
40,000 
2,600 (6%) 
6,500 (16%) 
7-RUMC 
Nijmegen 
and 
PedBIG 
sites 
Nijmegen 
Netherlands 
12,000 
3,600 (30%) 
1,500 (12%) 
8- SEGAS 
Hospital Universitario de 
Santiago de Compostela 
Santiago 
de 
Compostela 
Spain 
33,000 
19,080 (57,8%) 
1,172 (3,6%) 
9-MUG 
Medical University of Graz 
Graz 
Austria 
18,000 
6,000 (33%) 
4,000 (22%) 
10- UKCL 
University Klinicini Centre 
Ljubljana 
Ljubljana 
Slovenia 
 
 
 
11-RSU 
Riga Stradins University 
Riga 
Latvia 
42,750 
 29,900(69,9%) 
4,865 (11.4%) 
12-UBERN 
Medizinische Kinderklinik, 
University of Bern 
Bern 
Switzerland 
20,000 
6,000 (25%) 
1,800 (9%) 
13-LMU 
Ludwig 
Maximilian 
University Munich 
Munich 
Germany 
20,000 
6,400 (33%) 
2,200 (11%) 

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/throat swabs, stools or urine tests and result), antibiotics prescribed and final diagnosis/working 
diagnosis at discharge.  
Data protection  
This study will use large-scale, pseudo-anonymized departmental data. Each febrile patient will be 
identified with a subject code in the ED (3 digit study center code- 5 digit individual patient code 
(starting at P00001 for each center)). Data analysis in MOFICHE will differ from an audit in that there 
will not be complete anonymisation of data analysed, in order that we can identify patients that 
make repeated ED visits, and match patients in MOFICHE with those in other studies. We will 
maintain pseudo-anonymization of the MOFICHE data (birthdate will be included), so that patients 
will not be fully anonymised.  
We will not seek informed consent from individual patients in the MOFICHE study, as the data will 
consist of routinely-collected departmental-level data on all febrile patients seen in that location. We 
will collect data for the MOFICHE study only at sites that have ethics committee and local clearance 
for this protocol, for the analysis of the pseudo-anonymized data.  
Working diagnosis and infectious focus  
Each patient will be coded in the following areas, using an electronic questionnaire filled in at the ED 
[Appendix 1/A]:  
• 
working diagnosis of the likely cause of the febrile illness (including definite/probable 
bacterial,  definite/probable viral, inflammatory causes, unknown (see figure 3)) 
• 
description of the focus and syndrome (for instance upper/lower respiratory infection, 
urinary tract infection, enteric infection, cutaneous infection, septicaemia/meningitis, 
osteomyelitis, other, no clear focus or spot diagnosis) 
• 
treatment received (including details of antibiotics used and duration)  
• 
follow-up at discharge of the ED (ambulant, hospitalization, intensive care, no follow-up, ED 
readmission within 5 days)  
Data quality 
Each individual WP1 participant will carry the responsibility to collect the required data in his/her ED 
setting. Moreover, each WP1 participant will develop a procedure to monitor the completeness of 
the data collection and the quality of the data. Furthermore, all centers will have a limited number of 
key parameters checked (sex, age, working diagnosis category; in 5% of inclusion in MOFICHE) 100% 
in other substudies) by an independent trained investigator. For publication of the results of the 
study, it is important that the process of data collection and quality assurance is documented by each 
WP1 participant.  
Description of the setting (to be completed once for each setting)  
In addition to individual patient-level data, we will collect general information on each setting for the 
comparative health service study including information on the organisation, management and 
delivery of acute and emergency paediatric services; human resources; summary data on paediatric 
attendances (where available); contextual information   (rural/ inner city, university/ teaching/ non-

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teaching hospital, catchment area, country, indications for hospitalization/short-stay). [Appendix 
1/B].  
OUTCOMES 
Primary outcomes: Antibiotic prescription, hospitalization and number/type of investigations. 
Number of children re-attending within 5 days of the first hospital presentation. 
The antibiotic prescription rate is defined as the number of children that receive antibiotics among 
the number of febrile children. We will use the Anatomical Therapeutic Chemical (ATC) classification 
system to group antibiotics into narrow and broad spectrum 12. Broad spectrum antibiotics include 
broad spectrum penicillins, cephalosporins, and macrolides (J01CR, J01DC, J01DD and J01F (without 
J01FA01). Narrow spectrum antibiotics include beta lactamase sensitive penicillins (J01CE), first 
generation cephalosporins (J01DB) and macrolides (J01FA01). 
Investigations are defined as routine (FBC, CRP, urine analyses) and extended diagnostic tests (Chest 
X-Ray, culture of blood or liquor, viral tests)  
Secondary outcome: number of prescriptions of broad spectrum antibiotics versus the number of 
prescription of narrow spectrum antibiotics (dose in 24 hours). Clinical and general characteristics of 
antibiotic prescription, hospitalization duration of stay and investigations. 
ANALYSIS 
We will describe clinical and general characteristics of the patients at the different ED’s. We will 
define the number of children with alarming signs for serious infections 6, 13, quantify a risk 
assessment 
for 
serious 
infections 
for 
each 
child, 
using 
the 
feverkidstool 
(http://www.erasmusmc.nl/formulieren/feverkidstool/?lang=en 13) and describe the number of 
investigations and hospitalizations. We will quantify antibiotic prescription duration, antibiotic 
prescription rate, defined as the number of children with antibiotics among the number of febrile 
children and the number of prescription of broad spectrum antibiotics versus the number of 
prescription of narrow spectrum antibiotics with 95% confidence interval in different settings and for 
different working diagnoses. For antibiotic prescription, diagnostic tests and hospitalization we will 
use logistic regression analyses to evaluate influencing factors as age, triage urgency, clinical 
symptoms/alarming signs, CRP and other diagnostic tests, working diagnosis, season and setting. 
SAMPLE SIZE ESTIMATION  
We expect to include nearly 50,000 febrile children in the study, ranging from 6,000 in Austria to 
50,000 in the UK, with at most 5% of missing data. Pilot data indicate an overall antibiotic 
prescription rate of 20%, a hospitalization rate of 10%, a CRP rate of 15% and a blood culture rate of 
5%. Applying a commonly used rule of thumb of 10 events per variable, we expect that the study 
sample is large enough to analyse 120 determinants for the most prevalent outcome of antibiotic 
prescription and 25 determinants (0.1 determinants per 5% of 6000 events) for the least prevalent 
outcome of blood culture with sufficient accuracy 14, 15.  
 

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c. 
Overview of Biomarker Validation Studies (BIVA):  
a) Overview of approach 
We will use prospective, observational studies to recruit children with infectious and inflammatory 
diseases. Research samples, including blood, urine, stool (in the case of gastroenteritis), 
nasopharyngeal/throat swab will be taken for detailed pathogen analysis, and for host DNA, RNA, 
protein and metabolomics analysis at the earliest time point possible as well as 48 hours later and at 
follow-up in order to assess how the biomarkers change with illness progression and/or treatment. 
We will use a variety of RNA and protein estimation techniques to validate biomarkers identified in 
the BIVABI study as predictive of bacterial infection, viral infection or inflammatory disease. We will 
also use sequencing techniques to validate the predictive value of host genetic changes in 
predisposition to illness.  
We envisage that a new diagnostic test that can discriminate between bacterial, viral and 
mycobacterial infections will play a key role in the management of febrile children in Emergency 
Deparments. In addition, a diagnostic marker that is able to discriminate between bacterial, viral and 
mycobacterial infections will be particularly useful in specific groups of children in whom clinical 
diagnosis is particularly difficult or identification of bactieral infection is particularly important to 
make.  This includes children on intensive care units who often develop fevers and end up on 2nd or 
3rd line antibiotics with no positive microbiological diagnosis; immunocompromised children who 
develop fevers often without localising signs; and children with a range of inflammatory diagnoses 
such as Kawasaki Disease and Juvenile Idiopathic Arthritis, whose initial presentation is difficult to 
discriminate from bacterial infection. We will conduct separate sub-studies specifically aimed at 
targeting these children: 
There are 4 related prospective studies. The studies are:  
• 
Biomarker Validation in Emergency Department (BIVA-ED) 
• 
Biomarker Validation in Paediatric Intensive Care (BIVA-PIC) 
• 
Biomarker Validation in High Risk patients (BIVA-HR) 
• 
Biomarker Validation in Inflammatory patients (BIVA-INF) 
The potential performance of the identified biomarkers (in the ED setting) will be interpreted using 
data on numbers and types of presentations established by the MOFICHE study, to better model the 
potential impact of the biomarkers on management of febrile children.   
b) Analysis approach 
We will use data from the biomarker validation studies to calculate sensitivity and specificity of the 
new biomarkers to discriminate viral and bacterial infections and inflammatory diseases. Using 
comparative methods, the added value of new biomarkers to usual diagnostic work-up will be 
estimated. Additionally, the MOFICHE study data will allow us to calculate positive and negative 
predictive values of the biomarkers in different healthcare settings, using information on the 
numbers of febrile children presenting, and the proportion with bacterial, viral and inflammatory 
conditions. 
c) Sample sizes  
We will recruit a minimum of 3,000 children to the BIVA-ED study, in order to capture sufficient 
children with confirmed bacterial infection.  

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In order to increase the number of children with less common but important febrile illnesses who 
will not be adequately recruited in an ED study, we will recruit harder-to-reach groups using targeted 
studies as follows:  
• 
We will recruit 500 additional critically ill children - Biomarker Validation in Paediatric Intensive 
Care (BIVA-PIC)  
• 
We will recruit 200 additional children at high-risk of bacterial illness through primary or 
secondary immunodeficiency - Biomarker Validation in High Risk patients (BIVA-HR) 
• 
We will recruit 150 additional children with a range of inflammatory diagnoses, whose initial 
presentation is difficult to discriminate from bacterial infection - Biomarker Validation in 
Inflammatory patients (BIVA-INF) 
A sample size calculation is shown for each study below.  
 
d) 
Laboratory methodologies 
We will undertake molecular diagnostics to improve distinction between bacterial and viral diseases 
in both the discovery and prospective validation work packages. We will use PCR to detect a panel of 
respiratory viruses on nasopharyngeal or throat swabs. We will use PCR to detect a panel of viral and 
bacterial pathogens in blood. In addition, all patients will have blood culture performed. These 
molecular assays both extend and complement the routine microbiological investigations undertaken 
in the participating hospitals. We will also undertake validation of candidate protein biomarkers 
using multiplex immunoassay methodology in the validation stage of the project.  
The proteomic analysis will receive plasma/urine samples from well-characterised patients and 
undertake shotgun and targeted proteomic analysis using LC-MS/MS in selected well defined groups 
of patients. Immunoassays will be developed against the protein biomarkers selected to finally test 
these in the validation phase.  
Furthermore, we will undertake RNA transcriptomic analysis by microarray, RNASeq analysis and 
analysis of micro RNA and non-coding RNAs from the RNASeq data. We will identify diagnostic host 
transcriptional profiles, and validate selected sets of these RNA biomarkers using RT-PCR and 
nanotechnology. We will concentrate investigations on mRNA, but other RNA species discovered 
during the sequencing (such as micro RNA), that have the potential for translation will also be 
investigated. 
Specific techniques will be: 
• RT-PCR, microarray and RNA-seq for RNA 
• Mass spectrometry-based metabolomics 
• Sequencing to analyse host DNA using targeted and high-throughput (whole exome sequence 
WES) approaches 
• Multiplex immunoassays once candidates have been identified 
 

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e) Detail of the Biomarker Validation Studies 
1. Biomarker Validation in Emergency Department (BIVA-ED) 
This study will recruit 3,000 febrile children attending the Emergency Department or primary care in 
selected centres in the PERFORM consortium. Participating sites will vary with the numbers of 
patients recruited and the prevalence of serious bacterial infection. 
SUMMARY 
The PERFORM consortium will recruit at least 3,000 febrile children attending ED, as part of an 
observational study. Patients will have a set of samples taken for research. We will take an approach 
to recruitment to ensure the study cohort is highly representative to the true population of children 
presenting with febrile illness. At each recruitment site, we will therefore recruit as comprehensively 
as possible all febrile children who attend the ED who are willing to participate.  
 
ENDPOINTS  
 
Children’s involvement in the study will last for the duration of their illness episode, until discharge, 
and they will be followed up at 48-72 hours and at 28 days (+/- 2 days) post-admission to document 
any re-attendances or re-admissions. 48 hours and 28-day follow up will be by visit if clinically 
needed, telephone interview or text messaging depending on the preference of the 
parent/guardian. 
OBJECTIVES 
Evaluation and validation of the diagnostic biomarkers in diagnostically challenging groups of 
patients presenting to the ED suspected of infections or febrile children presenting to the ED. 
Development of a management algorithm for febrile children including use of the novel diagnostic 
marker and evaluation of its performance in the settings of different European countries. 
STUDY DESIGN 
A prospective observational study of febrile children attending the Emergency Department in 
selected centres in the PERFORM consortium.  
STUDY POPULATION 
All febrile children <18 years (16 years depending in the setting) of age presenting to Emergency 
Departments at participating sites: 
Inclusion Criteria 
• 
All children <18 years presenting to the Emergency department with fever >38ºC, or a history 
of fever (within 3 days), in whom the attending clinician determines the need for blood 
sampling or whom parents give consent for bloods taken for research purposes 
• 
All children <18 years presenting to the Emergency department suspected of infection. We 
will include children with the full spectrum of disease severity, and will include children with 
co-morbidities, as these children are at higher risk of antibiotic treatment and are therefore a 
particular target for study.  

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• 
We will collect samples from afebrile control children who are having blood tests for reasons 
other than for investigation of infectious or inflammatory illness.  
Exclusion Criteria  
• 
Children from whom parent/legal guardian signed consent is not received 
• 
For healthy control children only: febrile illness or vaccination within the last 3 weeks.  
 
STUDY PROCEDURES  
See 8.b ENROLLING AND CONSENTING PATIENTS INTO PROSPECTIVE STUDIES section.  
A complete history and physical examination will be performed. Information on degree and duration 
of fever, previous antibiotic use and previous existing co-morbidities will be collected (including also 
all items for the febrile children in the MOFICHE study). If the attending paediatrician decides that a 
full sepsis work-up is indicated (full blood count, CRP, blood culture, urine, lumbar puncture), then a 
nasopharyngeal or throat swab and research samples will be taken in addition. Research samples will 
include blood, a sample for respiratory secretions (nasopharyngeal aspirate or throat swab) a urine 
sample, and in the case of gastroenteritis, a stool sample. A full description of the research samples 
taken is shown in section 8.c.  
 
We will use a variety of recruitment approaches in order to achieve our aim of maximising the 
comprehensive recruitment of febrile children.  
• 
Where febrile patients are having blood tests as part of their clinically directed assessment, 
we will align research blood sample collection with the same blood draw used for clinically 
indicated tests 
• 
Only in the following situations will we approach patients and their families to consider 
research blood sampling that is not taken simultaneously with clinically indicated tests:  
o Patients with central access lines from which blood can be drawn painlessly, without 
needle puncture 
o Patients in whom antibiotics were prescribed following clinical assessment, but in 
whom no clinically indicated bloods were deemed necessary. It is important for the 
study to include these patients, in order to understand whether blood biomarkers 
might identify what proportion of these patients did actually need antibiotic 
treatment 
• 
We will take written consent and assent from older children and their families prior to 
sample collection when there are personnel from the research team available to speak to 
families, in a non-emergency situation and where the research approach will not disrupt the 
clinical management of the patient. 
• 
In circumstances where the child’s clinical condition necessitates immediate investigation 
and management, we will use a ‘deferred consent approach’, such that blood tests are 
carried out before written consent is obtained, through collection of research samples at the 
same time as the first clinical samples are taken. Parents and children will be approached at 
the earliest opportunity after sample collection to seek consent and assent from the children 
and their families. If no consent is given, all samples taken will be destroyed. 

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Where written informed consent is obtained, our study of biomarkers will be carried out on blood 
left over from the initial clinical sample once all necessary clinical tests have been performed.  
 
 
 
Figure 4.- Schematic of the flow chart of the study 
 

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TIME POINTS  
Patients will be followed up to discharge to ensure collection of all relevant clinical and laboratory 
data. Furthermore, there will be a text/phone/visit 48-72 hours after discharge follow-up and an 
optional 28 days follow-up if the patient remains feverish or illness is on-going. 
The majority of patients in the BIVA-ED study will have a single set of research samples, which will be 
collected at their point of entry to the study. Where children recruited to the BIVA-ED study are 
followed-up in convalescence, and are having clinically-indicated bloods taken, we will request 
permission to take a 2nd set of research samples at the time of follow up (Discharge/Day 28).  
Patients may withdraw from the study at any stage and a withdrawal CRF should be completed. Data 
collected up to the time of their withdrawal from the study will be included in the analysis. Clinical 
samples for those patients who withdraw or do not give deferred consent will be destroyed. 
OUTCOMES 
Primary:  
The validation of clinical, proteomic and transcriptomic biomarkers for diagnosis of febrile illness, 
including markers of bacterial and viral infection (confirmed by culture and/or molecular 
microbiology) and inflammatory conditions.  
Secondary: 
The identification of biomarkers predictive of disease severity in children with bacterial and viral 
infection, using a combination of clinical phenotypic markers and proteomic and transcriptomic 
biomarker signature discovery. 
ANALYSIS 
Sensitivity and specificity of the new biomarkers and the added value of new biomarkers to usual 
clinical and diagnostic work up to discriminate viral and bacterial infections. 
SAMPLE SIZE ESTIMATION 
Using pilot data on gene expression microarrays, we have calculated that transcript biomarkers can 
distinguish bacterial from viral illness with a sensitivity of 95% and a specificity of 95% (Herberg et al. 
JAMA submitted). Assuming that validation will be undertaken using the same platform, we have 
based sample size calculations on the performance (sensitivity and specificity) of the biomarkers in 
the pilot study.  
We aimed to validate the performance of the candidate biomarker, with 95% confidence intervals of 
<5%. Assuming there will be similar biomarker performance in the pilot and validation datasets, a 
sample size of 150 bacterial patients and 150 viral patients would provide 95% confidence intervals 
spanning 3.8% for the point estimate of sensitivity and 3.8% of the point estimate for specificity, 
calculated using the binomial distribution.  

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As SBI is estimated to represent 5% (5-20%)16 of illnesses in the group of febrile children (fever over 
38 ºC), we would need to recruit 3000 febrile children, to study 150 children with SBI. Based on 
studies successfully completed at these sites, we expect to meet the target of 3000 recruited febrile 
children in 36 months, and this is our recruitment target for the BIVA-ED study.  
Additionally, if we assume a prevalence of SBI of 5%, based on pilot data, we would expect a test 
with 95% specificity and sensitivity to have a negative predictive value (NPV) of 99%. In order to 
show with high probability (0.9) that NPV would not be lower than 99%, a total sample size of at 
least 1404 patients is required, with a prevalence of SBI of 0.05, assuming binomial sampling. This 
total sample size we expect to meet and exceed. We will recruit 3000 children in the BIVA-ED study 
as a minimum. Additional recruitment above 3000 children will improve stratified analyses based on 
clinical syndrome and disease severity. 
 
 
 

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2. Protocol for Biomarker Validation in Paediatric Intensive Care (BIVA-PIC) 
This study will recruit children admitted to Intensive Care Units with severe infection. Selected 
centres in the PERFORM consortium will recruit 500 subjects. 
SUMMARY 
We will recruit at least 500 children admitted to Intensive Care Units with fever, suspected or 
confirmed infection. We will also recruit children suffering with healthcare-associated infections and 
non-infectious children (children after operative procedures) to use as an internal control group of 
critically ill children without infection. Written consent from parents will be taken prior to sample 
collection. In an emergency situation we will use deferred consent. Patients and parents will be 
approached at the earliest opportunity. Research samples will be taken immediately after the first 
clinical samples are taken.  
BACKGROUND 
Paediatric Intensive Care Units take care of the most severely ill children in hospital. Due to high 
suspicion or fear of severe bacterial infection in febrile PICU patients, broad spectrum antibiotics are 
given frequently, whilst awaiting the results of a sepsis work-up, as inadequate treatment of sepsis 
further endangers essential organs i.e the kidneys. On the other hand, unneccesary antibiotic 
treatment in febrile children with viral illness can be damaging to patients, for instance causing drug 
reactions, nephrotoxicity and propagating antibiotic resistance.  
The routine sepsis work-up includes full blood count and other blood markers including CRP, blood 
culture, urine, throat swabs and often lumbar puncture to obtain cerebrospinal fluid. We plan to 
conduct an observational study and collect comprehensive information about clinical and laboratory 
data generated during routine care, treatment and follow-up of febrile children and children 
suspected of infection at the PICU. 
These data will be used to validate biomarkers that distinguish severe bacterial infections from other 
life threatening causes of fever and could result in the development of a new diagnostic approach, 
which will reduce the quantity of antibiotic use, preserve organ functions and reduce antibiotic 
resistance.   
OBJECTIVE 
Identify discriminators of bacterial and viral infection in children with a suspected/confirmed severe 
infection presenting with febrile illness.  
To evaluate the diagnostic value of Sepsis-3 Criteria in children compared to existing sepsis scores 
and to develop adjusted Sepsis-3 Criteria based on the needs of a paediatric population. 
STUDY DESIGN 
A prospective observational study of data and sample collection of children admitted to Intensive 
Care Units with fever, suspected or confirmed infection in selected centres in the PERFORM 
consortium 

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STUDY POPULATION 
All febrile children or children with suspected infection aged from birth to 18 years of age admitted 
into the paediatric intensive care units (PICUs) at participating sites. 
Inclusion Criteria 
Any patient, aged birth to 18 years, who requires a blood test for clinical reasons or consents for 
research bloods to be taken, AND who has: 
a) Fever and/or 
b) Suspected viral or bacterial infection and/or 
c) Healthcare-associated infection and/or 
d) Inflammatory disease requiring PICU admission and/or 
e) Immunodeficiency (primary and secondary (steroids, chemotherapy, biological, post-
transplant) and 
f) Already been admitted to intensive care unit and/or  
g) Illness of sufficient severity that would warrant admission to an intensive care unit and 
Informed consent in writing from parent(s) or legally-acceptable representative(s) and, 
informed assent from the subject (if age appropriate according to local requirements) 
h) Patients admitted for elective surgey will act as controls 
Exclusion Criteria  
• 
Patients who do not give consent or withdraw 
STUDY PROCEDURES 
See 8.b ENROLLING AND CONSENTING PATIENTS INTO PROSPECTIVE STUDIES section.  
After written informed consent from parents/guardians and patients, when applicable, data of 
history and physical examination will be collected. Information on degree and duration of fever, 
previous antibiotic use, previous existing co-morbidities, analytic results as well as PICU specific data 
(severity, organ support, outcomes) information will be collected.  
In circumstances where the child’s clinical condition necessitates immediate investigation and 
management, such that blood tests are carried out before written consent is obtained (at sites where 
deferred consent is approved by local ethics boards), parents and children will be approached at the 
earliest opportunity. Where written informed consent is obtained, our study of biomarkers will be 
carried out on blood left over from the initial sample once all necessary clinical tests have been 
performed.  
A 2nd set of research samples (as taken at first presentation) will be taken 48 hours after admission.  
Children will be followed up for at least 28 days after recruitment or until 28 days post admission. If 
reasonable a clinical visit will be preferred, otherwise, the parents will be phoned. Outcome at that 
time will be recorded (readmissions, hospital acquired infection, ongoing disease, mortality). If 

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clinical samples are taken at this point, a 3rd set of research samples (as taken at first presentation) 
will be collected for research. 
Patients may withdraw from the study at any stage and a withdrawal CRF should be completed. Data 
collected up to the time of their withdrawal from the study will be included in the analysis. Clinical 
samples for those patients who withdraw or do not give deferred consent will be destroyed. 
OUTCOMES 
The identification of serious bacterial or viral infection (confirmed by culture and/or molecular 
microbiology), or inflammatory disease. 
ANALYSIS 
We will calculate the sensitivity and specificity of the new biomarkers and the added value of new 
biomarkers to usual diagnostic work up (see case definitions for defined gold standards) to 
discriminate severe viral and bacterial infections in children admitted to Intensive Care Units.17,18  
SAMPLE SIZE ESTIMATION 
The BIVA-PIC study aims to evaluate the performance of our biomarkers in the group of patients that 
need admission to PICU.  Our pilot transcript biomarkers had a sensitivity of 95% and a specificity of 
95% (Herberg et al. JAMA submitted). However, we have based our sample size calculations for BIVA-
PIC on a sensitivity of 90% and a specificity of 90%, to allow for inferior biomarker performance in 
severly ill patients, while assuming that validation will be undertaken using the same platform. We 
aimed to recruit a sample size which would allow validation of the sensitivity and specificity with 
<10% margin of error.  
Basing calculations on the binomial distribution (α=0.05), for a biomarker with sensitivity of 90% and 
a specificity of 90%, we would require a sample size of 70 bacterial patients and 70 non-bacterial 
patients to be able to calculate sensitivity and specificity in the prospective dataset, with 95% 
confidence intervals spanning the point estimate + or - 7%.  
As SBI is estimated to represent 14% of the patients admitted in PICU with disease due to an 
infectious agent  (EUCLIDS data Graz 2015), we would need to recruit 500 febrile children, to study 
70 children with SBI. Based on studies successfully completed at our sites, we expect to meet the 
target of 500 recruited children.  
 
 
 

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3. Protocol for Biomarker Validation in High Risk patients (BIVA-HR) 
This study will recruit children who are immunocompromised. Selected centres in the PERFORM 
consortium will recruit 200 subjects  
SUMMARY 
The PERFORM consortium will recruit at least 200 episodes of febrile children who are 
immunocompromised attending ED or admitted on the appropriate wards, as part of an 
observational study. Patients will have a set of samples taken for research at presentation, at the 
same blood draw alongside their usual clinical tests; a 2nd set of research samples at 48 hours after 
admission and a 3rd set of research samples at least 28 days after recruitment or until 28 days post 
admission if clinical samples are taken at this point. 
We will take an approach to recruitment that maximises the representation of the recruited sample 
to the true population of immunocompromised children presenting with febrile illness. At each 
recruitment site, we will therefore recruit as comprehensively as possible all febrile 
immunocompromised children having bloods taken for clinical reasons, using methods as described 
for BIVA-ED. 
Patients can participate for multiple episodes. Consent will be taken with the first episode, to include 
future episodes with the same underlying condition, for the duration of the PERFORM project. 
ENDPOINTS 
Children will be followed up for 48-72 hours and at least 28 days after recruitment or until 28 days 
post admission. Outcome at that time will be recorded (cure, ongoing disease, mortality). When 
clinical samples will be taken at that point, we will also take a further set of research samples, as at 
enrollment. 
BACKGROUND 
Immunocompromised children (either primary or secondary) are at increased risk of infection with a 
variety of pathogens. These require a more vigilant approach than otherwise healthy individuals. 
Presenting symptoms can be minor and atypical. This results in frequent use of broad spectrum 
antibiotics and/or antifungals. Also antivirals are used, particularly in primary immunodeficient 
patients requiring haematopoietic stem cell transplant. While providing broad cover to treat 
potential infection, these drugs have toxic side effects. Challenges in diagnostics in this group include 
the use of prophylactic antibiotics making it difficult to identify a causative pathogen using 
conventional diagnostics. As these patients are more likely to experience several episodes with fever 
and to receive antimicrobial therapy, they are at increased risk of developing antimicrobial-resistant 
micro-organisms. Improved targeting of antimicrobial therapy might help prevent this. However, 
development of diagnostic biomarkers in this group is made more difficult by the changing dynamics 
in the immune system at different stages of treatment. 
 
 

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OBJECTIVE 
Evaluation and validation of the diagnostic biomarkers in diagnostically challenging groups of 
immunocompromised patients with fever  
Development of a management algorithm for febrile children including use of the novel diagnostic 
marker and evaluation of its performance in the settings of different European countries. 
STUDY DESIGN 
A prospective observational study of data and sample collection of immunocompromised in selected 
centres in the PERFORM consortium 
STUDY POPULATION 
All febrile immunocompromised children from birth to 18 years of age presenting to Emergency 
Departments or admitted to appropriate wards at participating sites. 
Inclusion Criteria 
All immunocompromised children from birth to 18 years presenting to the Emergency department or 
admitted in other appropriate wards or ICU, with fever, or a history of fever, or suspected of infection 
in whom the attending clinician determines the need for blood sampling or consent for research 
bloods to be taken has been achieved.  
Exclusion Criteria  
• 
Patients who withdraw or do not give consent 
STUDY PROCEDURES 
See 8.b ENROLLING AND CONSENTING PATIENTS INTO PROSPECTIVE STUDIES section.  
After written informed parental consent, a complete history and physical examination will be 
performed. Information on degree and duration of fever, previous antibiotic use and previous 
existing co-morbidities will be collected. If the attending paediatrician decides that a full sepsis work-
up is indicated (full blood count, CRP, blood culture, urine), then a nasopharyngeal swab/throat 
swab, urine and blood sample for other markers of SBI will also be performed.  
In circumstances where the child’s clinical condition necessitates immediate investigation and 
management, such that blood tests are carried out before written consent is obtained, parents and 
children will be approached at the earliest opportunity. Where written informed consent is obtained, 
our study of biomarkers will be carried out on blood left over from the initial sample once all 
necessary clinical tests have been performed.  
Patients will be followed up to discharge to ensure collection of all relevant clinical and laboratory 
data. 

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Patients may withdraw from the study at any stage and a withdrawal CRF should be completed. Data 
collected up to the time of their withdrawal from the study will be included in the analysis. Clinical 
samples for those patients who withdraw or do not give deferred consent will be destroyed. 
OUTCOMES 
The identification of serious bacterial or viral infection (confirmed by culture and/or molecular 
microbiology). 
ANALYSIS 
Sensitivity and specificity of the new biomarkers and the added value of new biomarkers to usual 
clinical signs and diagnostic work up to discriminate viral and bacterial infections in diagnostically 
challenging groups of immunocompromised patients with fever  
SAMPLE SIZE ESTIMATION  
The BIVA-ΗR study aims to evaluate the performance of our biomarkers in the challenging group of 
immunocompromised patients. Our pilot transcript biomarkers have a sensitivity of 95% and a 
specificity of 95% for detection of bacterial infection (Herberg et al. JAMA submitted). However, we 
have based the sample size calculations for BIVA-HR on sensitivity of 90% and specificity of 90% to 
allow for inferior biomarker performance in immunocompromised patients, while assuming that 
validation will be undertaken using the same platform. We have considered the feasibility to recruit 
large numbers of patients in this hard-to-reach group, based on the number of children seen by the 
consortium members, and have considered the number needed for successful validation of 
sensitivity and specificity. Based on studies successfully completed at our sites, we expect to meet 
the target of 200 recruited children in 36 months.  
As SBI is estimated to represent ~15% (literature reports 9-18%)19-22 of febrile immunocompromised 
patients, therefore with 200 febrile children recruited, we would expect to study 30 children with 
SBI. Basing calculations on the binomial distribution (α=0.05), for a biomarker with sensitivity of 90% 
and a specificity of 90%, with a sample size of 30 bacterial patients and 30 non-bacterial patients, we 
would be able to calculate sensitivity and specificity in the prospective dataset, with 95% confidence 
intervals spanning the point estimate + or - 11%.  
 
 

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4. Protocol for Biomarker Validation in Inflammatory patients (BIVA-INF) 
This study will recruit children who have inflammatory/rheumatological disorders that mimic 
infection. Selected centres within the PERFORM consortium as well as in centres of collaborating 
partners (IKDGC and UCAN) who have agreed to share data and materials will recruit 200 subjects. 
SUMMARY 
The PERFORM consortium will recruit at least 200 episodes of children who have a treatment-naïve 
presentation of inflammatory/rheumatological disorders attending ED or admitted on the 
appropriate wards, as part of an observational and/or local treatment study protocol. Patients will 
have a set of samples taken for research at presentation, at the same blood draw alongside their 
usual clinical tests; a 2nd set of research samples at 48 hours after admission and a 3rd set of research 
samples at least 28 days after recruitment or until 28 days post admission if clinical samples are 
taken at this point. Since some will have fever during admission and others a history of febrile 
episodes, the number of true febrile cases will be about 25%. The inflammatory markers and 
signatures will be required to consolidate the inflammatory signatures within the cohort (including 
children diagnosed with Kawasaki disease, systemic JIA or treatment-naïve JIA).  
 
We will take an approach to recruitment that maximises the representation of the recruited sample 
to the true population of inflammatory/rheumatological disorders in children presenting with febrile 
illness, using methods as described for BIVA-ED. 
Patients can participate for multiple episodes. Consent will be taken with the first episode, to include 
future episodes with the same underlying condition, for the duration of the PERFORM project. 
ENDPOINTS 
Children will be followed up for 48-72 hours and at least 28 days after recruitment or until 28 days 
post admission for the PERFORM study (telephone/text/visit if discharged). Outcome at that time 
will be recorded (cure, ongoing disease, mortality). When clinical samples will be taken at that point, 
we will also take samples for research. 
BACKGROUND 
Inflammatory/rheumatological disorders are a great mimic of infection. Several clinical and 
laboratory features are common to both inflammatory/rheumatological disorders and infection. 
Constitutional symptoms such as fever, malaise, arthralgia, myalgia and weight loss, and laboratory 
features such as normocytic normochromic anaemia, peripheral blood leucocytosis, thrombocytosis, 
raised erthyrocyte sedimentation rate (ESR) and C reactive protein (CRP) are encountered in both. 
Some patients, especially early in the course of their illness, present with isolated systemic 
manifestations posing a diagnostic challenge. Conversely, systemic inflammatory response is not 
seen in most patients with localised forms of inflammatory/rheumatological disorders. 
The treatment of inflammatory/rheumatological disorders entails the use of immunosuppressive 
drugs, and the consequences of not recognising infection can be disastrous. Thus, it is mandatory to 
perform 
a 
full 
and 
appropriate 
infection 
screen 
in 
all 
patients 
with 
suspected 

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inflammatory/rheumatological disorders especially those who present with systemic inflammatory 
features.  
OBJECTIVE 
Evaluation and validation of the diagnostic biomarkers in diagnostically challenging groups of 
patients with inflammatory/rheumatological disorders presenting with fever  
STUDY DESIGN 
A prospective observational study of data and sample collection of patients with 
inflammatory/rheumatological disorders in selected centres in the PERFORM consortium 
 STUDY POPULATION 
All febrile children from birth to 18 years of age with inflammatory/rheumatological disorders 
presenting to Emergency Departments or admitted on appropriate wards at participating sites. 
Inclusion Criteria 
All children with (or suspicion of) inflammatory/rheumatological disorders from birth to 18 years 
presenting to the Emergency department or admitted in other appropriate wards with fever, or a 
history of fever, in whom the attending clinician determines the need for blood sampling because of 
disease diagnosed or consent for research bloods to be taken has been achieved. 
Exclusion Criteria  
• 
Patients who withdraw or do not give consent 
STUDY PROCEDURES 
See 8.2 ENROLLING AND CONSENTING PATIENTS INTO PROSPECTIVE STUDIES section.  
After written informed parental consent, a complete history and physical examination will be 
performed. Information on degree and duration of fever, previous antibiotic use and previous 
existing co-morbidities will be collected. If the attending paediatrician decides that a full sepsis work-
up is indicated (full blood count, CRP, blood culture, urine), then a nasopharyngeal/throat swab, 
urine and blood sample for other markers of SBI will also be performed.  
In circumstances where the child’s clinical condition necessitates immediate investigation and 
management, such that blood tests are carried out before written consent is obtained, parents and 
children will be approached at the earliest opportunity. Where written informed consent is obtained, 
our study of biomarkers will be carried out on blood left over from the initial sample once all 
necessary clinical tests have been performed.  
Patients will be followed up to discharge to ensure collection of all relevant clinical and laboratory 
data. 

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Patients may withdraw from the study at any stage and a withdrawal CRF should be completed. Data 
collected up to the time of their withdrawal from the study will be included in the analysis. Clinical 
samples for those patients who withdraw or do not give deferred consent will be destroyed. 
OUTCOMES 
The identification of inflammatory syndromes, consistent with international agreed definitions. 
ANALYSIS 
Sensitivity and specificity of the new biomarkers and the added value of new biomarkers to usual 
clinical signs and diagnostic work up to discriminate severe viral and bacterial infections in children 
admitted with inflammatory/rheumatological disorders presenting with fever. 
SAMPLE SIZE ESTIMATION 
The BIVA-INF study aims to evaluate the performance of our biomarkers in patients with a range of 
inflammatory/rheumatological disorders. Our pilot transcript biomarkers have a sensitivity of 90% 
and specificity for discrimination of bacterial infection from JIA and Henoch Schonlein Purpura, and 
94% sensitivity and specificity for discrimination of bacterial infection and Systemic Lupus 
Erythematosus (using adjusted thresholds). We will carry out a case-control study, using targeted 
recruitment of children referred to tertiary rheumatological centres.  
We have based the sample size calculations for BIVA-INF on the lower estimates of sensitivity and 
specificity (90%), whilst assuming that validation will be undertaken using the same platform. We 
have considered the feasibility to recruit large numbers of patients with inflammatory conditions, 
based on the number of children seen by the consortium members, and have considered the number 
needed for successful validation of sensitivity and specificity. We aim to recruit 150 recruited 
children in 36 months, to match the 150 children with bacterial infection recruited in the BIVA-ED 
study.  
Basing calculations on the binomial distribution (α=0.05), for a biomarker with sensitivity of 90% and 
a specificity of 90%, with a sample size of 150 bacterial patients and 150 inflammatory patients, we 
would be able to calculate sensitivity and specificity in the prospective dataset, with 95% confidence 
intervals spanning the point estimate + or - 5%.  
 
 

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f) STUDY DATA BIVA studies 
 
a. CLINICAL RECORD FORMS AND DATA COLLECTION  
A unique PERFORM study number will be given to all patients included in the study. The study 
number and date of birth will be used to identify patients. 
A patient identification log will be kept in the site file to allow future location of the patient. This 
information will be only accessible to the local clinical research team: 
• 
Hospital number  
• 
Patient name 
• 
Date of birth 
• 
Date of consent 
• 
PERFORM study number 
All necessary documents (informed consent forms, paper clinical record forms, protocol, etc) will be 
available with local R&D approval. A paper copy of the clinical record form will be used to collect 
prospective data. The fields will match those on the database.  
 
b. INTERNET DATABASE 
A web-based database (Redcap) will be developed that incorporates EU security requirements for 
data protection and storage.  
Different levels of access will be established with personal login/password. The responsibilities for 
different personnel with respect to the database are as follows; 
The Principal investigator of each node must sign off each recruit with respect to data entry including 
phenotype.  
The Principal investigator and subinvestigator will also be able to see/modify/ his/her locally 
recruited patients and export their own anonymised existing dataset into a different programme 
(such as Excel) 
The Coordinator will be allowed to see / modify / export all anonymised information from the node 
and will also be allowed to open/close the patient records and to generate queries regarding patient 
data. 
The data entry (study nurses/datamangers) will also be able to see/modify/ and enter data for 
his/her locally recruited patients. 
Administrator: allowed to see and modify all, generate queries, export anonymised global data 
Study User: allowed to see all, generate queries, export anonymised global data 

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Pop-up menus with the definitions for each of the items requested in the electronic case report form 
will be available.   
7. IMPLEMENTATION 
The implementation stage of the study will run concurrently with the recruitment of the validation 
cohort although it is anticipated that implementation will extend beyond the recruitment stage. The 
potential healthcare benefits of the RNA signatures and protein biomarkers will be identified and 
their role and effect on patient care will be evaluated.  
The guidelines developed by the EMA on clinical evaluation of diagnostic agents www.ema.europa.eu 
and the draft Guidance for industry FDA staff and clinical laboratories: Framework for Regulatory 
Oversight of Laboratory Developed Tests (LDTs) www.fda.gov will be followed for the implementation 
of diagnostic tests and clinical guidelines. 
Public health management (including a cost effectiveness analysis) and the effect of introduction of 
the new diagnostic approach on healthcare in the diverse healthcare setting of Europe will also be 
evaluated.  
Industrial and biotechnology partners will be engaged to develop a clinically applicable prototype 
test and model its effect on healthcare, to provide information to national and international agencies 
on roll out of an improved model of healthcare for febrile children across Europe.  
 
 

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8. REGULATORY AND ADMINISTRATIVE ISSUES 
a. 
ETHICAL APPROVAL 
The Chief Investigator will obtain approval from the National Research Ethics Committee (NRES).  
Each participating site will obtain written local Trust approval (SSA), a copy of which will be sent to 
the Chief Investigator before accepting participants into the study.  The study will be conducted in 
accordance with the recommendations for physicians involved in research on human subjects 
adopted by the 18th World Medical Assembly, Helsinki 1964 and later revisions. 
Other sites from outside the UK (including West Africa) will obtain their own national ethical 
approval and local site agreements to implement the study protocol. 
b.  
ENROLLING AND CONSENT 
Patients will be enrolled from the Emergency Department, hospital wards and paediatric intensive 
care if they fulfil the study inclusion criteria for the biomarker group.  
Consent for children to enter any of the biomarker studies will be sought from parents or guardians 
only after a full explanation has been given, an information sheet offered and time allowed for 
consideration and to make questions. Informed consent can be obtained by any member of the 
clinical research team. 
Signed parental (or guardian) and (where appropriate) patient consent will be obtained for all 
children for agreement to take part in the study.  The right of the participant and their parent/legal 
guardian to refuse to participate without giving reasons will be respected.  
Once the child's parents or guardians have been informed of the study they will have time to discuss 
with each other and ask the research team any questions about the research. After having adequate 
time to consider the study and they are happy to take part, written documented consent will be 
obtained. A copy of the signed informed consent form will be given to parents/legal guardian.  
 
We declare that: 
1. 
Physicians, researchers and supporting staff involved in the research have the knowledge 
and skills to perform this research;  
2. 
All staff contributing to this research have received sufficient information about the 
study;  
3. 
The infrastructure at the 12 hospitals is suitable to perform the research adequately;  
4. 
No other research projects will be hindered by this research; and     
5. 
Enough patients can be included in the study 
 
a) DEFERRED CONSENT 
For optimal respect of patient’s autonomy, seeking consent before study participation is preferable. 
However, in emergency research this is often not possible. Emergency research represents an 
exceptional situation in which the mechanisms of the consent process may need to be modified, but 
the social contract between researcher and research subject must be respected in order to provide a 
safeguard against unethical research. If data obtained in emergency situations without consent 

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cannot be used, and selection bias is thus introduced, study results may be less robust and future 
patients may not benefit. While this premise cannot provide an argument for including data when 
research subjects expressly deny consent, it does make an ethically valid case for including samples 
or data collected before consent has been taken, and where such explicit denial of consent does not 
exist. 
There is a lack of significant evidence in many aspects of PICU and Emergency care management, due 
to paucity of randomised controlled trials in this setting. This means that the sickest children are 
subjected to evolved, anecdotal medical practice. It is essential that new research and therapeutic 
strategies are based on the most robust scientific understanding of critical illness, which is what our 
study aims to advance. In particular, our study uses state-of-the-art techniques to identify the 
pathogens causing critical illness. Without study samples taken before or close to the time of 
antibiotic treatment, pathogen identification is made difficult.  
Our study also aims to define potential biomarkers associated with critical illness, and deriving a 
robust marker of critical illness requires that samples are collected before treatments (such as fluids, 
inotropes) are initiated which may affect the results. We aim to recruit patients as close to 
presentation as possible. As the blood sample volumes in this study are small and would not 
constitute a harmful intervention, we will implement a system of deferred consent in emergency 
admissions if parents are not initially present, or a critical situation makes discussion of research 
inappropriate. If parents do not agree to participate they only have to indicate it to the personnel of 
the study. In the same way if parents require additional information, this will be provided by research 
team and as consequence informed consent will be required. This is an approach which has 
been used successfully in several other UK observational and interventional studies of childhood 
critical illness in Emergency Departments and in paediatric intensive care units. This process has 
been successful in increasing the number of recruits.  
We expect that our deferred consent approach will improve the representation of the full range of 
febrile illnesses in the study, and thus allow us to carry out a more meaningful validation of our 
bacterial and viral diagnostic biomarkers, discovered in the BIVABI study.  
Refer to the findings from the CONNECT study:  
https://www.liverpool.ac.uk/media/livacuk/instituteofpsychology/Research,without,prior,consent,in,
trials,investigating,the,emergency,treatment,of,critically,ill,children,CONNECT,guidance,July,2015.pdf 
Research or clinical staff trained in the project will collect baseline samples alongside clinical 
samples. No data or further sample would be collected until consent has been obtained. Parents or 
guardians will be approached for consent at the earliest appropriate opportunity during their 
hospital admission. If consent is given the child would be enrolled in the study, given a study number 
and subsequent research samples and data will be collected. If parents/guardians do not wish or are 
unable to consent, or study staff is unable to make contact with parents/guardians, the samples will 
be destroyed. Centres will take consent according to their country’s legislation. 
b) WAIVER OF INFORMED CONSENT FOR MOFICHE STUDY 
We believe that the rules laid down in the Medical Research Involving Human Subjects Act do not 
apply to this research proposal because the research does not involve any analysis, intervention, 
strategy or any other act, beside routine care. 

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The MOFICHE study is an international observational study. It involves the creation of a large, 
multicenter database with routinely collected electronic patient information. During the study, 
patients will not undergo any intervention beside routine care at the ED. Data will be shared with the 
scientific coordinator of the project. The database will be pseudo-anonymised (no personal 
information of the patients is available beside a unique identifier code), but not fully anonymised, in 
order that we can identify patients that make repeated ED visits, and match patients in MOFICHE 
with those in other studies. The data in MOFICHE will be transferred through an encrypted file to 
ensure safety.  
 
c) INFORMED CONSENT FOR FUTURE RESEARCH  
The PERFORM study will use samples obtained in previous ethically approved studies or 
prospectively in the PERFORM study. Samples from earlier studies will be used only if written 
consent for future use of samples was obtained at the time of patient recruitment into previous 
studies.  
We will request written consent for future research use of any leftover research samples for children 
prospectively recruited into PERFORM. All future such research will be approved by an ethics 
committee. 
Standards of operating practices (SOPs) for sample and data management will be used to both 
safeguard subject’s identity and to ensure samples are uniformally collected, processed and stored. 
c. SAMPLE TYPES 
Children taking part in the study will have the best available diagnostic assessment through a 
combination of clinical diagnostic laboratory tests and research tests. Tests performed in the clinical 
laboratory will be targeted towards the presenting syndrome, for instance, children with suspected 
urinary tract infection will have urine culture, children with lower respiratory tract infections will 
undergo chest radiographs. All febrile children included in the study will have blood cultures and 
inflammatory markers (including full blood count and CRP) performed by the clinical diagnostic 
laboratories. In addition we aim to collect the following research sample set on patients recruited to 
the biomarker validation studies:  
• 
Blood culture 
• 
Blood for RNA collected in RNA-preserving medium (1.0ml-2.5ml depending on body weight) 
• 
EDTA blood, which will be divided into sub-aliquots as follows (1.0ml-2.5ml on body weight):  
o  microbiological work-up using molecular approaches (whole blood) to identify 
bacterial and viral pathogens in blood (200µl aliquot) 
o disease biomarker studies based on host markers including proteins (plasma) (100µl 
aliquots after centrifugation) 
o correlation of clinical syndrome with host genetic markers using host DNA (extracted 
from the cell pellet) 
• 
non-invasive sample for respiratory secretions (nasopharyngeal or throat swab) 

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• 
urine sample for host metabolomic biomarker studies (using left-over of clinical samples 
where possible) 
• 
stool sample for pathogen detection (using left-over of clinical samples where possible) 
 
d. BLOOD SAMPLING VOLUME RESTRICTIONS 
 Blood sampling volume restrictions will comply with the recommendations of the European Union 
for the blood loss associated with pediatric research (European Commission, 2008) and the World 
Health Organization guidelines "Blood Sample Volumes in Child Health Research: Review of Safe 
Limits" 23. 
Volumes will be calculated based on patient’s weight and only term neonates will be suitable for 
recruitment as follows 23: 
 
Body weight (in kg) 
Volume of blood allowed for extraction 
2.4 - ≤ 3.7 
2ml 
3.7 - ≤ 4.9 
3ml 
4.9 - ≤ 6.2 
4ml 
>6.2 
5ml 
 
 
e. CONFIDENTIALITY 
The Principal Investigator will preserve the confidentiality of participants taking part in the study and 
will follow the Data Protection Act 1998. 
PERFORM project will respect the subjects’ rights to privacy and will ensure the confidentiality of 
their medical information in accordance with all applicable laws and regulations. The Declaration of 
Helsinki and the convention of the Council of Europe on Human Rights and Biomedicine, the ICH-GCP 
guidelines and national guidelines will be carefully respected.  
All personal data and individual clinical information will be retained within the standard information 
system, and all analysis of clinical and outcome data will be conducted using codified identifiers and 
without reference to individual patient information. The identified code list of each project will be 
retained in the research centre with limited access to researchers involved in the project. Once each 
project has ended that codification list will be safeguarded by the principal investigator during legally 
required time.  
All of the databases utilised by the study will be password protected and encrypted in conformance 
with current national and EU standards for data handling. 
The confidentiality of records that could identify subjects will be protected at all times. To ensure the 
confidentiality of records they will be kept in secure storage areas.  
All analysis performed on patient samples will be conducted on codified samples, identifiable only by 
a study number, and with all source data and clinical records held separately. 

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f. 
INDEMNITY 
Imperial College London holds insurance policies which apply to the design and management of the 
study.  The NHS indemnity scheme and professional indemnity will apply to the conduct of the study. 
g. 
SPONSOR 
Imperial College London will act as the main sponsor for this study.  
h. 
FUNDING 
European Commission (H2020) 
Grant Number- 668303 Personalised Risk assessment in Febrile illness to Optimise Real-life 
Management across the European Union (PERFORM) 
i. 
DATA PROTECTION, HANDLING AND RETENTION 
i. 
Data protection 
Genetic analysis of patient samples will be conducted on pseudo-anonymised samples, identifiable 
only by a study number and with all source data and clinical records held separately. The link 
between patient and samples will only be known to the local study data management team. All 
personal data and individual clinical information will be retained within the standard information 
systems of each participating hospital, all analysis of clinical and outcome data will be conducted 
using subject codes and without reference to individual patient information. All the partner 
institutions will follow their own established procedures for data protection and this study will 
conform to the national and European standards required for each partner institution. 
 
ii. 
Data handling 
All databases utilised by the study will be password protected, encrypted and conform with current 
national and EU standards for data handling. Imperial College is the co-ordinating centre for data 
handling and all data will be handled in keeping with Imperial College’s Data protection policy and 
codes of practice. The College Data Protection Officer has been informed about the data and 
measures to ensure that confidentiality of electronic data is maintained. 
iii.  
Data retention 
Data and all appropriate documentation will be stored for a minimum of 10 years after the 
completion of the study 
 
j. 
AUDITS 
The Chief Investigator of the study will delegate recruitment at other sites in the UK to local principal 
investigators. This may be delegated further at local sites to research nurses, data managers and sub-
investigators. Any delegation will be recorded on delegation logs. It will be the responsibility of local 
PI’s to ensure individuals are trained adequately. In addition to the protocol, standard operating 

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procedures will be created for specific tasks including sample collection, processing and shipment. 
Source documents (hospital record or Electronic Patient Record) will be kept at local sites as 
described previously. Data input will be monitored at each site and globally between sites to ensure 
accuracy. Documented audits will be performed regularly by a member of the research team. 
In addition, The Joint Research Office, part of Imperial Academic Health Science Centre, audits the 
studies it sponsors ensure adherence to GCP and compliance with the NHS Research Governance 
Framework for Health and Social Care (2nd edition).  
10. STUDY MANAGEMENT  
The day-to-day management of the study will be co-ordinated through the Chief Investigator. Local 
issues will be managed by PIs. 
11.  PUBLICATION POLICY 
All publications and presentations relating to the study will be authorised by the Chief Investigator 
and according to the publication policy of Imperial College and of the other universities involved. If 
there are named authors, these will include at least the trial’s Chief Investigator and contributors will 
be cited by name if published in a journal where this does not conflict with the journal’s policy. 
Authorship of parallel studies initiated outside of the main research team will be according to the 
individuals involved in the project but must acknowledge the contribution of any of the main study 
researchers involved. 
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23. Howie SRC. Blood Sample Volumes in Child Health Research: Review of Safe Limits. Bull World 
Health Organ 2011;89:46–53  

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APPENDIX 1A – MOFICHE and common information for biomarker studies CRF  
*** Green only apply for BIVA-ED and other biomarker studies 
Signed consent/assent Date___/___/___ Time: __:__ 
Number of parents signed: ___ 
 
 
Study samples  
- PAXgene RNA volume: ...ml 
      1st sample  Date of collection  ___/___/___Time: __:__    Tª:__ºC 
2nd sample Date of collection  ___/___/___Time: __:__    Tª:__ºC 
3rd sample  Date of collection  ___/___/___Time: __:__    Tª:__ºC 
 
- EDTA (volume: ...ml)   
 
      1st sample  Date of collection  ___/___/___Time: __:__    Tª:__ºC 
 finger prick   
      2nd sample Date of collection  ___/___/___Time: __:__    Tª:__ºC 
3rd sample  Date of collection  ___/___/___Time: __:__    Tª:__ºC 
 
- Nasopharyngeal/throat swab 
      1st sample   Date of collection  ___/___/___Time: __:__    Tª:__ºC 
2nd sample Date of collection  ___/___/___Time: __:__    Tª:__ºC 
3rd sample  Date of collection  ___/___/___Time: __:__    Tª:__ºC 
 
- Urine  
 
 
      1st sample   Date of collection  ___/___/___Time:__:__     Tª:__ºC 
2nd sample Date of collection  ___/___/___Time: __:__    Tª:__ºC 
3rd sample  Date of collection  ___/___/___Time: __:__    Tª:__ºC 
 
- Stools  
 
 
      1st sample   Date of collection  ___/___/___Time:__:__     Tª:__ºC 
2nd sample Date of collection  ___/___/___Time: __:__    Tª:__ºC 
 
General questions 
• Code of hospital, name and country* (automatically inserted)  
• Subject number and episode number. 
• Date of registration ddmmyyyy, automatically inserted 
• Date of visit ddmmyyyy 
• Sought medical/nurse care in previous 48 hours  no/yes/unknown-not stated  
• Date of previous ED visit for the same complaint in the previous 5 days no visit/[date]/unknown-
not stated  
• Gender child [required field] male/female 
• Date of birth [required field] ddmmyyyy 
• Referral GP/self/emergency medical service/other hospital or specialist/ unknown-not stated, if 
other, please specify 
• Comorbidity**none/cardiac/pulmonary/immunodeficiency/malignancy/neurologic/psychomot
or retardation/prematurity [gestational age]/unknown or not stated/other.  
Please specify comorbidity when present……….**Comorbidity as stated in the ED charts or previous history form . 
Defined as a chronic underlying condition that is expected to last at least 1 year. Adapted from Simon et al., Pediatrics, 2014. 
• Triage code immediate, very urgent, urgent, standard, non-urgent/unknown-not stated 
 
*SMH, London, UK 
LIV, Liverpool, UK 
UNEW, Newcastle, UK 
EMC, Rotterdam, the Netherlands 
NKUA, Athens, Greece 
RUMC, Nijmegen, the Netherlands 
SERGAS, Santiago de Compostela, Spain 
MUG, Graz, Austria 
RSU, Riga, Latvia 
AMC, Amsterdam, the Netherlands 
LMU, München, Germany 
UKCL, Ljubljana, Slovenia 

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 Specific disease questions (history based) 
• Antibiotic use in the last 7 days? no/yes/unknown-not stated 
• Duration of fever: < 24 hours, 24-48, longer than 48 hours [specify in hours/days] unknown-not 
stated 
• Duration of fever if less then 48 hours:…..hours:  
• Parental concern no/yes/unknown-not stated 
• Rigors no/yes/unknown-not stated 
• Myalgia no/yes/unknown-not stated 
• Pallor according to caregiver no/yes/unknown-not stated 
• Decreased intake no/yes/unknown-not stated 
• Wheeze no/yes/unknown-not stated 
• Cough no/yes/unknown-not stated 
• Difficulty breathing no/yes/unknown-not stated 
• Sore throat no/yes/unknown-not stated 
• Ear ache no/yes/unknown-not stated 
• Chest pain no/yes/unknown-not stated 
• Abdominal pain no/yes/unknown-not stated 
• Vomiting in the last 48 hours no/yes/unknown-not stated 
• Diarrhea in the last 48 hours no/yes/unknown-not stated 
• Dysuria no/yes/unknown-not stated 
• Seizures no/yes/focal/status epilepticus/unknown-not stated 
• Headache no/yes/unknown-not stated  
• Drowsiness no/yes/unknown-not stated 
• Irritability/inconsolable crying no/yes/unknown-not stated 
• Rash no/yes/unknown-not stated 
• Painful or swollen joints/extremities/not using an extremity no/yes/unknown-not stated 
• Other no/yes/unknown-not stated 
 
Vital signs (please fill in the first value that was measured). 
• Heart rate [value] per minute/unknown-not stated/recorded as normal, no number available    
• Respiratory rate [value] per minute/unknown-not stated/recorded as normal, no number 
available.   
• Cutaneous saturation [value] in %/unknown-not stated/recorded as normal, no number 
available) in room air/with oxygen supplied 
• Systolic blood pressure [value] in mm Hg/Unknown-not stated/recorded as normal, no number 
available 
• Body temperature [value] in °C/ unknown-not stated/recorded as normal, no number available  
Method: oral / rectal / tympanic / axillary / temporal 
 
Physical examination 
• Ill appearance no/yes/unknown-not stated 
• Color of skin, lips or tongue normal/cyanotic/pale/mottled/ashen 

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• Signs of dehydration no / yes (definition : dry mucous membranes /sunken eyes / reduced skin 
turgor , no tears) 
• Increased work of breathing defined as presence of  
o chest wall retractions no/mild / severe /unknown-not stated 
o nasal flaring no/ mild/ severe /unknown-not stated 
o grunting no/ mild/ severe /unknown-not stated 
o apnoea no/yes/unknown-not stated 
• Air entry normal / abnormal/unknown-not stated 
• Breath sounds normal / stridor / crackles / wheeze /unknown-not stated 
• Location of abnormal breath sounds RLL / RUL / LLL / LUL/generalised/ unknown-not state  
• Abnormal ENT signs no/yes/unknown-not stated 
• Peripheral capillary refill normal = <3 sec/prolonged>3sec/unknown-not stated 
• Oedema no / yes/unknown-not stated 
• Heart sounds normal / murmur / other/unknown-not stated 
• Abdominal pain no / yes if yes: RUQ, LUQ, RLQ, LLQ. /unknown-not stated 
• If yes, pain score ….. Used score system NIPS / FLACC / VAS/unknown-not stated 
• Liver enlarged no/yes [cm below RCM] /unknown-not stated 
• Spleen enlarged no/yes [cm below LCM] /unknown-not stated 
• Jaundice no / yes/unknown-not stated 
• Meningeal signs (defined as presence of Kernig, Brudzinski, tripod phenomenon, neck stiffness, 
bulging fontanelle for <1 year) no/yes/unknown-not stated 
• Focal neurological signs no/yes/unknown-not stated  
• Pupils PERL/abnormal, if abnormal please describe/unknown-not stated 
• Fundoscopy not performed / normal/abnormal / Blurred disc margins / Papilloedema | 
Haemorrhage / Not visualised/unknown-not stated 
• Consciousness A=alert, R=responsive to verbal stimulation, P=responsive to pain, 
U=unresponsive / unknown-not stated 
• Activity level 
o normal/not responding to normal social clues/no response to social clues 
o smiles / no smile / appears ill to a healthcare professional 
o Stays awake or awakens quickly / wakes only with prolonged stimulation / does not 
wake or if roused  does not stay awake 
o Strong normal cry or not crying / decreased activity / weak, high-pitched or 
continuous cry 
• Rash none/petechiae/other/unknown-not stated  
• If other: purpuric/other non-blanching/maculopapular/pustulous/desquamation/ 
itching/mucosal laesion/strawberry tongue/red lips/conjuncitivitis/involvement of hands or feet 
(target lesions, erythema palmare-plantare)/other 
•  Lymphadenopathy no / yes, if yes: cervical / submandibular / axillary / inguinal / epitrochlear, 
nuchal. Largest size of involved lymph nodes: …. Cm/unknown-not stated  
• Painful joints [number] /unknown-not stated 
• Swollen joints [number] /unknown-not stated 
 

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Diagnostics  In the final form local units for all lab tests will be pre-filled in. Only when performed at this ED visit. 
Please fill in the results if they become available within a few days after presentation. 
• 
CRP performed no/yes [value]/unknown-not stated 
• 
White bloodcell count performed no/yes [value]/unknown-not stated 
• 
Absolute neutrofile count: no/yes [value]/unknown-not stated 
• 
PCT performed no/yes [value]/unknown-not stated 
• 
Chest X-ray performed no/yes: normal/local infiltrate/diffuse abnormalities /other:  
unknown-not stated 
• 
Urinalysis performed no/yes: normal/no leucoyturia or nitrite/leucocyturia positive/nitrite 
positive/ leucocyturia  and nitrite positive/unknown-not stated  
• 
Urine culture performed no/yes [value]/unknown-not stated 
• 
Stools culture/rapid viral Ag test performed no/yes [value]/unknown-not stated.  
• 
Blood culture performed no/yes [value]/unknown-not stated 
• 
Rapid Strep Test no/yes: positive/negative/unknown-not stated 
• 
Lumbar puncture performed no/yes unknown-not stated: white blood cell count 
[value]/unknown-not stated glucose level [value]/unknown-not stated /protein level 
[value]/unknown-not stated culture [value]/unknown-not stated 
• 
Nasopharyngeal resp viral tests performed no/RSV/influenza/other/negative /unknown-not 
stated  
 
 
 
 
Treatment 
• 
Antibiotics prescribed at this ED visit or first day of admission no/yes/unknown-not stated 
o If yes, type/generic name: .......................................... 
o If yes, duration 1,2,3,4,5,6,7,10,14, other ………days  
o Prescription mode none /oral/ intravenous/intramuscular/topical/unknown-not stated   
• 
Time and date first antibiotic dose given [24 hour clock]  
• 
Oxygen therapy: no/yes/unknown-not stated 
• 
Inhaled medication: none/ ipratropium/salbutamol or albuterol/epinefrine/budesonide/ 
unknown-not stated  
 
Immediate lifesaving interventions: no/yes/unknown-not stated. If yes: 
1. 
Airway/breathing support: 
- non rebreathing mask no/yes/unknown-not stated  
- intubation/ventilation. no/yes/unknown-not stated 
2. Haemodynamic support (significant iv fluid > 20ml/kg) no/yes/unknown-not stated 
if yes please specify for first 6 hours: normal saline/albumin/gelofusin/blood/platelets/fresh 
frozen plasma 
3. 
Other emergent procedures or medication no/yes/unknown-not stated like 
       Electrical therapy (defibrillation)  
       Emergency procedures (e.g. chest needle decompression, pericardiocentesis, or open 
thoracotomy)   
       Emergency medications: (atropine, adenosine, inotropics, epinephrine, nalaxone, 
dextrose in case of hypoglycaemia) /other 
 

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Evolution 
• 
Destination: discharged home with out-patient clinic follow-up/ discharged home with out-
patient clinic telephone follow-up/ discharged home with follow-up by GP/ discharged home 
without follow-up / discharged home with follow-up unknown/ admission <24h/ admission 
to ward / admission to ICU/ dead in ED/unknown-not stated. 
• 
Time spent at the ED* (total duration): < 1 hour/ 1-2 hours /2-3 hours /3-4 hours />4 hours 
• 
Date of hospital admission [date]/unknown-not stated 
• 
Date of hospital discharge [date] /unknown-not stated 
• 
Duration of hospital stay in days ……/unknown-not stated 
• 
Duration of PICU/NICU days …../unknown-not stated 
• 
Days on oxygen …../unknown-not stated 
• 
Days on invasivie ventilation …../unknown-not stated 
• 
Days on non-invasive ventilation …../unknown-not stated 
• 
Days on inotropes …../unknown-not stated 
• 
Days on antibiotics …../unknown-not stated 
• 
Patient died no/yes. If yes, date of death  [date] /unknown-not stated 
 
*Definition: time of arrival until time of discharge or admission 
 
Working diagnosis and final diagnosis 
• 
Focus [required field] upper respiratory/lower respiratory/urinary 
tract/enteric/cutaneous/bones & joints/sepsis/meningitis/fever without focus/unknown-not 
stated 
If other, specify:…………………………………… 
• 
Final diagnosis* definite bacterial /probable bacterial / definite viral/probable 
viral/uncertain/unknown-not stated 
 
*Use the same definition as in BIVA ED, a link will be provided in the final form. 
Please use information from cultures if available. 
 
 
 
 
 
 
 
 

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This document is part of a project that has received funding from the European Union’s 
Horizon 2020 research and innovation programme, under grant agreement nr. 668303  
 
 
                    
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APPENDIX 1B – MOFICHE QUESTIONNAIRE: DESCRIPTION OF THE SETTING  
Filled in once for every emergency department 
 
Description of the setting 
Information about hospital, ED and healthcare organisation 
 
Date of registration (ddmmyyyy)  
Code of hospital, name and country (in final web form each participating hospital will be included in 
a list where to choose from) 
Adherence area (number of inhabitants < 16 year)   
Setting (inner city/ rural/mix) 
Socioeconomic status of the population in the adherence area (generally high/generally low / 
mixture 
of 
high 
and 
low 
SES 
/ 
other) 
Type (academic/teaching/ non-teaching) 
Number of paediatric emergency care admittance annually from the ED (number)  
Number of paediatric emergency visits annually (number)  
Type of triage system used (none/MTS/CTCS/ESI/other), If other: please specify ....................  
How is the temperature measured in general: oral / rectal / ear / axillary / temporal (forehead)  / 
other? 
Total number of inpatient beds for paediatric patients …….. 
Is there a paediatric ICU at this hospital?  
What is approximately the percentage of hospitalisation of paediatric ED patients (<5%, 5-10%, 10-
20%, 20-50%, >50%). 
What is the age-range at your ED defined as paediatric 
Is the emergency department in your hospital mixed adult and paediatric ED or an ED with paediatric 
patients only? 
What is approximately the percentage of self-referrals visiting the ED:  
(<5 %, 5-10%, 10-20%, > 20%)  
Who is the first person in charge of paediatric patients visiting the ED (multiple answers possible):  
Resident …. % 
Paediatrician …. % 
Surgeon …. %  
Emergency physician …. % 
Paediatric emergency physician …. % 
Other: paediatric specialist (e.g. neurologist, cardiologist)  …. % 
During day shifts a supervising specialist is available for * 
During out of hours  a supervising specialist is available for * 
 
*Direct Supervision – the supervising physician is physically present onsite with the resident and 
patient.  
*Indirect Supervision: (i) with direct supervision immediately available – the supervising physician is 
physically within the hospital and is immediately available to provide Direct Supervision.  
*Indirect Supervision: (ii) with direct supervision available – the supervising physician is not physically 
present within the hospital or other sites of patient care, but is immediately available by means of 
telephonic and/or electronic modalities, and is available to provide Direct Supervision in person 
within 20-30 minutes at all times.  
*Oversight – the supervising physician is available to provide review of procedures/encounters with 
feedback provided after care is delivered.  
 

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The majority of children visiting the ED has a primary care physician (yes / no) 
Primary care physicians deliver acute primary care during office hours in the adherence area of your 
ED (yes/no)  
Primary care physicians deliver acute primary care outside office hours  (yes/no) 
Patients can (officially) attend the ED without a referral from their GP  
Availability of specific guidelines: 
a) Guideline for children with fever (NICE/local/other/none)  
if other, please specify:................................. 
b) Guideline for children with urinary tract infections (NICE/local/other/none)  
if other, please specify:..................................... 
c) Guideline for children with respiratory infections (British thoracic society/NICE/local/other/none).  
If 
other, 
please 
specify:.......................................... 
d) Guideline for children suspected for meningitis/sepsis (NICE/local/other/none) 
If other, please specify:........................................  
e) 
Other 
guidelines 
(yes/no). 
If 
other, 
please 
specify:.......................................................  
The national immunization schedule    included 
 
a)  National immunization coverage: ...% 
 
 
b)  DKTP availability yes/no; administration schedule....;   
 
 
c)  HIB availability yes/no; administration schedule....;:... 
 
 
d)  S. Pneumoniae availability yes/no; administration schedule....; name vaccine:... 
 
 
e)  N. Meningococcus C availability yes/no; administration schedule....; name 
vaccine:... 
 
f)  Hepatitis B availability yes/no; administration schedule....; name vaccine:...  
g) N. meningococcus B availabiltity yes/no; administration schedule….: name vaccine:….. 
introduced…. 
 
Is there a paediatric haem/onc department at this hospital?  
How many patients with haematological malignancies are: 
Diagnosed on a yearly bases 
In treatment on a yearly basis 
In follow up on a yearly basis 
How many episodes of febrile neutropenia are seen on an annual basis 
 
Is there a paediatric stem cell transplant unit at this hospital? 
How many patients are being transplanted on a yearly basis? 
How many are haem/onc 
How many are primary immunodeficiencies 
How many are other (e.g. autoimmune) 
How many episodes of suspected infection are seen on annual basis? 
 
Is there a paediatric rheumatology department at this hospital?  
How many patients are seen with autoinflammatory diseases on an annual basis? 
How many patients are on biologicals on an annual basis 

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Checklist quality control variables  
  
General characteristics 
-        Are all patients at your ED registered  
  
Patients signs and symptoms 
-        Is working diagnosis assessed at the end of the ED visit  
-        Is working diagnosis made by the treating doctor  
o   If not, is it made by the treating nurse  
- 
Are vital signs truly measured yes/no 
Can vital signs be estimated? Yes / no 
 
Diagnostics and interventions 
- 
Are diagnostic tests recorded in the patient ED file? 
- 
In approximately what percent of the cases? (evt keuze-categorieen toevoegen) are they recorded 
in the patient file? 
- 
In approximately what percent of the cases is this information complete? 
- 
Who records this information? Physician / nurse / student / other.  
- 
Are diagnostic tests recorded in the patient file during the ED visit or is this done retrospectively? 
- 
In what percent of the cases  is this done during the ED visit 
- 
If recorded retrospectively, what could be a reason for this? E.g.: urgency of the patient, ED 
crowding, other reason.  
- 
If recorded retrospectively, is there a risk of bias? 
-        Are interventions recorded during the ED visit or is this done retrospectively?  
o   same questions as for diagnostic tests 
-        Are immediate life-saving interventions reported during the ED visit or is this done retrospectively.  
o    same questions as for diagnostic tests 
 
Follow-up 
o   same questions as for diagnostic tests 
Is hospitalization > 24 uur mainly (> 90..%) for medical reasons ?  
.  
Are children admitted to the ward if the stay at the ED takes longer than advised by local guidelines?  
-        Is ICU admission mainly (> 90..%) for lifethreatening diseases or abnormal vital signs including 
conscientiousness  ? 
If ICU admission occurs for other reasons, please specify: e.g. monitoring of vital signs, not enough 
beds at the regular department, other. 
- 
Is mortality at ED reported in the patient ED file? 
 
Time points 
-        Is the duration of ED visit measured electronically or noted?  Please specify.  
-        Are time points measured accurately? yes/no 
Is the time between arrival and triage recorded? 
 
In what percent of the cases is this recorded accurately? 
Is the time between arrival and discharge recorded? 
 
 
In what percent of the cases is this recorded accurately? 
Are there guidelines on the timeframe within triage has to be performed? Please specify  
 
 
 

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APPENDIX 2- BIVA-PIC CRF   
 
 
                               Subject num: _______ 
 
 
Episode num._____ 
If Neonate: Gestational age: _____ weeks 
 
 
 
Weight: _______kg  
 
 
 
 
 
 
 
 
 
 
INVESTIGATIONS 
 
Classification (tick all that apply) 
 Child with Fever 
 
 
 
 Suspected Sepsis 
 
 
   
 Healthcare-associated Infection (HAI)  
 Surgical Patient 
 
 
 
If HAI:  
• 
Surgical site infections  
 
 
 
Y 
N 
• 
Catheter-associated Urinary Tract Infections 
Y 
N 
• 
Ventilator-associated Pneumonia  
 
Y 
N 
• 
Central Line-Associated Bloodstream Infections 
Y 
N 
• 
Laboratory-Confirmed Bloodstream Infection 
Y 
N 
• 
Others _______________ (specify) 
 
Y 
N 
 
If Surgical Patient: 
• 
Specify kind of Operation: 
_________  
• 
Date of Operation  
 
___/___/___ 
 
HAEMATOLOGY 
Units 
Units 
(alt.) 
1st research 
bloods 
BASELINE 
2nd research 
bloods 
 48h after 1st 
Follow Up 
28d after 1st 
DATE: 
 
 
 
 
 
Hb 
g/dL 
 
 
 
 
WCC 
10^9/L 
 
 
 
 
Plt 
10^9/L 
 
 
 
 
Hct 
% 
 
 
 
 
Neutrophils 
10^9/L 
 
 
 
 
Lymphocytes 
10^9/L 
 
 
 
 
Monocytes  
10^9/L 
 
 
 
 
Eosinophils  
10^9/L 
 
 
 
 
Basophils  
10^9/L 
 
 
 
 
PT 
% 
 
 
 
 
INR 
- 
 
 
 
 
APTT 
sec 
 
 
 
 
Fibrinogen 
mg/dL 
 
 
 
 
D-Dimer 
µg/ml 
µg/l 
 
 
 
 
 
 
 
 
 
BIOCHEMISTRY 
 
 
 
 
 
Crea 
mg/dL 
 
 
 
 
Urea 
mg/dL 
 
 
 
 
Na 
mmol/
L 
 
 
 
 
K 
mmol/
L 
 
 
 
 
Cl 
mmol/
L 
 
 
 
 
Calcium 
mmol/
L 
 
 
 
 

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GMSPS (MENINGOCOCCAL ONLY) 
                                                                                                                                                       Y    N                                          
 
Hypotension (SBP < 75 if less than 4 years, < 85 if older) 
  
 
Core - peripheral temperature gap > 3 degrees  
  
 
Base deficit < -8 
  
 
Coma score < 8 at any time or deteriorated > = 3 in last hour  
  
 
Lack of meningism 
  
 
Parental opinion that child is worse over last hour 
  
 
Widespread ecchymoses 
  
 
 
PRISM SCORE  
Respiratory rate: 
 
PaO2 (kPa): 
 
Apnea:  
Y     N 
PaCO2 (kPa): 
 
GCS (total) 
 
HCO3 (mEq/L): 
 
- Eyes 
 
 
 
- Verbal 
 
 
 
- Motor 
 
 
 
Pupils fixed and dilated: 
Y    N 
Glucose 
 
Systolic BP (mm Hg):  
 
PT/PTT>1.5 
x 
contro 
(mmol/dL): 
 
Diastolic BP (mmHg):  
 
Bilirubin > 60 μmol/L if > 1 
month: 
Y      N 
Heart rate (bpm): 
 
Potassium (mEq/L):  
 
FiO2: 
 
Calcium (mmol/L): 
 
 
 
Gluc 
mg/dL 
 
 
 
 
Alb 
g/dL 
 
 
 
 
 
 
 
 
 
 
CRP 
mg/L 
mg/dL 
 
 
 
CRP max. 
mg/L 
mg/dL 
 
 
 
ProcalcitoninPCT 
ng/mL 
 
 
 
 
IL-6 
ng/L 
 
 
 
 
LDH 
U/L 
 
 
 
 
 
 
 
 
 
 
Alanine Transaminase 
U/L 
 
 
 
 
Bilirubine 
mg/dL 
 
 
 
 
CK 
U/L 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Ferritin 
µg/l 
 
 
 
 
 
 
 
 
 
 
BLOOD GAS ANALYSIS 
 
 
 
 
 
Arterial PaO2 
mmHg 
 
 
 
 
CO2 
mmHg 
 
 
 
 
HCO3 
mmol/
L 
 
 
 
 
BE 
mmol/
L 
 
 
 
 
Lactate 
mmol/
L 
 
 
 
 
PRISM data recorded should be worst in first 24hours of PICU admission 
PRISM data recorded should be worst in first 24hours of PICU admission 

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SEPSIS-3-
CRITERIA 
Value 
Units 
Units (alternative) 
Comments 
PaO2  
 
kPa 
mmHg 
 
Platelets 
 
×103/μL 
 
 
Bilirubin 
 
mg/dL 
μmol/L 
 
MAP 
 
mmHg 
 
 
Dobutamine 
Y       N 
 
 
 
Dopamine  
 
μg/kg/min 
 
for at least one hour 
GCS 
 
 
 
 
Creatinine 
 
mg/dL 
μmol/L 
 
Urine output 
 
mL/d 
 
 
 
 
Microbiology: 
 
Test methods: Culture | PCR | CF (Complement fixation) | EIA (Rag/IF) 
Material/Analysis/  Date: 
Test method (C,P,CF,E) 
Results 
 
Urinanalysis 
 
 
Leuc               Y / N 
Nit                 Y / N 
Urine micro 
 
 
 
 
Urine culture 
 
 
Pos / Neg / ND 
Organism: 
 
Blood culture 
 
 
Pos / Neg / ND 
Organism: 
 
Throat swab 
 
 
Pos / Neg / ND 
Organism: 
 
CSF micro 
 
 
 
 
CSF 
 
 
Pos / Neg / ND 
Organism: 
 
NPA 
 
 
Pos / Neg / ND 
Organism: 
 
BAL 
 
 
Pos / Neg / ND 
Organism: 
 
 Pernasal Swap 
 
 
Pos / Neg / ND 
Organism: 
 
Endotracheal 
Aspirate 
 
 
Pos / Neg / ND 
Organism: 
 
Stool 
 
 
Pos / Neg / ND 
Organism: 
 
PCR material 
 
 
Pos / Neg / ND 
Organism: 
 
Other 
pathogen 
identified: 
 
 
 
 
 
Screening for MRE 
|  
MRSA 
| 
MRGN/ESBL 
 
 
 
 
 

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THERAPIES / TREATMENT 
Urinary Catherisation 
 
 
 
Y 
N 
Central Line Catheter Implantation 
 
Y 
N 
Renal Replacement Therapy  
 
 
Y 
N 
Chest Drains 
 
 
 
Y 
N 
ECMO 
 
 
 
Y 
N 
 
 
 
 
 
 
 
 
 
 
Route: IV=intravenous, O=oral, Neb=Nebulised, IM=intramuscular, T=topical, IT=intrathecal, SDD= selective 
decontamination of digestive tract 
 
Imaging / Others – if conclusive for diagnosis 
Method 
Performing Date 
Results 
X-Ray 
 
 
MRT 
 
 
CT 
 
 
Sonography 
 
 
EEG 
 
 
others 
 
 
Antibiotics 
Route 
Start date (dd/mm/yy) 
Stop date (dd/mm/yy) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Virostatics 
Route 
Start date (dd/mm/yy) 
Stop date (dd/mm/yy) 
 
 
 
 
 
 
 
 
 
 
 
 
Antifungals 
Route 
Start date (dd/mm/yy) 
Stop date (dd/mm/yy) 
 
 
 
 
 
 
 
 
Steroids 
Route 
Start date (dd/mm/yy) 
Stop date (dd/mm/yy) 
 
 
 
 
 
 
 
 
 
 
 
 
IVIG 
Route 
Start date (dd/mm/yy) 
Stop date (dd/mm/yy) 
 
 
 
 
 
 
 
 
Other Fluids/Treatments 
Route 
Start date (dd/mm/yy) 
Stop date (dd/mm/yy) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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OUTCOME 
Full recovery  
 
 
 
Y  
N 
UNKNOWN 
Skin graft  
 
 
 
Y  
N 
UNKNOWN 
Amputation 
 
 
 
Y  
N 
UNKNOWN 
Hearing loss >40dB 
 
 
 
Y  
N 
UNKNOWN 
Disability (non/mild/moderate/severe/coma or vegative state/brain death/unknown) 
Other: 
 
___________________________ 
 
Severity of Illness: 
 
First results 
1st research bloods  
2nd RB 
further 
timepoint 
DATE: 
 
 
 
 
 
 
yes no 
yes no 
yes no 
yes no 
In oxygen 
  
 
  
 
  
 
  
 
Invasive 
ventilation 
 
  
 
 
  
 
 
  
 
 
  
 
 
Non-invasive 
ventilation  
  
 
  
 
  
 
  
 
 
(CPAP, 
CPAP/PS, 
BiPAP, 
IPPV/similar, 
HFOV, face mask, 
nasal prongs) 
……………………. 
……………………. 
……………………. 
……………………. 
 
 
 
 
 
Inotropes 
yes no 
  
 
yes no 
  
 
yes no 
  
 
yes no 
  
 
 
Sepsis:  
(see definitions) 
Focal infection 
Y 
N 
 
Suspected Sepsis          
Y 
N 
Proven Sepsis                      
Y 
N 
Severe Sepsis                      
Y 
N 
Shock  
Y 
N 
 
Organ dysfunctions/failure: 
(see definitions) 
Renal failure                      
Y 
N 
Liver failure                       
Y 
N 
Respiratory failure   
Y 
N 
DIC                          
Y 
N (pTT > 1,5 x normal, platelets < 100 000/µl and elevated D-dimeres) 
Seizures 
Y 
N 
 
 

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APPENDIX 3– BIVA-HR CRF  
 
 
                          Subject num: _______ 
 
 
 
 
 
 
 
Episode num. _______ 
 
Data to be collected in addition to BIVA-ED 
 
Lymphocyte count ………. mm3/ml 
Neutrophil count If neutropenic:  
since when 
Underlying diagnosis 
Treatment protocol 
Transplant yes/no 
If yes: pretransplant/conditioning for transplant/post transplant. If post transplant: 
Day … post transplanttransplant 
Neutrophil engraftment no/yes; if yes: day …. Post transplant 
Lymphocyte engraftment no/yes; if yes: day …. Post transplant 
 
GVHD: grade….. 
 
treatment 
Immunoglobulins schedule: no/yes : weekly/three weekly  
Immunoglobulins route: iv/sc 
Immunoglobulinsdose: …..g/kg 
 
Current medication 
PCP prohylaxis (yes/no); if yes what: septrin/pentamidine/atovaquone/otherAntiviral prophylaxis (): 
acyclovir/valaciclovir/other ie..... 
Antifungal prophylaxis (yes/no) ambisome/voriconazol/caspofungin/itraconazole/other 
Antimycobacterial prophylaxis: rifampicin yes/no, isoniazid yes/no, other yes…/no 
 
Antibiotics: () 
Antibiotic 1 dose, frequency since 
Antibiotic 2 dose, frequency since 
Antibiotic 3 dose, frequency since 
 
Antifungal 1 type, dose, frequency since 
Antifungal 2 type, dose, frequency since 
Antifungal 3 type, dose, frequency since 
 
Antiviral 1 type, dose, frequency since 
Antiviral 2 type, dose, frequency since 
Antiviral 3 type, dose, frequency since 
 
Immunesuppression yes/no: if yes: 
Steroids : (yes/no) 
Type: 
Systemic: dose mg/kg/day equivalent 
Topical: 
Type: hydrocorticone/emuvate 
 
Tacrolimus (yes/no) 
Cyclosporine (yes/no) 
Others yes/no, if yes… 

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Antacids (yes/no) 
 
Defibrotide (yes/no) 
 
G-CSF (yes/no) 
 
Central line (yes/no) 
 If yes: type: hickmann/portacath  
Number of lumen: 1/2/3 
Note for these patients culture form central line is allowed please indicate if bloodculture was taken 
from: peripheral stab / CVL  
 

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This document is part of a project that has received funding from the European Union’s 
Horizon 2020 research and innovation programme, under grant agreement nr. 668303  
 
 
                    
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APPENDIX 4– BIVA-INF CRF  
 
 
 
             Subject num: _______ 
 
 
 
 
 
 
 
Episode num. _______ 
 
Data to be collected in addition to BIVA-ED 
 
Underlying diagnosis (Kawasaki, Systemic JIA, treatment-naïve JIA)  
If included for another rheumatic disease, mention which: (SLE, PFAPA, FMF, MVD, TRAPS, CAPS, 
MWS, CINCA/NOMID, FCAS, Blau syndrome) 
Genetic confirmation no/yes; if yes: gene mutation 
Treatment protocol (local center ---) 
 
Year/month/Day of diagnosis:  
Assumed day of onset:  
 
Treatment with antibiotics (last 10-14 days) yes/no 
If yes, mention which antibiotics:   
 
 
Current medication: no/yes, if yes: 
IVIG: no/yes    dose: …..g/kg  
schedule weekly/three weekly  route: iv/sc 
 
Day of administration:  
 
NSAIDs: yes/no   
If yes, since when: mm/yyyy  
Colchicine yes/no   
If yes, since when: mm/yyyy  
Azathioprin yes/no   
If yes, since when: mm/yyyy  
DMARDs (MTX): yes/no  
If yes, since when: mm/yyyy  
Cyclophosphamide yes/no If yes, since when: mm/yyyy  
Infliximab yes/no 
If yes, since when: mm/yyyy  
Rituximab yes/no 
If yes, since when: mm/yyyy  
Tacrolimus yes/no 
If yes, since when: mm/yyyy  
Cyclopsorin yes/no  
If yes, since when: mm/yyyy  
 
 
Biological (anti-TNF blockade): yes/no If yes, since when: mm/yyyy  
Started during current admission: yes/no 
 
Anti-IL-1 drug (anakinra): yes/no 
 If yes, since when: mm/yyyy 
Started during current admission: yes/no 
 
Anti IL6yes/no  
If yes, since when: mm/yyyy 
 
Steroids: yes/no 
Methylprednisolone 3 day course (15-30 mg/kg): yes/no 
Started during current admission: yes/no 
Oral prednisone: yes/no   
If yes, since when: mm/yyyy  
 
Disease considered to be controlled: yes/no 
 
 

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Organ systems involved: 
Skin 
Joints 
Muscle 
Cardiac 
Lungs 
CNS 
Vasculitis 
Serositis 
Eyes 
Hearing loss 
GI Liver 
GI other