Development of Non-invasive Biomarkers to Direct Individualised Management of Preschool Wheeze
Conditions: Wheezing
Sponsor: Imperial College London

 
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Development of non-invasive biomarkers to direct individualised 
management of preschool wheeze 
 
 
 
 
V2 10th November 2023 
 
 
 
 
MAIN SPONSOR: Imperial College London 
FUNDERS: Action Medical Research Charitable Foundation & The Masonic Charitable Foundation 
STUDY COORDINATION CENTRE: Royal Brompton Hospital & National Heart and Lung Institute 
 
IRAS Project ID: 328947 
REC reference: xxx 
 
 
 
Protocol authorised by: 
 
Name & Role 
Date 
Signature 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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Study Management Group 
 
Chief Investigator: Prof Sejal Saglani 
 
Co-investigators: Dr Kushalinii Hillson, Prof Andrew Bush, Prof Adnan Custovic 
 
Statistician: Dr Sara Fontanella 
 
Study Management: Prof Sejal Saglani      
 
 
Clinical Queries 
 
Clinical queries should be directed to Dr Kushalinii Hillson who will direct the query to the 
appropriate person. 
 
 
Sponsor 
 
Imperial College London is the main research Sponsor for this study.  For further information 
regarding the sponsorship conditions, please contact the Head of Regulatory Compliance at: 
 
 
 
Research Governance and Integrity Team 
Imperial College London and Imperial College Healthcare NHS Trust 
Room 215, Level 2, Medical School Building 
Norfolk Place 
London, W2 1PG 
Tel: 0207 594 1862 
Imperial College - Research Governance and Integrity Team (RGIT) Website 
 
 
Funder 
 
Action Medical Research Charitable Foundation & The Masonic Charitable Foundation 
 
 
This protocol describes the “Non-invasive biomarkers for preschool wheeze” study and provides 
information about procedures for entering participants.  Every care was taken in its drafting, but 
corrections or amendments may be necessary. These will be circulated to investigators in the study.  
Problems relating to this study should be referred, in the first instance, to the Chief Investigator.  
 
This study will adhere to the principles outlined in the UK Policy Framework for Health and Social 
Care Research. It will be conducted in compliance with the protocol, the Data Protection Act and 
other regulatory requirements as appropriate.  
 
 
 

 
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Table of Contents                                                             Page No 
 
1. 
INTRODUCTION 
8 
1.1. 
BACKGROUND 
8 
1.2. 
RATIONALE FOR CURRENT STUDY 
8 
2. 
STUDY OBJECTIVES 
12 
3. 
STUDY DESIGN 
13 
3.1. 
STUDY OUTCOME MEASURES 
14 
4. 
PARTICIPANT ENTRY 
15 
4.1. 
PRE-REGISTRATION EVALUATIONS 
15 
4.2. 
INCLUSION AND EXCLUSION CRITERIA 
15 
5. 
ADVERSE EVENTS 
15 
5.1. 
DEFINITIONS 
15 
5.2. 
REPORTING PROCEDURES 
16 
6. 
ASSESSMENT AND FOLLOW-UP 
17 
7. 
STATISTICS AND DATA ANALYSIS 
21 
8. 
REGULATORY ISSUES 
21 
8.1. 
ETHICS APPROVAL 
21 
8.2. 
CONSENT 
21 
8.3. 
CONFIDENTIALITY 
21 
8.4. 
INDEMNITY 
21 
8.5. 
SPONSOR 
21 
8.6. 
FUNDING 
21 
8.7. 
AUDITS 
21 
9. 
STUDY MANAGEMENT 
23 
10. 
PUBLICATION POLICY 
22 
11. 
REFERENCES 
22 
 
 
 
 
 
 

 
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GLOSSARY OF ABBREVIATIONS 
ICS 
inhaled corticosteroids 
PCR 
polymerase chain reaction 
BDR 
bronchodilator reversibility 
FOT 
forced oscillation technique 
WBC 
White blood cell 
BAL 
Bronchoalveolar lavage 
PHC 
preschool healthy controls 
PSW 
preschool wheezers 
GP 
General practitioners 
dBNs 
dynamic Bayesian networks 
BNs 
Bayesian Networks 
 
 
 
 
 
 
 
 
 
 
 
 

 
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KEYWORDS 
preschool children, recurrent wheezing, blood eosinophils, lung function, infection, inflammation, 
treatment, non-invasive tests 
 
STUDY SUMMARY 
 
TITLE Development of non-invasive biomarkers to direct individualised 
management of preschool wheeze 
 
DESIGN 
(i) 
Cohort observational study with collection of clinical details, 
blood samples, sputum, oropharyngeal swabs and airway 
samples from children.  
 
(ii) 
Laboratory study using blood and airway samples collected to 
assess inflammation and infection. 
 
(iii) 
Proof-of-concept feasibility study in a sub-group of the cohort to 
investigate biomarker guided targeted treatment for preschool 
wheeze. This will enable sample size calculation for a larger 
clinical trial 
AIMS Aim 1: 
 Investigate the sensitivity and specificity of:  
(a) aeroallergen sensitisation and blood eosinophils in reflecting lower 
airway eosinophils;  
(b) blood neutrophil phenotype and chemotaxis in reflecting lower airway 
neutrophils; and  
(c) induced sputum and oropharyngeal and nasal swabs in reflecting 
lower airway infection 
in preschool children with severe, recurrent wheeze undergoing clinically 
indicated bronchoscopy (n=50).  
 
Aim 2:      
Determine the feasibility of undertaking 3 to 6 monthly assessments of the 
proposed non-invasive biomarker tests listed below in preschool 
wheezers, and relate the results to symptoms, wheeze attacks and 
prescribed medication.  
1. skin prick tests for aeroallergen sensitisation (will only be done once),  
2. blood eosinophils (using a point-of-care test),  
3. blood neutrophil function (using a chemotaxis assay and flow cytometry),  
4. bacterial detection (pathogen specific PCR from oropharyngeal and 
nasal swabs) and induced sputum,   
5. lung function with bronchodilator reversibility (using forced oscillation 
technique [FOT]). (n=100)  
 

 
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Aim 3: Undertake a proof-of-concept feasibility study to see if 
parents/caregivers will agree to take part in a stratified treatment clinical 
trial of biomarker guided therapy in recurrent preschool wheeze (n=15).  
 
OUTCOME 
MEASURES 
Primary outcomes: Specificity, sensitivity, longitudinal stability and 
acceptability 
of 
non-invasive 
tests 
(biomarkers) 
of 
eosinophilic 
inflammation, phenotype of airway neutrophils, airway infection and lung 
function in children with preschool wheeze.  
 
Secondary outcomes: Relationship of the tests below to wheeze attacks 
and symptom control (TRACK questionnaire) when assessed 3 monthly for 
over one year in children with preschool wheeze. 
1. Lung function assessed using FOT (Rrs: respiratory system 
resistance; Xrs: respiratory system reactance; AX: area of 
reactance) 3 monthly 
2. Inhaled corticosteroid dose and oral prednisolone dose 3 monthly 
3. Induced sputum cultures at baseline and 12 months 
4. Blood eosinophil count 3 monthly  
5. Neutrophil function and phenotype in relation to infection status, 
clinical symptoms, and prescribed medication 3 monthy 
6. Nasal and oropharyngeal swab for infection 6 monthly 
7. Assessment of symptom burden to caregivers using the validated 
Paediatric Asthma Caregivers Quality of Life Questionnaire: 
PACQLQ 
POPULATION Preschool children with recurrent wheeze aged between 1-5 years 
 
ELIGIBILITY Children aged 1-5 years with either  
(i) >2 hospitalisations for acute wheeze; or  
(ii) at least 1 admission requiring high dependency unit or intravenous 
bronchodilator therapy;  
during the previous year. 
 
For Aim 1 only - children undergoing clinically indicated bronchoscopy, as 
determined by their existing medical team, as part of their standard 
management 
 
DURATION 36 months 
 
 
 
 
 
 
 

 
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REFERENCE DIAGRAM 
 
FIGURE 1: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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1. INTRODUCTION  
 
1.1. 
BACKGROUND 
 
Healthcare utilisation and morbidity in preschool children with wheezing: Wheezing illnesses and 
asthma are the most common non-communicable diseases of childhood worldwide. Approximately 
half of children in the UK experience at least one wheezing episode by age 6 years, and children 
under 6 years account for 75% of UK childhood hospital admissions for acute wheeze attacks. The 
prevalence of preschool wheeze in England was 8% in 2017, and of those initially presenting to 
primary care, 15.8% attended an emergency department, and 13.9% had a hospital admission over 
the subsequent 2 years.  Recurrent symptoms, multiple attacks and hospital admissions in 
preschool wheezers account for one-third of healthcare costs for childhood asthma and are 
associated with long-term diminished lung function and increased morbidity and mortality in early 
adulthood4. The unchanged disease burden over the last 20 years, both in the UK and USA 
suggests current management is failing to reduce numbers of acute wheeze attacks in preschool 
age, and we urgently need a step-change in our treatment approach to prevent recurrent attacks of 
preschool wheeze. 
 
1.2. 
RATIONALE FOR CURRENT STUDY 
The poorly understood pathophysiology of preschool wheeze to date has led to a “one size fits all”, 
standardised treatment of all preschool wheezers using inhaled corticosteroids (ICS) as 
maintenance therapy to prevent attacks. The justification for this approach has been an 
extrapolation of the treatment used in school-age asthma. However, most school-age children have 
allergic, eosinophilic asthma associated with type 2 immunity. In contrast, only a sub-group of 
preschool wheezers have airway eosinophilia and aeroallergen sensitisation (~25%), and a 
randomised, double-blind clinical trial (INFANT study) in this age group has shown that these are 
the group most likely to benefit from ICS. However, children without these features have no or poor 
response to any currently available treatment. Preschool wheeze is now known to be 
heterogeneous, but the current management approach purely based on parental reporting of 
symptoms, which we know cannot distinguish between underlying airway pathologies, likely 
accounts for the ineffectiveness of this approach in preventing wheezing attacks.   
Even when ICS are ineffective and could be causing harm, treatment is rarely discontinued because 
of symptom burden, physician insecurity and parental reluctance to stop therapy. We know that 
when they are targeted to the eosinophilic subgroup, ICS are highly beneficial in preschool 
wheezers. But our current approach only considers symptom patterns reported by parents, without 
objective tests to define the underlying airway phenotype leading to symptoms in each patient. 
Biomarkers and treatment options to identify and prevent wheeze attacks in preschool children 
without allergic sensitization or blood eosinophilia are lacking. Therefore, there is an urgent unmet 
need for appropriate and clinically feasible biomarkers that will allow identification of effective 
treatment strategies for individual patients. 
 
 
 

 
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Review of existing literature 
A novel approach for biomarker-guided management of preschool wheeze 
Pathological clusters of preschool wheeze: The Saglani group have recently shown that 
preschool wheeze is a heterogenous condition by identifying 4 pathologically distinct clusters in 
children with severe, recurrent preschool wheeze: 1. Atopic, 2. Non-atopic with low infection rate 
and high ICS use, 3. Non-atopic with high viral and bacterial infection rate and airway neutrophilia, 
and 4. Non-atopic with low infection rate and not prescribed ICS. The variables which distinguished 
the preschool wheeze clusters included allergic sensitisation, blood eosinophils, lower airway 
neutrophils, and the pattern of airway bacterial infection6. However, these findings have their 
limitations as they were from invasive, lower airway bronchoscopic samples, which can only be 
obtained on a single occasion from very severe wheezers. Symptom pattern (episodic vs persistent), 
which is the current way of distinguishing pathology and determining treatment, was shown to not 
be predictive of lower airway inflammation or infection. These findings have now identified the need 
for a change in physician mindset, and that management strategies for preschool wheeze needs to 
be based on objective biomarkers which predict response to treatment. To facilitate clinical 
translation and targeted treatments for all preschool wheezers with disease of all severity and in all 
healthcare settings, these limitations need to be addressed by identifying non-invasive biomarkers 
of lower airway infection and inflammation, assessing their stability and feasibility over time, and 
comparing their accuracy to lower airway samples. 
These newly described pathological preschool wheeze clusters, based on blood and airway 
inflammation and infection, suggest that a biomarker guided treatment approach may prove more 
successful than current standard care in preventing preschool wheeze attacks. My project aims to 
extend these findings to bedside clinical tests that can be used for targeted treatment. I will 
investigate the feasibility, sensitivity, specificity, and longitudinal variability of 1. skin prick tests (to 
detect allergen sensitisation), 2. blood eosinophils (using a point-of-care test), 3. blood neutrophil 
function (using a chemotaxis assay and flow cytometry), 4. bacterial detection (pathogen specific 
PCR from oropharyngeal and nasal swabs) and induced sputum, and 5. lung function with 
bronchodilator reversibility (forced oscillation technique [FOT]), as biomarkers that can be applied 
in future stratified interventional clinical trials. 
Proposed non-invasive biomarkers: 
1. Non-invasive tests for allergic sensitisation and eosinophilia 
The previous analyses identified that 25% of wheezers were in the atopic cluster, and both 
observational and clinical trial data have identified atopy (aeroallergen sensitisation) and blood 
eosinophilia as biomarkers of preferential response to ICS7. Based on the known action of ICS 
targeting eosinophilia, I propose that ICS would be an appropriate treatment for children in the atopic 
cluster. This suggests every preschool wheezer who is considered for ICS treatment should have 
phenotyping with aeroallergen sensitisation and blood eosinophil as a minimum before a decision 
is made to commence ICS. 
Skin prick tests for allergen sensitisation are simple, non-invasive and can be performed in clinic. 
However, performing blood test in preschool children is challenging. Venepuncture is a difficult 
procedure in this age group, time consuming and the result is not immediately available, and 

 
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frequently declined by parents. The Saglani group have obtained preliminary feasibility and 
acceptability data for a finger prick blood sample, using a point-of-care device (HemoCue WBC DIFF 
analyser for WBC) that measures differential white blood cell count, providing a result within a few 
minutes. Children and parents/ carers were approached in clinic and 84% agreed to have the test, 
with the main reason for refusal being needle anxiety. Of those that had the test, both parents (98%) 
and children found the test acceptable, and most were happy to have the test performed routinely 
(81%) (Table 1) suggesting this is a pragmatic approach that can be adopted. 
Table 1. Feasibility and acceptability of finger prick blood eosinophil test in children with 
wheeze and asthma 
Number of children whose parents/carers were approached 
  67 
Number of children whose parents/carers agreed to participate 
  56 (84%) 
Number of children who had the test (8 had to leave clinic before 
test was done) 
  48/56 (86%) 
Age (years); median (range) 
  4.3 (1-5) 
Median child rating score: How they found it* 
  2 
Median parent rating score: Acceptable method to obtain blood 
sample** 
  5 (98% scored 5) 
Median parent rating score: Acceptable for child to have this done 
routinely** 
  5 (81% scored 5) 
Successful sample obtained first attempt (out of 48) 
  45 (94%) 
Successful sample obtained >1 attempt (out of 48) 
  2 (4%) 
Fingerprick blood eosinophil count (109/L); median (range) 
  0.40 (0-3.8) 
*Children rated score from a 2-point smiley Likert scale (1 – sad face; 2 – happy face) 
**Parent rated score from a 5-point Likert scale (1 – worst; 5 – best) 
2. Blood biomarkers to identify phenotype and function of airway neutrophils 
Biomarkers and treatment to prevent wheeze attacks in preschool children without allergic 
sensitisation / blood eosinophilia remain elusive. The Saglani group have obtained preliminary data 
showing that the phenotype of airway neutrophils is distinct in the different preschool wheeze 
clusters. Wheezers as a group had a predominance of CD62Lhi regulatory neutrophils, however only 
those without bacterial infection had significantly more CD62Lhi neutrophils. Those with bacterial 
infection in their airways had significantly more CD62Llo inflammatory neutrophils (Figure 2). A flow 
cytometry panel has been established in the lab to phenotype neutrophils from 200mcl of blood 
taken during finger prick sampling, and I will use this to relate blood-derived data to lower airway 
neutrophil phenotyping in order to identify peripheral biomarkers of neutrophil activation status. 

 
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The Saglani lab has also investigated the function of blood neutrophils with the use of TAXIScan 
chemotaxis apparatus which allows dynamic measurements of leukocyte migration with as few as 
500 cells/data point. The assay generates a rich data set regarding the migration of individual 
neutrophils along a gradient of chemoattractant. Analysis of the cell tracks allows measurement of 
the velocity (V) and directionality (D) of each cell and to combine this in a V-D plot. Setting relevant 
threshold values for both parameters directional migration amongst the population of neutrophils 
can be accurately measured. Using this assay, preliminary data show reduced chemotaxis of blood 
neutrophils to CXCL8 from preschool wheezers compared to healthy controls (Figure 3). 
 
 
 
 
 
In this study, using small 100mcL blood samples, I will correlate neutrophil function and phenotype 
with infection status, clinical symptoms, and prescribed medication in children with recurrent 
preschool wheeze.  
3. Non-invasive tests for airway bacterial infection 
Preschool wheeze Cluster 3 (non-atopic, high infection rate) had the highest prevalence of airway 
bacterial and viral infection, and Moraxella catarrhalis, Haemophilus influenzae and Streptococcus 
pneumoniae made up 80% of the bacteria identified, suggesting these children may benefit from 
targeted antibiotics. Induced sputum testing is feasible and safe in young children but is time 
consuming and requires specialist training. However, a pathogen specific PCR to detect these three 
bacteria is also available. Bacterial detection in induced sputum, or by PCR, may be an additional 
biomarker for stratification of targeted antibiotic treatment. It has been shown that oropharyngeal 
swabs reflect lower airway microbes in children with different chronic lung diseases and 
nasopharyngeal samples also reveal Moraxella catarrhalis, Haemophilus influenzae and 
Streptococcus pneumoniae as the commonest organisms in preschool children’s respiratory 
systems during wheeze episodes. 
Figure 2. Proportion of CD62L+ 
neutrophils in bronchoalveolar 
lavage (BAL) from preschool 
wheeze (W) compared to non-
wheeze related disease (NWRD), 
and wheezers with (+) and without 
(-) airway bacterial infection. 
CXCL8 
PHC 
PS
W
PHC
PSW
0
20
40
60
80
% of Cells in
 +ve Gate
✱
A 
B 
C 
Figure 3. Chemotaxis of blood 
neutrophils from preschool healthy 
controls 
(PHC) 
compared 
to 
preschool 
wheezers 
(PSW) 
to 
CXCL8. (A) reduced % chemotaxis, 
(B&C) cell tracks towards CXCL8. 

 
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Preliminary data from the Saglani group comparing the pathogen specific PCR in oropharyngeal 
swabs to broncho-alveolar lavage (BAL) PCR and BAL culture results have shown complete 
agreement between BAL culture and oropharyngeal swab PCR.  Additional organisms were 
detected by the PCR compared to culture in 26/64 (41%) samples, and the PCR failed to detect a 
cultured organism in 3/64 (4%) patients. This suggests that the PCR test is accurate, and I propose 
that throat and nasal swabs will prove to be feasible as non-invasive tests to detect lower airway 
bacterial infection and identify children who may benefit from targeted antibiotics. 
4. Lung function tests to assess airway obstruction and bronchodilator reversibility (BDR) 
For recurrent wheezers without atopy, peripheral eosinophilia, or airway infection, the treatment of 
choice may be as required bronchodilators and/or regular long-acting muscarinic antagonists. 
However, to understand the benefit of these treatments, lung function testing and demonstration of 
bronchodilator reversibility is required. The Saglani group have generated preliminary data in fifteen 
preschool children showing feasibility of the forced oscillation technique (FOT), a recognised test 
for airway function, and have demonstrated BDR (Figure 4). However, feasibility in a larger number 
and for longitudinal assessments needs to be determined. 
 
 
 
 
 
 
Research question:  
Can non-invasive tests (biomarkers) of allergen sensitisation, eosinophilic inflammation, phenotype 
and function of airway neutrophils, airway infection and lung function be used to personalise therapy 
for recurrent preschool wheeze? 
Hypothesis:  
Non-invasive biomarkers are stable, feasible to measure longitudinally, with high sensitivity and 
specificity, and can be used to personalise therapy of recurrent preschool wheeze. 
2. STUDY OBJECTIVES 
Primary Objectives: 
Aim 1. Investigate the sensitivity and specificity of: (a) aeroallergen sensitisation and blood 
eosinophils in reflecting lower airway eosinophils; (b) blood neutrophil phenotype and chemotaxis in 
reflecting lower airway neutrophils; and (c) induced sputum and oropharyngeal and nasal swabs in 
Figure 4: Correlation between respiratory 
resistance at 8 Hz (R8) z-score obtained 
using the forced oscillation technique and 
blood eosinophils using finger prick testing 
(%) in preschool children aged 1-5 years.  

 
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reflecting lower airway infection in preschool children with severe, recurrent wheeze undergoing 
clinically indicated bronchoscopy. 
Aim 2. Determine the feasibility of undertaking 3 to 6 monthly assessments of the proposed non-
invasive biomarker tests listed below in preschool wheezers, and relate the results to symptoms, 
wheeze attacks and prescribed medication. 
Aim 3. Undertake a proof-of-concept feasibility study to see if parents/caregivers will agree to take 
part in a stratified treatment clinical trial of biomarker driven therapy in recurrent preschool wheeze. 
Secondary Objectives: 
1. To determine the severity of preschool symptoms using a validated symptom questionnaire 
(TRACK) and relate the results to the tests above and assessment of symptom burden to 
caregivers using the validated Paediatric Asthma Caregivers Quality of Life Questionnaire: 
PACQLQ. 
 
2. To determine how much treatment is required for preschool wheeze, including calculating 
the steroid burden (inhaled corticosteroid and oral prednisolone) the child requires, and the 
number of unscheduled healthcare visits. 
  
3. To determine lung function in the preschool wheeze cohort, including bronchodilator 
response.   
 
4. To explore the relationship between airway inflammation and infection and serum 
inflammation and inflammatory markers. 
 
5. To explore the accuracy of oropharyngeal and/or nasal swabs in detecting lower airway 
infection in preschool wheezers.  
 
3. STUDY DESIGN 
 
Study design: 
 
1. Single centre, basic science study with human biological sample collection. 
 
2. Laboratory studies of biological samples collected. 
 
3. Proof-of-concept, feasibility study in a subgroup of the whole cohort. 
 
This study has three aims and the children may be recruited into one or more of the aims.  
 
Recruitment: 
 
Recruitment will be from the Royal Brompton Hospital which is a tertiary referral centre for 
respiratory paediatrics, and also sees patients who are directly referred by their GPs from Northwest 
London. This target population includes children from all sociodemographic and ethnic backgrounds 
as referrals are received from across London and the Southeast region. 

 
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Children eligible for participation will first be identified in clinic by their existing clinical care teams. 
The potential participants’ parents or guardian will only be approached by members of the research 
team: Clinical Fellows, Research Nurses or the Principal Investigator (PI), face to face in the clinic 
to explain the study. If they are interested, then parents will be given the Patient information Sheet 
(PIS). We will then give them at least 48 hours and up to two weeks to decide. We will schedule a 
time to make a phone call, if they consent, to answer any potential queries. We will then follow this 
up within two weeks, but this can be extended by mutual agreement if families feel they need longer 
to consider.  
 
If they agree to take part, we organise the first study visit to fit in with their next outpatient 
appointment, where the consent form will be signed. All subsequent visits will be scheduled to tie in 
with their outpatient appointments, so no extra study visits will be required. Research participants 
will not receive any payments, reimbursement of expenses or any other benefits or incentives for 
taking part in this study during their outpatient appointments. However, in the event that additional 
study appointments are required, travel and car parking expenses will be reimbursed. Individual 
researchers will not receive any personal payment over and above normal salary, or any other 
benefits or incentives, for taking part in this research. 
 
Aim 1: Children participating in this aim are recruited from the cohort of preschool wheezers who 
have been identified by their existing clinical teams as needing a bronchoscopy, as part of their 
standard clinical management. We will aim to use surplus samples taken during the bronchoscopy 
and endobronchial biopsy for the study, with parental consent. Number of children: 50. 
 
Aim 2: Children participating in this aim are recruited from the respiratory clinics who meet the 
criteria listed below. Children recruited for Aim 1 can be recruited into Aim 2. Number of children: 
100 
 
Aim 3: Children participating in this aim can be recruited from Aims 1 or 2, as this is a small proof-
of-concept study to investigate biomarker-driven targeted therapy for preschool wheeze. Number of 
children: 15 
 
Duration:  
 
Over 36 months, with each child being followed-up for 12 months after recruitment. 
 
 
3.1. 
STUDY OUTCOME MEASURES 
 
Primary outcomes: 
 
Specificity, sensitivity, longitudinal stability and acceptability of non-invasive tests (biomarkers) of 
allergen sensitisation, eosinophilic inflammation, phenotype and function of airway neutrophils, 
airway infection and lung function (FOT) in objective phenotyping of preschool wheeze and 
investigating the relationship between these objective tests and symptom control (TRACK 
questionnaire) and wheeze attacks. 
 

 
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Secondary outcomes:  
 
Relationship of the tests below to wheeze attacks and symptom control, using the respiratory and 
asthma control in kids (TRACK): a caregiver-completed validated questionnaire for preschool-
aged children, 3 monthly for a year. 
1. Lung function (Rrs: respiratory system resistance; Xrs: respiratory system reactance; AX: 
area of reactance) 3 monthly 
2. Inhaled corticosteroid dose and oral prednisolone dose 3 monthly 
3. Induced sputum cultures at baseline and 12 months 
4. Blood eosinophil count 3 monthly  
5. Neutrophil function and phenotype in relation to infection status, clinical symptoms, and 
prescribed medication 
6. Nasal and oropharyngeal swab for infection 6 monthly 
7. Assessment of symptom burden to caregivers using the validated Paediatric Asthma 
Caregivers Quality of Life Questionnaire: PACQLQ 
 
 
4. PARTICIPANT ENTRY 
 
4.1. 
PRE-REGISTRATION EVALUATIONS  
 
Participants do not need any pre-registration evaluations before being considered for recruitment.  
Parent / guardian must be able to give informed consent to participate in the study.  
Child and accompanying parent / guardian are contactable by telephone and able to attend 3 
monthly visits, which will be scheduled at the same time as their existing clinic appointment.  
Child and parent/guardian are able to comply with the requirements and restrictions listed in the 
consent form. 
 
4.2. 
INCLUSION and EXCLUSION CRITERIA   
 
Table 2: Inclusion and Exclusion criteria for each aim 
 
Aims  
Inclusion Criteria 
Exclusion Criteria 
Aim 1  
Children aged 1-5 years, undergoing clinically 
indicated flexible bronchoscopy, for recurrent 
preschool wheeze, as determined by their existing 
clinical team as part of their routine medical 
management 
Children with a known 
alternative respiratory 
diagnosis such as cystic 
fibrosis or bronchiectasis. 
A child will not be eligible for 
inclusion in this study if 
following medical 
assessment, physical 
examination or initial 
investigations, the physician 
Aim 2  
 
 
 
Children aged 1-5 years attending the paediatric 
respiratory clinics at the Royal Brompton Hospital with 
either  
(i) >/=2 hospitalisations for acute wheeze; and/or  
Aim 3 

 
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(ii) at least 1 admission requiring high dependency unit 
or intravenous bronchodilator therapy; during the 
previous year. 
 
considers the child to be unfit 
for the study. 
 
 
Withdrawal criteria: If the person with parental responsibility loses capacity, the child will be 
withdrawn from the study. Any identifiable data or tissue already collected with consent will be 
retained and used for the study, but no further data collection or procedures would be undertaken. 
5. ADVERSE EVENTS  
 
5.1. 
DEFINITIONS  
 
Adverse Event (AE): any untoward medical occurrence in a patient or clinical study subject.   
 
Serious Adverse Event (SAE): any untoward medical occurrence or effect that: 
• 
Results in death 
• 
Is life-threatening – refers to an event in which the subject was at risk of death at the 
time of the event; it does not refer to an event which hypothetically might have caused 
death if it were more severe 
 
• 
Requires hospitalisation, or prolongation of existing inpatients’ hospitalisation 
• 
Results in persistent or significant disability or incapacity 
• 
Is a congenital anomaly or birth defect 
 
Medical judgement should be exercised in deciding whether an AE is serious in other situations.  
Important AEs that are not immediately life-threatening or do not result in death or hospitalisation 
but may jeopardise the subject or may require intervention to prevent one of the other outcomes 
listed in the definition above, should also be considered serious. 
 
5.2. 
REPORTING PROCEDURES  
 
All adverse events should be reported.  Depending on the nature of the event the reporting 
procedures below should be followed.  Any questions concerning adverse event reporting should 
be directed to the Chief Investigator in the first instance.   
 
5.3.1 Non serious AEs 
 
All such events, whether expected or not, should be recorded  
 
5.3.2 Serious AEs 
 
An SAE form should be completed and emailed to the Chief Investigator within 24 hours.  However, 
relapse and death due to preschool wheeze, and hospitalisations for elective treatment of a pre-
existing condition do not need reporting as SAEs. 
 
All SAEs should be reported to the <name of REC> where in the opinion of the Chief Investigator, 
the event was: 
• 
‘related’, ie resulted from the administration of any of the research procedures; and 

 
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• 
‘unexpected’, ie an event that is not listed in the protocol as an expected occurrence 
 
Reports of related and unexpected SAEs should be submitted within 15 days of the Chief 
Investigator becoming aware of the event, using the NRES SAE form for non-IMP studies.  The 
Chief Investigator must also notify the Sponsor of all related and unexpected SAEs. 
 
Local investigators should report any SAEs as required by their Local Research Ethics Committee, 
Sponsor and/or Research & Development Office. 
 
Contact details for reporting SAEs 
RGIT@imperial.ac.uk 
Prof Sejal Saglani 
s.saglani@imperial.ac.uk 
Fax:02075943119, attention Sejal Saglani 
Please send SAE forms to: Sejal Saglani 
Tel: 0207 5943167 (Mon to Fri 09.00 – 17.00) 
 
 
6. ASSESSMENT AND FOLLOW-UP  
 
Children participating in Aim 1: One-off assessment carried out on the day of the bronchoscopy and 
no follow-up needed. Assessments carried out are listed below in Table 4. We will aim to do the 
oropharyngeal swab, skin prick testing (if not done in the last 6 months) and finger prick blood test 
under general anaesthetic, during the bronchoscopy procedure. 
 
A bronchoscopic programme which enables research samples to be collected, with parental 
informed consent, from children undergoing clinically indicated bronchoscopy for severe recurrent 
wheeze is established at the Royal Brompton Hospital. The Saglani group have shown that it is safe 
to take additional samples and this does not add significant time.  
 
Table 3: Assessments and investigations for Aim 1 only: 
 
Assessments/ Investigations 
Study visit  
Vital Signs measurements (Temp, BP, Pulse and respiration) 
X 
Height and weight 
X 
Physical Examination 
X 
Medical History 
X 
Informed consent  
X 
Validated symptom questionnaire (TRACK) 
X 
Paediatric Asthma Caregivers Quality of Life Questionnaire: PACQLQ  X 
Forced oscillation technique and bronchodilator reversibility test 
X 
Oropharyngeal and nasal swabs for pathogen specific PCR to detect 
Moraxella/Streptococcus/Haemophilus 
X 
Skin prick test or blood aeroallergen IgE (house dust mite, cat, dog, 
grass, tree pollen and mixed moulds) 
X 

 
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Sputum induction for bacterial culture and pathogen specific PCR to 
detect Moraxella/Streptococcus/Haemophilus 
X 
Blood sample collection (Finger prick) for point of care testing and 
laboratory studies 
X 
Bronchoscopy – bronchoalveolar lavage and endobronchial biopsy 
(part of routine clinical management and surplus sample used for 
study with parental consent) 
X 
 
Children participating in Aim 2 and Aim 3: Children in these two aims do not need bronchoscopy. 
They will have the panel of non-invasive tests listed in Table 5, at the intervals below. These children 
will be seen for the research study at the same time as their routine outpatient clinic follow-up visits, 
which will be every three months over one year (Visits 1-4), and therefore no separate research 
visits are required. The tests listed below in Table 5 will be undertaken at the times indicated.  
 
Table 4: Assessments and Investigations for Aims 2 and 3. 
 
Assessments/ Investigations 
Study visits (every 3 months for 12 months) 
 
Baseline 
1 
2 
3 
4 
Vital Signs measurements (Temp, BP, 
Pulse and respiration) 
X 
X 
X 
X 
X 
Height and weight 
X 
X 
X 
X 
X 
Physical Examination 
X 
X 
X 
X 
X 
Medical History 
X 
X 
X 
X 
X 
Informed consent  
X 
X 
X 
X 
X 
Validated symptom questionnaire 
(TRACK) 
X 
X 
X 
X  
X 
Paediatric Asthma Caregivers Quality of 
Life Questionnaire: PACQLQ 
X 
X 
X 
X 
X 
Forced oscillation technique and 
bronchodilator reversibility test 
X 
X 
X 
X 
X 
Oropharyngeal and nasal swabs for 
pathogen specific PCR to detect 
Moraxella/Streptococcus/Haemophilus 
X 
 
X 
 
X 
Skin prick test (house dust mite, cat, dog, 
grass, tree pollen,and mixed moulds) 
X 
 
 
 
 
Sputum induction for bacterial culture and 
pathogen specific PCR to detect 
Moraxella/Streptococcus/Haemophilus 
X 
 
 
 
X 
Blood sample collection (finger prick) for 
point of care testing and laboratory 
studies 
X 
X 
X 
X 
X 
 
Children participating in Aim 3 only: Our experience is that intervention studies are challenging 
in preschool wheezers. Parents are often hesitant about proposed changes to treatment, even 
though existing treatment may be ineffective, and they may not adhere to prescribed therapy. Aim 
3 is to undertake a proof-of-concept feasibility study for using biomarker results to determine 
management. Children in this Aim will be recruited from the cohort of children in Aim 2, and therefore 

 
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will have all the tests carried out at the same intervals. If parents consent to taking part, children will 
have the following treatment adjustments based on the results of their tests (Table 5): 
 
 
 

 
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Table 5: Proposed biomarker guided management for proof-of-concept feasibility study 
 
Biomarker result at baseline assessment 
Treatment 
Allergen sensitisation +/- blood eosinophilia (>0.3x109/L) Regular inhaled 
corticosteroids 
Non-atopic*; positive bacterial detection in sputum 
4 weeks of targeted 
antibiotics 
Non-atopic*; negative bacterial infection 
Short acting 
bronchodilators as 
required 
*non-atopic is defined as blood eosinophil of <0.3x109/L + no evidence of allergen sensitisation 
 
This proof-of-concept study will be undertaken to understand whether parents will agree to their 
children taking part in an interventional trial, and to assess the feasibility of the proposed targeted 
treatment approach in a subgroup of wheezers. Wheeze attacks and symptoms over the course of 
a year will be compared to the children in Aim 2 as a reference control cohort. This aim will enable 
a more accurate sample size calculation and design for a future interventional trial.  
 
The sputum samples, oropharyngeal swabs and finger prick blood test will be collected by a clinical 
research fellow or research nurse. Bronchoscopy samples (bronchoalveolar lavage and 
endobronchial biopsy) will be collected by a clinician responsible for the patient, as part of the 
samples collected for the patient's clinical management and surplus samples will be used for this 
study, with parental consent. 
 
In all three aims, parents and the child can refuse any test at any visit and continue to take part 
in this study. This will be made very clear at recruitment and during each study visit. One of our key 
aims is to look at the acceptability of the proposed non-invasive tests to both parents and the child, 
and therefore, the reason for refusal will be recorded. This will help us understand the acceptability 
of particular tests and the frequency of testing that is deemed acceptable. We will also perform a 
visual analogue score for every test done on the child to assess tolerance and acceptability. 
 
The participants will have a thorough respiratory assessment (including lung function tests, skin 
prick allergy testing) and the results will be made available immediately to the participants and 
caregivers, as part of the study. Incidental findings, during the course of the assessment, would be 
communicated to the families, and their GP and clinical care team - examples may include incidental 
bacterial infection, for which the participants will be prescribed appropriate treatment.  
 
In some cases, children may have an incidental finding during the study, such as identified as having 
respiratory disease or symptoms that requires further referral or treatment, and in such cases their 
families and their usual health care providers (GP and clinical care team) will be informed.  
 
End of study is defined as last research visit of the last subject recruited. 
 
 
 

 
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7. STATISTICS AND DATA ANALYSIS 
 
Sample size calculations: 
 
Aims 1 and 2 are observational, thus sample size is opportunistic and pragmatic. 
 
Aim 1: Approximately 4 wheezers/month undergo bronchoscopy, with recruitment over 24 months 
and based on past experience, a conservative 60% acceptance rate for inclusion, we estimate 50 
children will be included in Aim 1. 
 
Aim 2: Based on previous data, a sample size of 44 participants (assuming 25% positive cases) will 
allow the detection of a moderate level of agreement, with expected sensitivity at 67% (95%CI: 23%- 
95%), at a significance level of 5% with 80% power. Children for Aims 2 and 3 need to be recruited 
in the first 2 years to allow 12 months follow-up. From the number of preschool wheezers seen in 
the clinic, we estimate recruitment of 4 children per month, allowing approximately 100 children to 
be included in Aim 2 over 24 months. 
 
Aim 3: 20% of families agreed to be included in a previous interventional trial undertaken by the 
Saglani group, therefore n=15 is my target for Aim 3. An important outcome of Aim 3 will be to 
understand how many agree to be included and reasons for declining, this will enable a realistic 
sample size calculation for a future interventional clinical trial. A sample size of 15 participants will 
allow the detection of a high level of agreement, with expected sensitivity at 95% (95%CI: 45%- 
100%), and at a significance level of 5% with 80% power. 
 
Data Analysis: 
 
Aim 1 
To assess the diagnostic accuracy of aeroallergen sensitisation and blood eosinophils in reflecting 
lower airway eosinophils; of blood neutrophil phenotype and chemotaxis in mirroring lower airway 
neutrophils; and of induced sputum and oropharyngeal swabs in replicating lower airway infection, 
I will compute several accuracy measures, such as sensitivity, specificity, positive and negative 
predictive values, and positive and negative likelihood ratios. Furthermore, to verify the validity of 
the standard cutoff and to eventually identify more appropriate cutoff values for the considered 
diagnostic tests, I will perform Receiver Operating Characteristic (ROC) curve analysis.  
 
Aims 2 and 3 
Information related to the feasibility of the intervention will be gathered using a combination of 
quantitative and qualitative methods. Descriptive statistics will be used to summarise the baseline 
demographics of all patients included in the feasibility analysis and to evaluate parental and 
children's satisfaction with the various diagnostic tests. To identify factors responsible for dropout, 
baseline characteristics and questionnaire scores of patients who agreed or did not agree to undergo 
follow-up will be compared using parametric and non-parametric tests, where appropriate.  
 
To understand the robustness of the proposed diagnostic approach, I will investigate the temporal 
variation in biomarker trajectories and their relation to clinical status by adopting longitudinal 
generalised linear models and dynamic Bayesian networks (dBNs). Bayesian networks (BN) are 
probabilistic graphical models that, using Bayes’ theorem, represent knowledge about a certain 
domain/disease by encoding conditional dependences among random variables in directed acyclic 

 
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graphs. A dBN is the extension of a BN applied to longitudinal data. The model describes the 
dependencies among the variables over time. This allows for encoding cycles and feedbacks 
between variables to represent their relationships over different time windows. The heterogeneity of 
the joint dynamics of wheeze recurrence and biomarker variation will be explored using joint latent 
class models for recurrent events, which will allow me to identify subgroups of children characterised 
by different disease risk profiles linked to specific biomarker variations. The groups are formed to 
minimise the within-class variability both with respect to the evolution of longitudinal markers and to 
the occurrence of an event.  
 
To identify differences between patients in the intervention study compared to those in the 
observational arm, I will perform differential longitudinal analysis. I will combine simple parametric 
and nonparametric hypothesis testing techniques with time-varying models based on time-series 
analysis and smoothing splines. Symptom scores will be recorded using a validated questionnaire 
at each visit for those in the intervention study and adjustments will be made for season. 
 
Data and all appropriate documentation will be stored for a minimum of 10 years after the completion 
of the study, including the follow-up period.   
 
 
8. REGULATORY ISSUES  
 
8.1. 
ETHICS APPROVAL  
 
The Study Coordination Centre has obtained approval from the xxx Research Ethics Committee 
(REC) and Health Research Authority (HRA). The study must also receive confirmation of capacity 
and capability from each participating NHS Trust before accepting participants into the study or any 
research activity is carried out. 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. 
 
8.2. 
CONSENT   
 
Consent to enter the study must be sought from each participant’s parents or care giver (with 
parental rights) only after a full explanation has been given, an information leaflet offered and time 
allowed for consideration.  Signed participant consent should be obtained.  The right of the 
participant to refuse to participate without giving reasons must be respected.  After the participant 
has entered the study the clinician remains free to give alternative treatment to that specified in the 
protocol at any stage if he/she feels it is in the participant’s best interest, but the reasons for doing 
so should be recorded.  In these cases the participants remain within the study for the purposes of 
follow-up and data analysis.  All participants are free to withdraw at any time from the protocol 
treatment without giving reasons and without prejudicing further treatment. 
 
8.3. 
 CONFIDENTIALITY  
 
The samples will be stored in a linked pseudonymised form. The Chief Investigator will preserve the 
confidentiality of participants taking par in the study and is registered under the Data Protection Act. 
 
Data will be pseudonymised. 

 
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8.4. 
INDEMNITY 
 
Imperial College London holds negligent harm and non-negligent harm insurance policies which 
apply to this study. 
 
8.5. 
SPONSOR  
 
Imperial College London will act as the main Sponsor for this study.  Delegated responsibilities will 
be assigned to the NHS trusts taking part in this study.   
 
8.6. 
FUNDING  
 
Action Medical Research and The Masonic Charitable Trusts are funding this study.   
 
8.7. 
AUDITS   
 
The study may be subject to audit by Imperial College London under their remit as sponsor and 
other regulatory bodies to ensure adherence to GCP and the UK Policy Frame Work for Health and 
Social Care Research.  
 
 
9. STUDY MANAGEMENT  
 
The day-to-day management of the study will be co-ordinated through the National Heart & Lung 
Institute, Imperial College London and department of respiratory paediatrics, Royal Brompton 
Hospital, London.   
 
 
10. 
PUBLICATION POLICY  
 
All results will be published in open access journals in Line with Imperial College London’s open 
access policy, and in line with the finder’s publishing policy. 
 
 
11. 
REFERENCES  
 
1. Bloom CI, Franklin C, Bush A, Saglani S, Quint JK. Burden of preschool wheeze and 
progression to asthma in the UK: Population-based cohort 2007 to 2017. The Journal of 
allergy and clinical immunology 2021; 147(5): 1949-58. 
2. Davies G, Paton JY, Beaton SJ, Young D, Lenney W. Children admitted with acute 
wheeze/asthma during November 1998-2005: a national UK audit. ArchDisChild 2008; 
93(11): 952-8. 
3. Ferreira de Magalhães M, Amaral R, Pereira AM, et al. Cost of asthma in children: A 
nationwide, population-based, cost-of-illness study. Pediatric allergy and immunology: 
official publication of the European Society of Pediatric Allergy and Immunology 2017; 
28(7): 683-91. 

 
Research Governance and Integrity 
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IRAS: 328947 
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Page 24 of 25 
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4. Agustí A, Noell G, Brugada J, Faner R. Lung function in early adulthood and health in later 
life: a transgenerational cohort analysis. The Lancet Respiratory medicine 2017; 5(12): 935-
45. 
5. Moorman JE, Akinbami LJ, Bailey CM, et al. National surveillance of asthma: United States, 
2001- 2010. Vital Health Stat 3 2012; (35): 1-58. 
6. Robinson PFM, Fontanella S, Ananth S, et al. Recurrent Severe Preschool Wheeze: From 
Prespecified Diagnostic Labels to Underlying Endotypes. American journal of respiratory 
and critical care medicine 2021; 204(5): 523-35. 
7. Fitzpatrick AM, Jackson DJ, Mauger DT, et al. Individualized therapy for persistent asthma in 
young children. The Journal of allergy and clinical immunology 2016; 138(6): 1608-18.e12. 
8. Kwong CG, Bacharier LB. Phenotypes of wheezing and asthma in preschool children. 
Current opinion in allergy and clinical immunology 2019; 19(2): 148-53. 
9. Fernandes RM, Wingert A, Vandermeer B, et al. Safety of corticosteroids in young children 
with acute respiratory conditions: a systematic review and meta-analysis. BMJ Open 2019; 
9(8): e028511. 
10. Brand PL, Caudri D, Eber E, et al. Classification and pharmacological treatment of preschool 
wheezing: changes since 2008. Eur Respir J 2014; 43(4): 1172-7. 
11. Saglani S, Bingham Y, Balfour-Lynn I, et al. Blood eosinophils in managing preschool 
wheeze: Lessons learnt from a proof-of-concept trial. Pediatric allergy and immunology: 
official publication of the European Society of Pediatric Allergy and Immunology 2022; 
33(1): e13697. 
12. Feleszko W, Jartti T, Bacharier LB. Current strategies for phenotyping and managing 
asthma in preschool children. Current opinion in allergy and clinical immunology 2022; 
22(2): 107-14. 
13. Raissy HH, Kelly HW, Harkins M, Szefler SJ. Inhaled corticosteroids in lung diseases. 
American journal of respiratory and critical care medicine 2013; 187(8): 798-803. 
  
14. Heffler E, Terranova G, Chessari C, et al. Point-of-care blood eosinophil count in a severe 
asthma clinic setting. Annals of allergy, asthma & immunology : official publication of the 
American College of Allergy, Asthma, & Immunology 2017; 119(1): 16-20. 
15. Kanegasaki S, Nomura Y, et al. A novel optical assay system for the quantitative 
measurement of chemotaxis. Journal of immunological methods. 2003 Nov 1;282(1-2):1-1. 
16. Schwerk N, Brinkmann F, Soudah B, Kabesch M, Hansen G. Wheeze in preschool age is 
associated with pulmonary bacterial infection and resolves after antibiotic therapy. 
PLoSOne 2011; 6(11): e27913. 
17. Jochmann A, Artusio L, Robson K, et al. Infection and inflammation in induced sputum from 
preschool children with chronic airways diseases. Pediatric pulmonology 2016; 51(8): 778-
86. 
18. Ahmed B, Cox MJ, Cuthbertson L, et al. Comparison of the upper and lower airway 
microbiota in children with chronic lung diseases. PloS one 2018; 13(8): e0201156. 
19. Bisgaard H, Hermansen MN, Bonnelykke K, et al. Association of bacteria and viruses with 
wheezy episodes in young children: prospective birth cohort study. BMJ 2010; 341: c4978 
20. Rosenfeld M, Allen J, Arets BH, et al. An official American Thoracic Society workshop 
report: optimal lung function tests for monitoring cystic fibrosis, bronchopulmonary 
dysplasia, and recurrent wheezing in children less than 6 years of age. Annals of the 
American Thoracic Society 2013; 10(2): S1-s11. 

 
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21. Murphy KR, Zeiger RS, et al. Test for respiratory and asthma control in kids (TRACK): a 
caregiver-completed questionnaire for preschool-aged children. The Journal of allergy and 
clinical immunology 2009; 123(4): 833-9.e9. 
22. Saglani S, Payne DN, Nicholson AG, Scallan M, Haxby E, Bush A. The safety and quality of 
endobronchial biopsy in children under five years old. Thorax. 2003 Dec;58(12):1053-7.