Identification and Characterization of Bacteria in the Lower Airways of Children Aged ≥ 6 Months to < 6 Years With Suspected Lower Respiratory Tract Infections (LRTI) in Spain.
Phase: NA
Conditions: Infections, Streptococcal
Sponsor: GlaxoSmithKline

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Study Protocol
Sponsor:
GlaxoSmithKline Biologicals
Rue de l’Institut 89,
1330 Rixensart, Belgium
eTrack study number and 
Abbreviated Title
115813 (EPI-STREP-064 BOD ES)
Date of protocol
Final: 25 May 2012
Date of protocol 
amendment
Amendment 1 Final: 08 March 2013
Amendment 2 Final: 05 October 2015
Title
Identification and characterisation of bacteria in the 
lower airways of children aged ! 6 months to < 6 years 
with suspected lower respiratory tract infections in 
Spain.
Detailed Title
A multi-centre, hospital-based, cross-sectional 
epidemiology study to identify and characterise 
bacteria in the lower airways of children aged ! 6 
months to < 6 years with suspected chronic lower 
respiratory tract infections (LRTIs) in Spain.
Co-ordinating author
, Scientific Writer
Contributing authors 
(Amended 05 October 2015)
∀
, Senior Manager, Epidemiology
∀
, Senior Manager, 
Epidemiology
∀
, Epidemiologist, Cromsource for 
GSK Biologicals
∀
, Epidemiologist, freelance for 
GSK Biologicals
∀
, Study Statistician, freelance for GSK 
Biologicals
∀
, Project Statistician
∀
, Study Delivery Lead
∀
, Study Delivery Manager
∀
, Global Study Manager 
(Consultant, Harrison Clinical Research)
∀
, Global Study Manager, Valesta 
for GSK Biologicals

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PPD
PPD
PPD
PPD
PPD
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eTrack study number and 
Abbreviated Title
115813 (EPI-STREP-064 BOD ES)
Date of protocol
Final: 25 May 2012
Date of protocol 
amendment
Amendment 1 Final: 08 March 2013
Amendment 2 Final: 05 October 2015
Detailed Title
A multi-centre, hospital-based, cross-sectional 
epidemiology study to identify and characterise 
bacteria in the lower airways of children aged ! 6 
months to < 6 years with suspected chronic lower 
respiratory tract infections (LRTIs) in Spain.
Contributing authors 
(Amended 05 October 2015)
∀
, Medical Director, Vaccines
∀
, Study Data Manager, TCS 
for GSK Biologicals
∀
, Study Data Manager, Business & 
Decision for GSK Biologicals
∀
, Study Data Manager
∀
, Safety Associate
∀
, Safety Physician
∀
, Senior Manager, Statistics, 
Epidemiology
∀
, GVCL Project Manager
∀
, GVCL Study Manager
∀
, Laboratory Study Manager, 
Business & Decision for GSK Biologicals
∀
, Laboratory Project Manager, 
Global Clinical Read-Outs
∀
, Local Delivery Lead
GSK Biologicals’ protocol template for observational studies and interventional studies 
without administration of medicinal products as described in a research protocol based 
on the Protocol Document Standard version 13.2
Copyright 2012-2015 of the GlaxoSmithKline group of companies. All rights reserved. Unauthorised 
copying or use of this information is prohibited.

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PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
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PPD

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Protocol Amendment 2 Sponsor Signatory Approval
eTrack study number and 
Abbreviated Title
115813 (EPI-STREP-064 BOD ES)
Date of protocol amendment
Amendment 2 Final: 05 October 2015
Detailed Title 
A multi-centre, hospital-based, cross-sectional 
epidemiology study to identify and characterise 
bacteria in the lower airways of children aged 6 
months to < 6 years with suspected chronic lower 
respiratory tract infections (LRTIs) in Spain.
Sponsor signatory
(Amended 05 October 2015)
Laurence Baril
Director, Head of Global Epidemiology
GlaxoSmithKline Biologicals
Signature
Date
 
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Protocol Amendment 2 Rationale
Amendment number:
Amendment 2
Rationale/background for changes:
The EPI-STREP-064 BOD ES protocol has been updated for the following reasons:

It has been clarified that:

the antibiotic susceptibility profile (including penicillin, erythromycin, 
azithromycin, tetracycline, levofloxacin, trimethoprim/ sulfamethoxazole, 
amoxicillin/ clavulanate) will be determined for Haemophilus influenzae (H. 
influenzae), Streptococcus pneumoniae (S. pneumoniae) and Moraxella 
catarrhalis (M. catarrhalis) identified not only from bronchoalveolar lavage 
(BAL) fluid but also from nasopharyngeal swab samples;

the beta-lactamase test will be performed for H. influenzae and M. catarrhalis
identified not only from BAL fluid but also from nasopharyngeal swab 
samples;

ampicillin resistance testing will be performed for beta-lactamase negative H. 
influenzae from BAL fluid and from nasopharyngeal swab samples.

In addition, inconsistencies between the secondary endpoints in the synopsis and 
the secondary endpoints in the body of the protocol amendment 1 have been
clarified.
Other changes have been made for simplification, clarification or consistency.
 
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Protocol Amendment 2 Investigator Agreement
I agree:

To conduct the study in compliance with this protocol, any mutually agreed future 
protocol amendments or protocol administrative changes, and with any other study 
conduct procedures and/or study conduct documents provided by GlaxoSmithKline 
Biologicals (GSK Biologicals).

To assume responsibility for the proper conduct of the study at this site.

That I am aware of, and will comply with, ‘Good Clinical Practice’ (GCP) or other 
applicable guidelines and all applicable regulatory requirements.

To ensure that all persons assisting me with the study are adequately informed about 
study-related duties and functions as described in the protocol.

To acquire the reference ranges for laboratory tests performed locally and, if required 
by local regulations, obtain the laboratory’s current certification or Quality 
Assurance procedure manual.

To ensure that no samples are retained onsite or elsewhere without the approval of
GSK Biologicals and the express written informed consent of the subject and/or the 
subject’s legally authorised representative.

To perform no other biological assays on the samples except those described in the 
protocol or its amendment(s).

To co-operate with a representative of GSK Biologicals in the monitoring process of 
the study and in resolution of queries about the data.

That I have been informed that certain regulatory authorities require the sponsor to 
obtain and supply, as necessary, details about the investigator’s ownership interest in 
the sponsor, and more generally about his/her financial ties with the sponsor. GSK 
Biologicals will use and disclose the information solely for the purpose of complying 
with regulatory requirements.
Hence I:

Agree to supply GSK Biologicals with any necessary information regarding 
ownership interest and financial ties (including those of my spouse and dependent 
children).

Agree to promptly update this information if any relevant changes occur during the 
course of the study and for one year following completion of the study. 

Agree that GSK Biologicals may disclose any information it has about such 
ownership interests and financial ties to regulatory authorities.

Agree to provide GSK Biologicals with an updated Curriculum Vitae and other 
documents required by regulatory agencies for this study.
 
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eTrack study number and 
Abbreviated Title
115813 (EPI-STREP-064 BOD ES)
Date of protocol amendment
Amendment 2 Final: 05 October 2015
Detailed Title
A multi-centre, hospital-based, cross-sectional 
epidemiology study to identify and characterise 
bacteria in the lower airways of children aged 6 
months to < 6 years with suspected chronic lower 
respiratory tract infections (LRTIs) in Spain.
Investigator name
Signature
Date
 
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SYNOPSIS
Detailed Title
A multi-centre, hospital-based, cross-sectional epidemiology 
study to identify and characterise bacteria in the lower airways 
of children aged 6 months to < 6 years with suspected 
chronic lower respiratory tract infections (LRTIs) in Spain.
Rationale for the 
study
(Amended 05 October 
2015)
The present study aims to identify and characterise bacteria 
present in the lower airways of children with suspected 
chronic LRTIs and for whom bronchoalveolar lavage (BAL)
is indicated by the clinician. BAL is generally indicated in 
cases of unresolved persistent or recurrent respiratory 
symptoms. Bronchoscopic exploration of the airways along 
with cytology and microbiology results and analysis of other 
constituents of the BAL fluid can provide meaningful 
information for clinical management. This study may provide 
a better understanding of the aetiology of chronic and 
recurrent chronic LRTIs in children for whom a BAL 
sampling procedure was indicated. The ultimate aim is to 
improve treatment recommendations and determine whether 
there are potential prevention strategies including vaccination.
In addition, it would be valuable to identify a reliable, less 
invasive procedure than BAL to describe the bacterial 
aetiology of chronic LRTIs. Linking evidence from BAL 
culture to nasopharyngeal swabs, combined with additional 
laboratory information (since nasopharyngeal swabs alone 
may not be specific enough) may allow for an evaluation of 
new approaches to estimate aetiology. If this could be 
validated, i.e. if a predictor of BAL culture results could be 
identified, then it may be possible to determine the aetiology 
of other, non-chronic or non-recurrent presentations of LRTIs 
for which an invasive procedure such as bronchoscopy would 
not be indicated to guide treatment and clinical management.
Objectives
(Amended 05 October 
2015)
Primary

To characterise the bacterial aetiology of BAL fluid 
samples in subjects visiting the hospital with suspected 
chronic LRTIs.
Secondary
In a hospital setting and among subjects with suspected 
chronic LRTIs:

To describe the bacterial load of H. influenzae, S. 
pneumoniae and M. catarrhalis detected by quantitative 
culture and by molecular techniques (PCR) in the BAL 
 
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fluid. 

To describe the presence of other bacterial pathogens 
detected by qualitative culture in the BAL fluid.

To describe the bacterial load of H. influenzae, S. 
pneumoniae and M. catarrhalis detected by quantitative 
culture and by molecular techniques (PCR) in the 
nasopharyngeal swab samples. 

To describe the presence of other bacterial pathogens 
detected by qualitative culture in the nasopharyngeal 
swab samples.

To describe colonisation of the upper airways by 
characterising H. influenzae, S. pneumoniae, M. 
catarrhalis and other pathogens in nasopharyngeal swab 
samples.

To determine the serotypes of H. influenzae and S. 
pneumoniae identified from BAL fluid samples.

To determine the serotypes of H. influenzae and S. 
pneumoniae identified from nasopharyngeal swab 
samples.

To determine the antibiotic susceptibility profile for H. 
influenzae, S. pneumoniae and M. catarrhalis identified 
from BAL fluid and nasopharyngeal swab samples.

To describe, according to microbiological results 
observed:

Age and gender

Clinical symptoms and radiological evidence

Pneumococcal conjugate vaccine, H. influenzae type 
b vaccine and influenza vaccine status

Medical history and co-morbidities

Information on feeding and day care practice, 
environmental exposure and smoking environment

History of antibiotic use in the past six months as 
well as other treatments for chronic lower respiratory 
disease

Laboratory results [including white blood cell counts, 
C-Reactive Protein (CRP) level and erythrocyte 
sedimentation rate, if available].
Study design

Type of design: Epidemiological, interventional, multi-
centre, hospital-based, cross-sectional study with 
prospective subject recruitment in Spain.
 
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
Study population: Subjects aged 6 months to < 6 years 
visiting the study hospital with suspected chronic LRTI 
and for whom a BAL sample will be collected as per the 
recommendation of the clinician.

Written informed consent will be obtained from the 
parent(s)/ legally acceptable representatives [LAR(s)] of 
those subjects who fulfil the inclusion/ exclusion criteria. 
Subject numbers will be assigned sequentially to subjects 
whose parent(s)/ LAR(s) consent for their participation in 
the study.

Parent(s)/ LAR(s) of the subjects will be interviewed to 
collect data on demographics (e.g. age, gender), clinical 
characteristics (including medical history, chronic LRTI 
symptoms), vaccination history, feeding and day care 
practice, environmental exposure and smoking 
environment, radiological and laboratory results, etc. 
Data on physical examination will also be collected from 
the medical record of the subject. If results of blood 
testing are available from routine practice, then data on 
white blood cell counts, CRP level and erythrocyte 
sedimentation rate, etc will be recorded in the electronic 
Case Report Form (eCRF). 

BAL fluid collection will follow routine procedures and 
will only be performed in children with a medical 
indication for this procedure. The first aliquot of BAL 
fluid will be used for local testing and case management, 
and the second aliquot of BAL fluid obtained will be sent 
to the GlaxoSmithKline (GSK) designated laboratory for 
microbiological analysis.

Nasopharyngeal swabs will be collected as part of this 
study. Collected samples will also be sent to the GSK 
designated laboratory for microbiological analysis.

Type of study: This will be a self-contained study.

Data collection: Remote Data Entry (RDE).

Duration of the study: For each subject, there will be a 
single study visit. The duration of the study will be 
approximately 2 years

Epoch 001: Prospective data collection.
Synopsis Table 1
Study group and epoch foreseen in the study
Study Group
Number of subjects
Age (Min/Max)
Epoch
Prospective
At least 150
6 months to < 6 years
Epoch 001
 
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Discussion of study 
design
A cross-sectional study design is the simplest approach to 
describe the burden of disease, i.e. bacterial aetiology of 
chronic LRTI symptoms. BAL is routinely performed for 
diagnosis of infection in children with such chronic 
symptoms, and it is unlikely to be performed repeatedly for 
otherwise healthy children. Thus, extended follow-up would 
not be possible.
Considering the invasive nature of the BAL fluid sampling 
procedure, it has been decided to enrol only those subjects 
with suspected chronic LRTIs who have BAL procedure 
indicated by their clinician as part of the local routine 
diagnostic procedure. This should facilitate subject 
recruitment and increase participation rates. Nasopharyngeal 
swabs may not be systematically collected in routine practice. 
However, since the patient will be under sedation to undergo 
BAL, he/she will not be subject to additional discomfort or 
risk due to the swabbing procedure. Thus, it is not expected to 
affect the willingness of the parent(s)/ LAR(s) to allow their 
child’s participation in this study.
Number of subjects
At least 150 subjects are planned to be enrolled in the study 
based on the sample size estimation.
Endpoints
(Amended 05 October 
2015)
Primary

Occurrence of H. influenzae, S. pneumoniae, M. 
catarrhalis and other bacteria in BAL fluid samples of 
subjects aged 6 months to < 6 years with suspected 
chronic LRTIs and an indication for BAL

H. influenzae, S. pneumoniae and M. catarrhalis 
confirmed by bacterial load >10^4 cfu/mL if present 
alone or 10^5 cfu/mL if present as co-infection.
Secondary
In a hospital setting and among subjects with suspected 
chronic LRTIs:

Bacterial load of H. influenzae, S. pneumoniae and M. 
catarrhalis in the BAL fluid as determined by 
quantitative culture and PCR. 

Description of other bacterial pathogens in the BAL 
fluid as determined by qualitative culture.

Bacterial load of H. influenzae, S. pneumoniae and M. 
catarrhalis in nasopharyngeal swab samples as 
determined by quantitative culture and PCR. 
 
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
Description of other bacterial pathogens in 
nasopharyngeal swab samples as determined by 
qualitative culture.

Occurrence of H. influenzae, S. pneumoniae, M. 
catarrhalis and other pathogens detected by culture in 
the nasopharyngeal swab samples. 

Occurrence of H. influenzae and S. pneumoniae 
serotypes identified from BAL fluid samples and 
nasopharyngeal swab samples. 

Occurrence of H. influenzae, S. pneumoniae and M. 
catarrhalis minimum inhibitory concentration (MIC) 
values identified from BAL fluid and nasopharyngeal 
swab samples.

Demographic characteristics (age, gender, etc), clinical 
characteristics (including medical history, LRTI 
symptoms, treatment history, vaccination status, etc), 
radiological evidence and laboratory results (including 
white blood cell counts, CRP level and erythrocyte 
sedimentation rate, if available).
 
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TABLE OF CONTENTS
PAGE
SYNOPSIS......................................................................................................................7
LIST OF ABBREVIATIONS...........................................................................................18
GLOSSARY OF TERMS ...............................................................................................19
1.
INTRODUCTION....................................................................................................21
1.1.
Background ................................................................................................21
1.2.
Rationale for the study................................................................................22
2.
OBJECTIVES.........................................................................................................23
2.1.
Primary objective ........................................................................................23
2.2.
Secondary objectives..................................................................................23
3.
STUDY DESIGN OVERVIEW ................................................................................24
3.1.
Discussion of study design .........................................................................25
3.2.
Case definitions ..........................................................................................25
4.
STUDY POPULATION...........................................................................................26
4.1.
Number of subjects/ centres .......................................................................26
4.2.
Inclusion criteria for enrolment....................................................................26
4.3.
Exclusion criteria for enrolment...................................................................26
5.
CONDUCT OF THE STUDY ..................................................................................27
5.1.
Regulatory and ethical considerations, including the informed 
consent process..........................................................................................27
5.2.
Subject identification...................................................................................28
5.3.
General study aspects ................................................................................28
5.4.
Outline of study procedures ........................................................................29
5.5.
Detailed description of study procedures ....................................................29
5.5.1.
Collection of aggregated data from logbooks...............................29
5.5.2.
Procedures prior to study participation.........................................30
5.5.2.1.
Check inclusion and exclusion criteria ........................30
5.5.2.2.
Informed consent........................................................30
5.5.3.
Procedures during study participation..........................................30
5.5.3.1.
Subject number attribution..........................................30
5.5.3.2.
Collect demographic data...........................................30
5.5.3.3.
Clinical characteristics and other information..............30
5.5.3.4.
Collect physical examination data...............................30
5.5.3.5.
Radiological and laboratory results.............................31
5.5.3.6.
Collection of BAL fluid samples and 
nasopharyngeal swabs...............................................31
5.5.3.7.
Recording of Serious Adverse Events ........................31
5.5.3.8.
Study conclusion ........................................................31
5.6.
Biological Sample handling and analysis ....................................................31
5.6.1.
Use of specified study materials ..................................................32
5.6.2.
Biological samples.......................................................................32
5.6.3.
Laboratory assays .......................................................................33
 
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6.
SERIOUS ADVERSE EVENTS..............................................................................34
6.1.
Safety definitions ........................................................................................34
6.1.1.
Definition of an SAE.....................................................................34
6.1.2.
Clinical laboratory parameters and other abnormal 
assessments qualifying as SAEs .................................................35
6.2.
Detecting and recording SAEs....................................................................35
6.2.1.
Time period for detecting and recording SAEs.............................35
6.2.2.
Evaluation of SAEs......................................................................35
6.2.2.1.
Active questioning to detect SAEs..............................35
6.2.2.2.
Assessment of causality .............................................36
6.2.2.3.
Assessment of outcomes............................................37
6.3.
Reporting of SAEs ......................................................................................37
6.3.1.
Prompt reporting of SAEs related to a study procedure to 
GSK Biologicals...........................................................................37
6.3.2.
Contact information for reporting SAEs to GSK Biologicals..........37
6.3.3.
Completion and transmission of SAE reports related to a 
study procedure to GSK Biologicals.............................................37
6.3.3.1.
Back-up system in case the electronic SAE 
reporting system does not work..................................38
6.3.3.2.
Updating of SAE information after freezing of 
the subject’s eCRF .....................................................38
6.4.
Follow-up of SAEs ......................................................................................38
7.
DATA EVALUATION: CRITERIA FOR EVALUATION OF OBJECTIVES...............39
7.1.
Endpoints....................................................................................................39
7.1.1.
Primary endpoint..........................................................................39
7.1.2.
Secondary endpoints ...................................................................39
7.2.
Sample size consideration ..........................................................................40
7.3.
Study cohorts to be evaluated.....................................................................40
7.3.1.
Screened cohort ..........................................................................40
7.3.2.
Total cohort..................................................................................40
7.3.3.
According-To-Protocol cohort ......................................................41
7.4.
Derived and transformed data.....................................................................41
7.5.
Conduct of analyses ...................................................................................41
7.5.1.
Sequence of analyses..................................................................41
7.5.2.
Statistical considerations for interim analyses..............................41
7.6.
Statistical methods......................................................................................42
7.6.1.
Analysis of demographics/ baseline characteristics .....................42
7.6.2.
Analysis of primary objective........................................................42
7.6.3.
Analysis of secondary objectives .................................................42
8.
ADMINISTRATIVE MATTERS ...............................................................................43
8.1.
Remote Data Entry instructions ..................................................................43
8.2.
Monitoring by GSK Biologicals....................................................................43
8.3.
Archiving of data at study sites ...................................................................44
8.4.
Audits .........................................................................................................45
8.5.
Posting of information on public registers....................................................45
8.6.
Ownership, confidentiality and publication ..................................................45
8.6.1.
Ownership ...................................................................................45
8.6.2.
Confidentiality ..............................................................................45
8.6.3.
Publication...................................................................................46
 
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8.6.4.
Provision of study results to investigators, posting to the 
clinical trials registers and publication..........................................46
9.
COUNTRY SPECIFIC REQUIREMENTS...............................................................46
10. REFERENCES.......................................................................................................47
 
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LIST OF TABLES
PAGE
Table 1
Bacterial aetiology of BAL fluid culture from children with 
respiratory symptoms (Amended 05 October 2015) ...............................22
Table 2
Study group and epoch foreseen in the study ........................................25
Table 3
List of study procedures .........................................................................29
Table 4
Biological samples .................................................................................32
Table 5
Laboratory assays (Amended 05 October 2015) ....................................33
Table 6
Timeframes for submitting SAEs related to a study procedure to 
GSK Biologicals .....................................................................................37
 
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LIST OF FIGURES
PAGE
Figure 1
Illustration of the lower (LL) and upper (UL) limits of the exact 
95%CI built around various observed proportions for a sample of 
150 subjects...........................................................................................40
 
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LIST OF APPENDICES
PAGE
APPENDIX A AMENDMENTS AND ADMINISTRATIVE CHANGES TO THE 
PROTOCOL...........................................................................................49
 
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LIST OF ABBREVIATIONS
AIDS
Acquired Immune Deficiency Syndrome
ATP
According-To-Protocol
BAL
Bronchoalveolar Lavage
CI
Confidence Interval
CRP
C-Reactive Protein
eCRF
electronic Case Report Form
GCP
Good Clinical Practice
GSK
GlaxoSmithKline
H. haemolyticus
Haemophilus haemolyticus
H. influenzae
Haemophilus influenzae
HIV
Human Immunodeficiency Virus
ICF
Informed Consent Form
ICH
International Conference on Harmonisation
IEC
Independent Ethics Committee
IRB
Institutional Review Board
LAR
Legally Acceptable Representative
LRTIs
Lower Respiratory Tract Infections
M. catarrhalis
Moraxella catarrhalis
MIC
Minimum Inhibitory Concentration
RDE
Remote Data Entry
SAE
Serious Adverse Event
SDV
Source Document Verification
SPM
Study Procedures Manual
S. pneumoniae
Streptococcus pneumoniae
 
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GLOSSARY OF TERMS
Child in care:
A child who has been placed under the control or 
protection of an agency, organisation, institution or entity 
by the courts, the government or a government body, 
acting in accordance with powers conferred on them by 
law or regulation. The definition of a child in care can 
include a child cared for by foster parents or living in a 
care home or institution, provided that the arrangement 
falls within the definition above. The definition of a child 
in care does not include a child who is adopted or has an 
appointed legal guardian.
Coded:
Information is associated with a subject number i.e. a 
code number. Coded information can only be linked back 
to the individual via a key code i.e. a listing of the 
research participants and their code. Within the 
pharmaceutical industry coding data is the usual 
mechanism used for protecting an individual’s research 
data. The key code is kept secure, usually by the 
investigator, and GSK researchers cannot identify the 
research individual other than in exceptional and 
controlled circumstances.
Eligible:
Qualified for enrolment into the study based upon strict 
adherence to inclusion/exclusion criteria.
Epidemiology study:
An observational study or an interventional study without 
administration of medicinal product(s) as described in a 
research protocol.
Epoch:
An epoch is a well defined part of a protocol that covers a 
set of consecutive time points. Generally, an epoch is 
self-contained and allows to perform a data analysis to 
address some of the trial objectives (e.g. primary, booster, 
yearly follow-ups, retrospective data collection, 
prospective data collection).
eTrack:
GSK’s tracking tool for clinical/epidemiology trials.
Evaluable:
Meeting all eligibility criteria, complying with the 
procedures defined in the protocol, and, therefore, 
included in the according-to-protocol (ATP) analysis (see 
Section 7.3 for details on criteria for evaluability).
Prospective study:
A study in which the subjects/cases are identified and 
then followed forward in time in order to address one or 
more study endpoints.
 
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Research protocol:
A document that describes the objective(s), design, 
methodology, statistical considerations, and organisation 
of a trial. The protocol usually also gives the background 
and rationale for the trial, but these could be provided in 
other protocol referenced documents.
Site Monitor:
An individual assigned by the sponsor who is responsible 
for assuring proper conduct of epidemiology studies at 
one or more investigational sites.
Study population:
Sample of population of interest.
Subject:
Term used throughout the protocol to denote an 
individual who has been contacted in order to participate 
or participates in the epidemiology study.
Subject number:
A unique number identifying a subject, assigned to each 
subject consenting to participate in the study.
 
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1.
INTRODUCTION
1.1.
Background
Lower respiratory tract infections (LRTIs) such as pneumonia and bronchitis, are 
important causes of childhood morbidity and mortality worldwide [Mulholland, 2007; 
Obaro, 2006; Rudan, 2004]. Determining the aetiology of LRTIs in children has always 
been a challenge due to the difficulty in obtaining a sample from the lungs. Streptococcus 
pneumoniae and Haemophilus influenzae are considered to be the main bacterial 
pathogens associated with lobar pneumonia, and their importance in disease was 
described in studies using transthoracic needle aspiration (lung tap), in which a sample 
was collected directly from the site of infection, i.e. the section of consolidation. Studies 
using this technique to describe non-consolidated infections such as broncho-pneumonia 
are less frequent. Lung taps are now rarely used because the potential risk to patients is 
considered unnecessary and LRTIs can be treated with empiric antibiotics [Hausdorff, 
2008]. Thus, there have been attempts to use other less-invasive techniques for describing 
bacterial aetiology. 
Among the non-invasive procedures, blood culture is the most widely used. It is routinely 
performed in patients hospitalised with moderate or severe pneumonia, but this procedure 
yields evidence of a pathogen in only 10-15% of the cases [Mccracken, 2000]. Moreover, 
since blood culture may not be performed for non-hospitalised cases, the role of un-
encapsulated (non-typeable) H. influenzae strains in causing LRTIs may be 
underestimated [Shann, 1999].
Sputum samples, which are considered the best non-invasive alternative for detecting 
bacterial causes of LRTIs in adults, can rarely be collected in children because they often 
have difficulties producing sputum. Thus, the simplest approach of describing the 
aetiology of LRTI has been through the collection of nasopharyngeal samples in 
symptomatic children. Nevertheless, because the main bacteria associated with infection 
are also common colonisers of the nasopharynx, the specificity of such samples alone as 
a measurement of aetiology is considered low. 
Another approach to obtaining a sample from the lower airways is bronchoalveolar 
lavage (BAL). In this procedure, a fiberoptic bronchoscope is introduced into a segmental 
or subsegmental bronchus until the airway is occluded proximally, and saline solution is 
instilled and subsequently aspirated. The fluid recovered can then be submitted for 
cytological and microbiological analysis. BAL is used as a diagnostic and research tool in 
adults with lung problems and in children and infants with underlying respiratory 
diseases [de Blic, 2000]. It is regarded as the best technique for microbacteriological 
surveillance of young children with cystic fibrosis, providing more reliable information 
than sampling of the upper airway with oropharyngeal/ nasopharyngeal swabs or nasal 
lavage [Brennan, 2008]. The main limitation of this procedure is in its invasive nature. As 
contamination from the upper respiratory tract flora during the procedure can sometimes 
occur, its capacity to describe LRTI aetiology may be challenged. Hence, a suitable cut-
off should be used to avoid making spurious conclusions [Woodhead, 2005].
 
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Recent studies reporting results from bacterial culture of BAL fluid in children with 
respiratory symptoms have highlighted the importance of H. influenzae, S. pneumoniae,
and Moraxella catarrhalis in chronic or recurrent presentations of the disease (Table 1).
Table 1
Bacterial aetiology of BAL fluid culture from children with 
respiratory symptoms (Amended 05 October 2015)
Reference
Population
Number of 
samples
H. influenzae S. pneumoniae M. catarrhalis
[De Schutter, 
2011]
Acute nonresponsive CAP (cut off 
≥10^4 CFU/mL)
127
26%
6.3%
8.7%
Recurrent CAP (cut off ≥10^4
CFU/mL)
123
51.2%
7.3%
21.1%
[Hare, 2010]
Bronchiectasis (cut off > 10^4
CFU/mL)
45
47%
18%
20%
[Marchant, 
2006]
Chronic cough of > 3 weeks 
duration of unknown etiology
20
47%
35%
26%
[Saito, 2006]
Persistent bacterial bronchitis 
(persistent, wet cough for >1month 
that resolves with appropriate 
antibiotic treatment)
19
26.3%
21%
42.1%
[Le Bourgeois, 
2002]
Severe recurrent wheezy 
bronchitis unresponsive to inhaled
steroids
30
30%
13.33%
10%
[Marguet, 1999]
5 groups: 
asthma
chronic cough
infantile wheeze
cystic fibrosis
control
72 (total)
12
9
23
10
10
8.33%
33.33%
30.43%
20%
30%
8.33%
22.22%
0.04%
10%
-
16.67%
11.11%
13.04%
-
-
[Schellhase, 
1998]
Recurrent wheezing
27
3.7%
-
7.4%
CAP = community-acquired pneumonia
1.2.
Rationale for the study
The present study aims to identify and characterise bacteria present in the lower airways 
of children with suspected chronic LRTIs and for whom BAL is indicated by the 
clinician. BAL is generally indicated in cases of unresolved persistent or recurrent 
respiratory symptoms. Bronchoscopic exploration of the airways along with cytology and 
microbiology results and analysis of other constituents of the BAL fluid can provide 
meaningful information for clinical management. This study may provide a better 
understanding of the aetiology of chronic and recurrent chronic LRTIs in children for 
whom a BAL sampling procedure was indicated. The ultimate aim is to improve 
treatment recommendations and determine whether there are potential prevention 
strategies including vaccination.
In addition, it would be valuable to identify a reliable, less invasive procedure than BAL 
to describe the bacterial aetiology of chronic LRTIs. Linking evidence from BAL culture 
to nasopharyngeal swabs, combined with additional laboratory information (since 
nasopharyngeal swabs alone may not be specific enough) may allow for an evaluation of 
new approaches to estimate aetiology. If this could be validated, i.e. if a predictor of BAL 
 
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culture results could be identified, then it may be possible to determine the aetiology of 
other, non-chronic or non-recurrent presentations of LRTIs for which an invasive 
procedure such as bronchoscopy would not be indicated to guide treatment and clinical 
management.
2.
OBJECTIVES
2.1.
Primary objective

To characterise the bacterial aetiology of BAL fluid samples in subjects visiting the 
hospital with suspected chronic LRTIs.
Refer to Section 7.1.1 for the definition of the primary endpoint.
2.2.
Secondary objectives
In a hospital setting and among subjects with suspected chronic LRTIs:

To describe the bacterial load of H. influenzae, S. pneumoniae and M. catarrhalis
detected by quantitative culture and by molecular techniques (PCR) in the BAL 
fluid. 

To describe the presence of other bacterial pathogens detected by qualitative culture 
in the BAL fluid.

To describe the bacterial load of H. influenzae, S. pneumoniae and M. catarrhalis
detected by quantitative culture and by molecular techniques (PCR) in the 
nasopharyngeal swab samples.

To describe the presence of other bacterial pathogens detected by qualitative culture 
in the nasopharyngeal swab samples.

To describe colonisation of the upper airways by characterising H. influenzae, S. 
pneumoniae, M. catarrhalis and other pathogens in nasopharyngeal swab samples.

To determine the serotypes of H. influenzae and S. pneumoniae identified from BAL 
fluid samples.

To determine the serotypes of H. influenzae and S. pneumoniae identified from 
nasopharyngeal swab samples.

To determine the antibiotic susceptibility profile for H. influenzae, S. pneumoniae
and M. catarrhalis identified from BAL fluid and nasopharyngeal swab samples.
(Amended 05 October 2015)

To describe, according to microbiological results observed:

Age and gender

Clinical symptoms and radiological evidence

Pneumococcal conjugate vaccine, H. influenzae type b vaccine and influenza 
vaccine status
 
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
Medical history and co-morbidities

Information on feeding and day care practice, environmental exposure and 
smoking environment

History of antibiotic use in the past six months as well as other treatments for 
chronic lower respiratory disease

Laboratory results [including white blood cell counts, C-Reactive Protein (CRP)
level and erythrocyte sedimentation rate, if available]. 
Refer to Section 7.1.2 for the definition of the secondary endpoints.
3.
STUDY DESIGN OVERVIEW

Type of design: Epidemiological, interventional, multi-centre, hospital-based, cross-
sectional study with prospective subject recruitment in Spain.

Study population: Subjects aged 6 months to < 6 years visiting the study hospital 
with suspected chronic LRTI and for whom a BAL sample will be collected as per 
the recommendation of the clinician.

Written informed consent will be obtained from the parent(s)/ legally acceptable 
representatives [LAR(s)] of those subjects who fulfil the inclusion/ exclusion criteria. 
Subject numbers will be assigned sequentially to subjects whose parent(s)/ LAR(s) 
consent for their participation in the study.

Parent(s)/ LAR(s) of the subjects will be interviewed to collect data on demographics 
(e.g. age, gender), clinical characteristics (including medical history, chronic LRTI 
symptoms), vaccination history, feeding and day care practice, environmental 
exposure and smoking environment, radiological and laboratory results, etc. Data on 
physical examination will also be collected from the medical record of the subject. If 
results of blood testing are available from routine practice, then data on white blood 
cell counts, CRP level and erythrocyte sedimentation rate, etc will be recorded in the 
electronic Case Report Form (eCRF). 

BAL fluid collection will follow routine procedures and will only be performed in 
children with a medical indication for this procedure. The first aliquot of BAL fluid 
will be used for local testing and case management, and the second aliquot of BAL 
fluid obtained will be sent to the GlaxoSmithKline (GSK) designated laboratory for 
microbiological analysis.

Nasopharyngeal swabs will be collected as part of this study. Collected samples will 
also be sent to the GSK designated laboratory for microbiological analysis.

Type of study: This will be a self-contained study.

Data collection: Remote Data Entry (RDE).

Duration of the study: For each subject, there will be a single study visit. The 
duration of the study will be approximately 2 years

Epoch 001: Prospective data collection.
 
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Table 2 presents the study group and epoch foreseen in the study.
Table 2
Study group and epoch foreseen in the study
Study Group
Number of subjects
Age (Min/Max)
Epoch
Prospec
At least 150
6 months to < 6 years
Epoch 001
3.1.
Discussion of study design
A cross-sectional study design is the simplest approach to describe the burden of disease, 
i.e. bacterial aetiology of chronic LRTI symptoms. BAL is routinely performed for 
diagnosis of infection in children with such chronic symptoms, and it is unlikely to be 
performed repeatedly for otherwise healthy children. Thus, extended follow-up would not 
be possible.
Considering the invasive nature of the BAL fluid sampling procedure, it has been decided 
to enrol only those subjects with suspected chronic LRTIs who have BAL procedure 
indicated by their clinician as part of the local routine diagnostic procedure. This should 
facilitate subject recruitment and increase participation rates. Nasopharyngeal swabs may 
not be systematically collected in routine practice. However, since the patient will be 
under sedation to undergo BAL, he/she will not be subject to additional discomfort or 
risk due to the swabbing procedure. Thus, it is not expected to affect the willingness of 
the parent(s)/ LAR(s) to allow their child’s participation in this study.
3.2.
Case definitions

Suspected chronic LRTIs where BAL is indicated:
Based on clinical and/or radiological symptoms, suspected chronic LRTI will be 
defined as follows:

Persistent or recurrent respiratory signs/ symptoms that do not respond to usual 
treatment:
i.
Wet cough lasting > 4 weeks without additional associated symptoms
ii.
Recurrent (3 episodes/ year) or persistent wheezing (lasting > 3 months)
iii.
Persistent pathologic auscultation (apart from wheezing) (lasting > 4 weeks).
AND/OR

Persistent (> 1 month) or recurrent (≥ 3 occurrences/ year) infiltrates/ atelectasis 
observed on chest radiograph.
Episode of Wheezing: Being previously healthy, and now presenting pathological 
auscultation with wheezing during long expiration as the main feature.
Evidence of infiltrate/ atelectasis: The presence of new radiological lung
condensation, following radiological evidence of normalisation (between two 
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4.
STUDY POPULATION
4.1.
Number of subjects/ centres
Overview of the recruitment plan:

This study will be conducted in at least 5 hospitals in Spain.

The study population will include subjects aged 6 months to < 6 years visiting the 
study hospital with suspected chronic LRTIs and for whom BAL sampling will be 
performed as per the recommendation of the clinician.

Information on demography and medical history of the subject will be collected. 
BAL samples and nasopharyngeal swabs will be obtained for this study.

At least 150 subjects are planned to be enrolled in the study based on the sample size
estimation. Refer to section 7.2 for a detailed description of the criteria used in the 
estimation of sample size.

The duration of the study will be approximately 2 years from the study initiation. 
There will be a single visit for each subject.

A GSK study staff member or delegate will be responsible for monitoring the study.
4.2.
Inclusion criteria for enrolment
All subjects must satisfy ALL the following criteria at study entry:

Subjects who the investigator believes that parent(s)/ LAR(s) can and will comply 
with the requirements of the protocol.

A male or female subject aged 6 months to < 6 years at the time of enrolment.

Subjects meet the case definition of suspected chronic LRTIs where BAL is 
indicated (as listed under Section 3.2).

Subject’s parent(s)/ LAR(s) agree to the collection of a nasopharyngeal swab from 
the subject.

Written informed consent obtained from the parent(s)/ LAR(s) of the subject.
4.3.
Exclusion criteria for enrolment
The following criteria should be checked at the time of study entry. If ANY exclusion 
criterion applies, the subject must not be included in the study:

Known cystic fibrosis, immunosuppression, or other severe immunodeficiencies 
such as agammaglobulinaemia, T cell deficiency or Human Immunodeficiency Virus 
(HIV)/ Acquired Immune Deficiency Syndrome (AIDS), chemotherapy treatment, 
etc. 
 
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
Exacerbation of persistent respiratory symptoms (cough, wheezing, difficulty in 
breathing, etc) in the previous 2 weeks. Please refer to Section 3.2 for the definition 
of exacerbation of persistent respiratory symptoms.

Antibiotic treatment in the 2 weeks prior to study entry. 

Concurrent participation in another study within 30 days prior to study entry or at 
any time during the study period, in which the subject has been or will be exposed to 
an investigational or a non-investigational product (pharmaceutical product or 
device).

Subjects having previously participated in this study.

Child in care. Please refer to the GLOSSARY OF TERMS for the definition of child 
in care.
A list of criteria that may eliminate subjects from according to protocol (ATP) analyses 
can be found in Section 7.3.
5.
CONDUCT OF THE STUDY
5.1.
Regulatory and ethical considerations, including the 
informed consent process
The study will be conducted in accordance with all applicable regulatory requirements.
The study will be conducted in accordance with the International Conference on 
Harmonisation (ICH) Guideline for Good Clinical Practice (GCP) or other applicable 
guidelines, all applicable subject privacy requirements and the guiding principles of the 
Declaration of Helsinki. 
The study has been designed and will be conducted in accordance with the ICH 
Harmonised Tripartite Guideline for clinical investigation of medicinal products in the 
paediatric population (ICH E11) and all other applicable ethical guidelines.
GSK will obtain favourable opinion/approval to conduct the study from the appropriate 
regulatory agency, in accordance with applicable regulatory requirements, prior to a site 
initiating the study in that country or will document that neither a favourable opinion nor 
an approval to conduct the study is needed.
Conduct of the study includes, but is not limited to, the following:

Institutional Review Board (IRB)/Independent Ethics Committee (IEC) review and 
favourable opinion/ approval of study protocol and any subsequent amendments.

Subject’s parent(s)/LAR(s) informed consent.

Investigator reporting requirements as stated in the protocol.
GSK will provide full details of the above procedures to the investigator, either verbally, 
in writing, or both.
 
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Freely given and written informed consent must be obtained from each subject’s 
parent(s)/LAR(s), as appropriate, prior to participation in the study. 
GSK Biologicals will prepare a model Informed Consent Form (ICF) which will embody 
the applicable ICH GCP and GSK Biologicals required elements. While it is strongly 
recommended that this model ICF be followed as closely as possible, the informed 
consent requirements given in this document are not intended to pre-empt any local 
regulations which require additional information to be disclosed for informed consent to 
be legally effective. Clinical judgement, local regulations and requirements should guide 
the final structure and content of the local version of the ICF. 
The investigator has the final responsibility for the final presentation of the ICF, 
respecting the mandatory requirements of local regulations. The ICF generated by the 
investigator with the assistance of the sponsor’s representative must be acceptable to 
GSK Biologicals and be approved (along with the protocol, and any other necessary 
documentation) by the IRB/IEC.
Prior to the BAL procedure, the investigator will also ask the subject’s parent(s)/LAR(s) 
to sign a hospital ICF with the details of the BAL sampling procedure. This hospital ICF 
is independent of GSK and allows a parent to decide on routine clinical care as separate 
from study participation.
5.2.
Subject identification
Subject numbers will be assigned sequentially to subjects who will be included in the 
study, according to the range of subject numbers allocated to each study centre.
5.3.
General study aspects
Supplementary study conduct information not mandated to be present in this protocol is 
provided in the accompanying Study Procedures Manual (SPM). The SPM provides the 
investigator and the site personnel with administrative and detailed technical information 
that does not impact the safety of the subjects.
 
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5.4.
Outline of study procedures
Table 3 presents the list of study procedures.
Table 3
List of study procedures
Epoch
Prospective data 
collection
Visit
VISIT 1
Timing
Day 0
Check inclusion and exclusion criteria
●
Informed consent and subject number attribution
●
Demographic data
●
Collection of data such as clinical characteristics (medical history, 
chronic LRTI symptoms), vaccination history, feeding and day care 
practice, environmental exposure and smoking environment
●
Physical examination data
●
Collection of radiological and laboratory results (if available)
●
Collection of BAL fluid samples that were obtained as per routine 
practice and nasopharyngeal swabs
●
Send BAL fluid and nasopharyngeal swab samples to designated 
laboratory for microbiological analysis
○
Reporting of SAEs related to the study procedure
●
Laboratory results transcription to database
○
Study conclusion
●
Collection of aggregated data from logbooks
●*
BAL: Bronchoalveolar lavage
● is used to indicate a study procedure that requires documentation in the individual eCRF.
○ is used to indicate a study procedure that does not require documentation in the individual eCRF.
*Aggregated data will be documented in the aggregated logbook module on a monthly basis
5.5.
Detailed description of study procedures
5.5.1.
Collection of aggregated data from logbooks
A logbook will be maintained at the study hospitals in which aggregated data on the 
following will be recorded during the entire study period by year of age, on a monthly 
basis:

Total number of children presenting to the hospital with an indication for BAL. 

Total number of children presenting to the hospital with an indication for BAL that 
may be attributable to chronic LRTI.

Total number of children presenting to the hospital with suspected chronic LRTI 
who have an indication for BAL and are not enrolled due to applying exclusion 
criteria.

Total number of children presenting to the hospital and not being enrolled in the 
study due to refusal of consent by their parent(s)/ LAR(s).
 
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5.5.2.
Procedures prior to study participation
5.5.2.1.
Check inclusion and exclusion criteria
Check all applicable inclusion and exclusion criteria as described in Sections 4.2 and 4.3
before enrolment. 
5.5.2.2.
Informed consent
Before performing any other study procedure, the signed informed consent of the 
subject’s parent(s)/ LAR(s) needs to be obtained. Refer to Section 5.1 for the 
requirements on how to obtain informed consent.
5.5.3.
Procedures during study participation
5.5.3.1.
Subject number attribution
Once a subject meets the eligibility requirements and has been included into the study, a 
unique subject number will be assigned. This subject number will be used to uniquely 
identify all data collected on the subject for the study.
Subject numbers will be assigned sequentially to all subjects, according to the range of 
subject numbers allocated to each study centre. 
5.5.3.2.
Collect demographic data
The parent(s)/ LAR(s) of the subject will be interviewed to collect demographic data such 
as age and gender.
Collected information needs to be recorded in the eCRF.
5.5.3.3.
Clinical characteristics and other information
For all subjects aged 6 months to < 6 years visiting the study hospital with suspected 
chronic LRTIs, the following information will be recorded, but will not be limited to:

Clinical characteristics (including medical history, chronic LRTI symptoms),

Vaccination history,

Feeding and day care practice, 

Environmental exposure and smoking environment.
5.5.3.4.
Collect physical examination data
A physical examination of the subject will be performed as per routine practice, which 
will include assessment of body temperature, heart rate, respiratory rate after at least 10 
 
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minutes of rest, blood pressure and oxygen saturation; the results will be collected as part 
of this study. 
Collected information needs to be recorded in the eCRF.
5.5.3.5.
Radiological and laboratory results
For all subjects aged 6 months to < 6 years visiting the study hospital with suspected 
chronic LRTIs, radiological and laboratory results will be collected, if available. Findings 
from the chest X-ray, (if present) will also be recorded.
If results of blood testing are available from routine practice, then data on white blood 
cell counts, CRP level and erythrocyte sedimentation rate, etc will be recorded in the 
eCRF.
5.5.3.6.
Collection of BAL fluid samples and nasopharyngeal swabs
Following routine BAL procedures at the hospital, the second aliquot of BAL fluid that is 
obtained will be sent to the GSK designated laboratory for microbiological analysis.
Nasopharyngeal swabs will be collected as part of this study. Collected samples will also 
be sent to the GSK designated laboratory for microbiological analysis.
5.5.3.7.
Recording of Serious Adverse Events

The subjects’ parent(s)/LAR(s) will be instructed to contact the investigator 
immediately should the subjects manifest any signs or symptoms they perceive as 
serious.
Refer to Section 6.2 for procedures for the Investigator to record serious adverse events 
(SAEs) that are related to study participation and to Section 6.3 for guidelines on how to 
report these SAEs to GSK Biologicals.
5.5.3.8.
Study conclusion
The investigator will review the data collected to ensure accuracy and completeness and 
will complete the Study Conclusion screen in the eCRF.
5.6.
Biological Sample handling and analysis
Please refer to the SPM for details of biospecimen management (handling, storage and 
shipment).
Samples will not be labelled with information that directly identifies the subjects but will 
be coded with the identification number for the subject (subject number). 
Collected samples may be used in other assays, for test improvement or test development 
of analytical methods related to the disease under study to allow to achieve a more 
 
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reliable measurement. Under these circumstances, additional testing on the samples may 
be performed by GSK Biologicals outside the scope of this protocol.
Information on further investigations and their rationale can be obtained from GSK
Biologicals.
Any sample testing will be done in line with the consent of the individual subject’s 
parent(s)/ LAR(s).
Any human pharmacogenetic testing will require specific consent from the individual 
subject’s parent(s)/ LAR(s) and the ethics committee approval. Any human 
immunodeficiency virus (HIV) testing will also require specific consent and ethics 
committee approval.
Refer also to the Investigator Agreement, where it is noted that the Investigator cannot 
perform any other biological assays except those described in the protocol or its 
amendment(s).
Collected samples will be stored for up to 15 years (counting from when the last subject 
performed the last study visit), unless local rules, regulations or guidelines require 
different timeframes or different procedures, which will then be in line with the subject 
consent. These extra requirements need to be communicated formally to and discussed 
and agreed with GSK Biologicals.
For routine sample collection when coded sample is shipped to GSK and needs to be 
linked to coded subject data, (re-) consent from the subjects and/or IEC/IRB approval 
will be sought. 
5.6.1.
Use of specified study materials
When materials are provided by GSK Biologicals, it is MANDATORY that all samples 
be collected and stored exclusively using those materials in the appropriate manner. The 
use of other materials could result in the exclusion of the subject from the ATP analysis 
(See Section 7.3 for the definition of study cohorts to be evaluated). The investigator 
must ensure that his/her personnel and the laboratory(ies) under his/her supervision 
comply with this requirement. However, when GSK Biologicals does not provide 
material for collecting and storing samples, appropriate materials from the investigator’s 
site must be used. Refer to the Module on Clinical Trial Supplies in the SPM.
5.6.2.
Biological samples
Table 4 presents the biological samples that will be collected for this study.
Table 4
Biological samples
Sample type
Quantity
Time point
BAL
At least 2 mL
Visit 1
Day 0
Nasopharyngeal swab
1 swab
 
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5.6.3.
Laboratory assays
Laboratory assays to characterise BAL fluid and nasopharyngeal swab samples including 
the detection of S. pneumoniae, H. influenzae and M. catarrhalis will be performed by 
standard techniques at GSK Biologicals’ laboratory or in a validated laboratory 
designated by GSK Biologicals using standardised and validated procedures (refer to 
Table 5).
Table 5
Laboratory assays (Amended 05 October 2015)
Pathogen/ 
Marker
Method
Scale
Sample type
Qualitative
Quantitative
BAL
NP
Identification and/ or quantification of bacteria
S. 
pneumoniae 
Culture
X
X
X
Molecular 
techniques (PCR)
X
H. influenzae
Culture
X
X
X
Molecular 
techniques (PCR)
(differentiation 
between Hi and 
Haemophilus 
haemolyticus)†
X
M. catarrhalis
Culture
X
X
X
Molecular 
techniques (PCR)
X
Other 
pathogens (if 
detected)
Culture
X
X
X
Serotyping of bacterial pathogens
S. 
pneumoniae
Agglutination 
assay or 
molecular 
techniques
X
X
X
H. influenzae 
Monovalent 
antisera 
X
X
X
Antibiotic sensitivity :
Including penicillin, erythromycin, azithromycin, tetracycline, levofloxacin, trimethoprim/ 
sulfamethoxazole, amoxicillin/ clavulanate susceptibility testing for H. influenzae, S. pneumoniae 
and M. catarrhalis isolated from BAL and NP swab samples;
and additionally a beta-lactamase test for H. influenzae and M. catarrhalis isolated from BAL and 
NP swab samples;
and ampicillin resistance testing by broth microdilution for beta-lactamase negative H. 
influenzae isolated from BAL and NP swab samples.
†: PCR differentiation between Hi and H. haemolyticus will be performed on isolates from BAL/ NP swab samples that 
are H. influenzae positive
Collected samples will be used for purposes related to the quality assurance of data 
generated within the scope of this protocol, such as for maintenance of assays described 
in this protocol and comparison between analytical methods and/or laboratories.
 
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6.
SERIOUS ADVERSE EVENTS
The procedure of nasopharyngeal sampling will be performed on subjects enrolled in the 
study to collect nasopharyngeal swab samples.
Only SAEs related to the nasopharyngeal sampling procedure that occur until the subject 
is discharged will be recorded. 
The investigator or site staff is/are responsible during the study for the detection and 
documentation of events meeting the criteria and definition of an SAE as provided in this 
protocol.
Each subject’s parent(s)/LAR(s) will be instructed to contact the investigator immediately 
should the subject manifest any signs or symptoms they perceive as serious.
6.1.
Safety definitions
6.1.1.
Definition of an SAE
An SAE is any adverse event that:
a.
Results in death,
b.
Is life-threatening,
NB: The term ‘life-threatening’ in the definition of ‘serious’ 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, had it been more severe.
c.
Requires hospitalisation or prolongation of existing hospitalisation,
NB: In general, hospitalisation signifies that the subject has been admitted at the 
hospital or emergency ward for observation and/or treatment that would not have 
been appropriate in the physician’s office or in an out-patient setting. 
Complications that occur during hospitalisation are also considered AEs. If a 
complication prolongs hospitalisation or fulfils any other serious criteria, the event 
will also be considered serious. When in doubt as to whether ‘hospitalisation’ 
occurred or was necessary, the AE should be considered serious.
Hospitalisation for elective treatment of a pre-existing condition (known or 
diagnosed prior to informed consent signature) that did not worsen from baseline is 
NOT considered an SAE.
d.
Results in disability/incapacity,
NB: The term disability means a substantial disruption of a person’s ability to 
conduct normal life functions. This definition is not intended to include experiences 
of relatively minor medical significance such as uncomplicated headache, nausea, 
vomiting, diarrhoea, influenza like illness, and accidental trauma (e.g. sprained 
 
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ankle) which may interfere or prevent everyday life functions but do not constitute a 
substantial disruption.
Medical or scientific judgement should be exercised in deciding whether reporting is 
appropriate in other situations, such as important medical events that may not be 
immediately life-threatening or result in death or hospitalisation but may jeopardise the 
subject or may require medical or surgical intervention to prevent one of the other 
outcomes listed in the above definition. These should also be considered serious. 
Examples of such events are invasive or malignant cancers, intensive treatment in an 
emergency room or at home for allergic bronchospasm, blood dyscrasias or convulsions 
that do not result in hospitalisation.
6.1.2.
Clinical laboratory parameters and other abnormal assessments 
qualifying as SAEs
Abnormal laboratory findings (e.g. clinical chemistry, haematology, urinalysis) or other 
abnormal assessments that are judged by the investigator to be clinically significant will 
be recorded as SAEs if they meet the definition of an SAE, as defined in Section 6.1.1. 
Clinically significant abnormal laboratory findings or other abnormal assessments that 
are detected during the study or are present at baseline and significantly worsen following 
the start of the study will be reported as SAEs. However, clinically significant abnormal 
laboratory findings or other abnormal assessments that are associated with the disease 
being studied, unless judged by the investigator as more severe than expected for the 
subject’s condition, or that are present or detected at the start of the study and do not 
worsen, will not be reported as SAEs.
The investigator will exercise his or her medical and scientific judgement in deciding 
whether an abnormal laboratory finding or other abnormal assessment is clinically 
significant.
6.2.
Detecting and recording SAEs
6.2.1.
Time period for detecting and recording SAEs
In order to fulfil international reporting obligations, SAEs that are related to study 
participation (i.e. protocol-mandated procedures, invasive tests, a change from existing 
therapy) or any fatal SAE will be collected and recorded from the time the parent(s)/ 
LAR(s) of the subject consents to allow participation in the study and will last until the 
subject is discharged.
6.2.2.
Evaluation of SAEs
6.2.2.1.
Active questioning to detect SAEs
Each subject’s parent(s)/ LAR(s) will be instructed to contact the investigator 
immediately should the subject manifest any signs and symptoms they perceive as 
serious.
 
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All SAEs either observed by the investigator or his/her staff or reported by the subject’s 
parent(s)/ LAR(s) spontaneously or in response to a direct question will be evaluated by 
the investigator. The nature of each event, data and time (where appropriate) of onset, 
outcome, intensity and relationship to the study procedures should be established.
When an SAE occurs, it is the responsibility of the investigator to review all 
documentation (e.g. hospital progress notes, laboratory, and diagnostics reports) relative 
to the event. The investigator will then record all relevant information regarding an SAE 
on the eCRF or SAE Report screens as applicable. It is not acceptable for the investigator 
to send photocopies of the subject’s medical records to GSK Biologicals instead of the 
appropriate completed SAE screens in the eCRF. However, there may be instances when 
copies of medical records for certain cases are requested by GSK Biologicals. In this 
instance, all subject identifiers will be blinded on the copies of the medical records prior 
to submission to GSK Biologicals.
The investigator will attempt to establish a diagnosis pertaining to the event based on 
signs, symptoms, and/or other clinical information. In such cases, the diagnosis should be 
documented as the SAE and not the individual signs/symptoms.
6.2.2.2.
Assessment of causality
The investigator should assess the causality of each SAE. The investigator will use 
clinical judgement to determine the relationship of SAEs to study procedures. Alternative 
causes, such as natural history of the underlying diseases, concomitant therapy and other 
risk factors will be considered and investigated.
There may be situations when an SAE has occurred and the investigator has minimal 
information to include in the initial report to GSK Biologicals. However it is very 
important that the investigator always makes an assessment of causality for every event 
prior to submission of the SAE to GSK Biologicals. The investigator may change his/her 
opinion of causality in light of follow-up information, amending the SAE information 
accordingly. 
If an event meets the criteria to be determined ‘serious’ (refer to Section 6.1.1), it will be 
examined by the investigator to the extent to enable determination of all contributing 
factors applicable to each SAE. 
Possible contributing factors include:

Medical history.

Concomitant medication.

Protocol required procedure.

Other procedure not required by the protocol.

Other cause (specify).
 
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6.2.2.3.
Assessment of outcomes
Outcome of any SAE reported during the entire study will be assessed as:
∀
Recovered/resolved.
∀
Recovering/resolving.
∀
Not recovered/not resolved.
∀
Recovered with sequelae/resolved with sequelae.
∀
Fatal (SAEs only).
6.3.
Reporting of SAEs
All SAEs occurring until the subject is discharged and considered related to the 
nasopharyngeal sampling procedure must be recorded on the SAE screen.
6.3.1.
Prompt reporting of SAEs related to a study procedure to GSK 
Biologicals
SAEs that occur in the time period defined in Section 6.2.1 will be reported promptly to 
GSK within the timeframes described in Table 6 once the investigator determines that the 
event meets the protocol definition of an SAE.
Table 6
Timeframes for submitting SAEs related to a study procedure to 
GSK Biologicals
Type of Event
Initial Reports
Follow-up of Relevant Information
on a Previous Report
Timeframe
Documents
Timeframe
Documents
SAEs related 
to a study 
procedure
24 hours*
SAE screen
24 hours*
SAE screen
* Timeframe allowed after receipt or awareness of the information.
6.3.2.
Contact information for reporting SAEs to GSK Biologicals
Please see the Sponsor Information Sheet for contact details. 
Back-up Study Contact for Reporting SAEs
24/24 hour and 7/7 day availability:
GSK Biologicals Clinical Safety & Pharmacovigilance
Fax: +
 or +
6.3.3.
Completion and transmission of SAE reports related to a study 
procedure to GSK Biologicals
Once an investigator becomes aware that an SAE has occurred in a study subject, the 
investigator (or designate) must complete the information in the SAE screens of the 
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eCRF WITHIN 24 HOURS. The SAE screens will always be completed as thoroughly as 
possible with all available details of the event. Even if the investigator does not have all 
information regarding an SAE, the SAE screens should still be completed within 24 
hours. Once additional information is received, the SAE screens in the eCRF should be 
updated WITHIN 24 HOURS.
The investigator will always provide an assessment of causality at the time of the initial 
report.
6.3.3.1.
Back-up system in case the electronic SAE reporting system does not 
work
If the electronic SAE reporting system does not work, the investigator (or designate) must 
complete, then date and sign a SAE Report Form and fax it to the GSK Biologicals 
Clinical Safety and Pharmacovigilance department within 24 hours. 
This back-up system should only be used if the electronic SAE reporting system is not 
working and NOT if the system is slow. As soon as the electronic SAE reporting system 
is working again, the investigator (or designate) must complete the SAE screens in the 
eCRF within 24 hours. The final valid information for regulatory reporting will be the 
information reported through the electronic SAE reporting system. 
6.3.3.2.
Updating of SAE information after freezing of the subject’s eCRF
When additional information is received on a SAE after freezing of the subject’s eCRF, 
new or updated information is to be recorded on a SAE Report Form, with all changes 
signed and dated by the investigator. The updated form should be faxed to the GSK 
Biologicals Clinical Safety and Pharmacovigilance department or to the Study Contact 
for Reporting SAEs (refer to the Sponsor Information Sheet) WITHIN 24 HOURS of 
receipt of the follow-up information.
6.4.
Follow-up of SAEs
After the initial SAE report, the investigator is required to proactively follow each subject 
and provide further information on the subject’s condition to GSK Biologicals.
All SAEs documented at the visit and designated as not recovered/not resolved or 
recovering/resolving will be reviewed until the end of the study.
Investigators will follow-up subjects:

With SAEs until the event has resolved, subsided, stabilised, disappeared, or until the 
event is otherwise explained, or the subject is lost to follow-up. 
Clinically significant laboratory abnormalities will be followed up until they have 
returned to normal, or a satisfactory explanation has been provided. Additional 
information (including but not limited to laboratory results) relative to the subsequent 
course of such abnormalities noted for any subject must be made available to the Site 
Monitor.
 
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GSK Biologicals may request that the investigator performs or arranges for the conduct 
of additional clinical examinations/tests and/or evaluations to elucidate as fully as 
possible the nature and/or causality of the SAE. The investigator is obliged to assist. If a 
subject dies during participation in the study or during a recognised follow-up period, 
GSK Biologicals will be provided with a copy of any available post-mortem findings, 
including histopathology.
7.
DATA EVALUATION: CRITERIA FOR EVALUATION OF 
OBJECTIVES
7.1.
Endpoints
7.1.1.
Primary endpoint

Occurrence of H. influenzae, S. pneumoniae, M. catarrhalis and other bacteria in 
BAL fluid samples of subjects aged 6 months to < 6 years with suspected chronic 
LRTIs and an indication for BAL

H. influenzae, S. pneumoniae and M. catarrhalis confirmed by bacterial load 
>10^4 cfu/mL if present alone or 10^5 cfu/mL if present as co-infection.
7.1.2.
Secondary endpoints
In a hospital setting and among subjects aged 6 months to < 6 years with suspected 
chronic LRTIs and an indication for BAL:

Bacterial load of H. influenzae, S. pneumoniae and M. catarrhalis in the BAL fluid 
as determined by quantitative culture and PCR.

Description of other bacterial pathogens in the BAL fluid as determined by 
qualitative culture.

Bacterial load of H. influenzae, S. pneumoniae and M. catarrhalis in nasopharyngeal 
swab samples as determined by quantitative culture and PCR.

Description of other bacterial pathogens in nasopharyngeal swab samples as 
determined by qualitative culture.

Occurrence of H. influenzae, S. pneumoniae, M. catarrhalis and other pathogens 
detected by culture in the nasopharyngeal swab samples. 

Occurrence of H. influenzae and S. pneumoniae serotypes identified from BAL fluid 
samples and nasopharyngeal swab samples. 

Occurrence of H. influenzae, S. pneumoniae and M. catarrhalis minimum inhibitory 
concentration (MIC) values identified from BAL fluid and nasopharyngeal swab
samples. (Amended 05 October 2015)

Demographic characteristics (age, gender, etc), clinical characteristics (including 
medical history, LRTI symptoms, treatment history, vaccination status, etc), 
 
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radiological evidence and laboratory results (including white blood cell counts, CRP 
level and erythrocyte sedimentation rate, if available).
7.2.
Sample size consideration
The proportions of S. pneumoniae and H. influenzae among all BAL fluid samples 
collected is expected to be between 4% and 35%, and between 7% and 51%, respectively 
[Schellhase, 1998; Marguet, 1999; Marchant, 2006; De Schutter, 2011].
Figure 1 provides the precision that can be expected around an estimated proportion of S. 
pneumoniae and H. influenzae for a sample size of at least 150 subjects. For example, the 
exact 95% confidence interval (CI) for an observed proportion of 11.3% is 6.7; 17.5. The 
exact 95% CI for an observed proportion of 33.3% is 25.9; 41.5.
Figure 1
Illustration of the lower (LL) and upper (UL) limits of the exact 95%CI 
built around various observed proportions for a sample of 150 
subjects
7.3.
Study cohorts to be evaluated
7.3.1.
Screened cohort
The screened cohort will include all subjects who visit the hospital with suspected 
chronic LRTIs and have an indication for BAL. All the information (anonymised) for 
these subjects will be collected in the aggregated logbook.
7.3.2.
Total cohort
The total cohort will include all enrolled subjects.
 
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7.3.3.
According-To-Protocol cohort
The ATP cohort will include all evaluable subjects (i.e. those meeting all eligibility 
criteria and complying with the procedures defined in the protocol during the study) with 
BAL fluid sampling and/ or nasopharyngeal swab sampling results available.
A detailed, comprehensive list of reasons for elimination from ATP analyses will be 
established at the time of data cleaning.
7.4.
Derived and transformed data
Age at time of enrolment in the study will be computed as the difference between the date 
of enrolment [date when the ICF was signed by the parent(s)/ LAR(s)] and the date of 
birth. The age will be expressed in months.
The age will be grouped into the following classes 6-11, 12-23, 24-35, 36-47, 48-59 and 
60-71 months.
The duration of hospitalisation will be computed as the difference between the date of 
discharge and the date of admission +1.
The pneumococcal conjugate vaccination status will defined as:

Vaccinated, if the subject either received at least two doses or received only one 
dose after one year of age;

Unvaccinated, if the subject did not receive any dose or received only one dose 
within the first year of life;

Unknown otherwise.
Temperature will be converted to axillary route temperature (if route is rectal or tympanic 
rectal, then value will be 0.5°C). If this derived value of temperature is ≥37.5°C, then the 
subject is considered to have fever.
7.5.
Conduct of analyses
Any deviation(s) or change(s) from the original statistical plan outlined in this protocol 
will be described and justified in the final study report.
7.5.1.
Sequence of analyses
The analyses, as described in Sections 7.6.2 and 7.6.3, will be performed in one step, 
when all data, will be available and cleaned. These analyses and associated individual 
data will be presented in a final study report.
7.5.2.
Statistical considerations for interim analyses
No interim analysis is planned for this study.
 
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7.6.
Statistical methods
Demographic, clinical and microbiological characteristics of subjects will be described
using percentages for categorical variables and mean (standard deviation) or median 
(min-max) for continuous variables. The same variables will be presented descriptively, 
overall and by microbiological results.
7.6.1.
Analysis of demographics/ baseline characteristics
The number of screened and enrolled subjects as well as the number excluded from ATP 
analyses will be presented. Demographic characteristics (age and gender) will be 
summarised using descriptive statistics. 
7.6.2.
Analysis of primary objective
The proportions and associated exact 2-sided 95% confidence intervals (CI) of H. 
influenzae, S. pneumoniae, M. catarrhalis and other bacteria confirmed by culture in 
BAL samples will be computed and presented for all study subjects. The denominators 
will be the number of tested subjects.
7.6.3.
Analysis of secondary objectives
The distribution (with number of non-missing observations, geometric mean, standard 
deviation, median, minimum and maximum and number of missing observations) of the 
bacterial load of H. influenzae, S. pneumoniae, M. catarrhalis and other pathogens will 
be described according to the testing method (culture or PCR) and to the samples (BAL 
fluid sample or nasopharyngeal swab sample).
The serotype distribution (number and percentage) of H. influenzae and S. pneumoniae
detected in BAL fluid will be presented in frequency tables. 
The bacterial aetiology and serotype distribution of nasopharyngeal samples will be 
described along with that of BAL fluid samples.
The proportions and associated exact 2-sided 95% CI of H. influenzae, S. pneumoniae, M. 
catarrhalis and other bacterial pathogens confirmed by culture in nasopharyngeal swab 
will be computed for all study subjects. 
For the antibiotic susceptibility, H. influenzae, S. pneumoniae and M. catarrhalis isolates 
(detected by culture of BAL fluid and nasopharyngeal swab), classified as susceptible, 
intermediate or resistant (following Clinical and Laboratory Standards Institute
guidelines [CLSI, 2015]) will be tabulated by bacterial pathogen and antimicrobial agent.
(Amended 05 October 2015)
 
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8.
ADMINISTRATIVE MATTERS
To comply with ICH GCP administrative obligations relating to data collection, 
monitoring, archiving data, audits, confidentiality and publications must be fulfilled. 
8.1.
Remote Data Entry instructions
Remote Data Entry (RDE), a validated computer application, will be used as the method 
for data collection. 
In all cases, subject initials will not be collected nor transmitted to GSK. Subject data 
necessary for analysis and reporting will be entered/transmitted into a validated database 
or data system. Clinical data management will be performed in accordance with 
applicable GSK standards and data cleaning procedures.
While completed eCRFs are reviewed by a GSK Biologicals’ Site Monitor at the study 
site, omissions or inconsistencies detected by subsequent eCRF review may necessitate 
clarification or correction of omissions or inconsistencies with documentation and 
approval by the investigator or appropriately qualified designate. In all cases, the 
investigator remains accountable for the study data. 
The investigator will be provided with a CD-ROM of the final version of the data 
generated at the investigational site once the database is archived and the clinical study 
report is complete and approved by all parties.
8.2.
Monitoring by GSK Biologicals
Monitoring visits by a GSK Site Monitor are for the purpose of confirming that GSK 
Biologicals’ sponsored studies are being conducted in accordance with the ethical 
principles that have their origins in the Declaration of Helsinki and that are consistent 
with Good Clinical practice (GCP) and the applicable regulatory requirement(s) 
(verifying continuing compliance with the protocol, amendment(s), verifying that the site 
staff and facilities continue to be adequate to conduct the study). 
The investigator must ensure provision of reasonable time, space and qualified personnel 
for monitoring visits.
Direct access to all study-site related and source data is mandatory for the purpose of
monitoring review. The monitor will perform an RDE review and a Source Document 
Verification (SDV). By SDV we understand verifying RDE entries by comparing them 
with the source data that will be made available by the investigator for this purpose. 
The Source Documentation Agreement Form describes the source data for the different 
data in the RDE. This document should be completed and signed by the site monitor and 
investigator and should be filed in the monitor’s and investigator’s study file. Any data 
item for which the RDE will serve as the source must be identified, agreed and 
documented in the source documentation agreement form. 
 
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For RDE, the monitor will mark completed and approved screens at the visit.
In accordance with applicable regulations, GCP, and GSK procedures, GSK monitors 
will contact the site prior to the start of the study to review with the site staff the protocol, 
study requirements, and their responsibilities to satisfy regulatory, ethical, and GSK 
requirements. When reviewing data collection procedures, the discussion will also 
include identification, agreement and documentation of data items for which the eCRF
entries will serve as the source document.
GSK will monitor the study to verify that, amongst others, the: 

Data are authentic, accurate, and complete.

Safety and rights of subjects are being protected.

Study is conducted in accordance with the currently approved protocol and any 
amendments, any other study agreements, GCP and all applicable regulatory 
requirements. 
The investigator and the head of the medical institution (where applicable) agrees to 
allow the monitor direct access to all relevant documents.
Upon completion or premature discontinuation of the study, the monitor will conduct site 
closure activities with the investigator or site staff, as appropriate, in accordance with 
applicable regulations, GCP, and GSK procedures.
8.3.
Archiving of data at study sites
Following closure of the study, the investigator must maintain all site study records in a 
safe and secure location. The records must be maintained to allow easy and timely 
retrieval, when needed (e.g. audit or inspection), and, whenever feasible, to allow any 
subsequent review of data in conjunction with assessment of the facility, supporting 
systems, and staff. Where permitted by applicable laws/regulations or institutional policy, 
some or all of these records can be maintained in a validated format other than hard copy 
(e.g. microfiche, scanned, electronic for studies with an eCRF); however, caution needs 
to be exercised before such action is taken. The investigator must assure that all 
reproductions are legible and are a true and accurate copy of the original and meet 
accessibility and retrieval standards, including re-generating a hard copy, if required. 
Furthermore, the investigator must ensure there is an acceptable back-up of these 
reproductions and that an acceptable quality control process exists for making these 
reproductions.
GSK will inform the investigator/institution of the time period for retaining these records 
to comply with all applicable regulatory requirements. However, the 
investigator/institution should seek the written approval of the sponsor before proceeding 
with the disposal of these records. The minimum retention time will meet the strictest 
standard applicable to that site for the study, as dictated by ICH GCP any institutional 
requirements or applicable laws or regulations, or GSK standards/procedures; otherwise, 
the minimum retention period will default to 15 years. 
 
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The investigator/institution must notify GSK of any changes in the archival 
arrangements, including, but not limited to, the following: archival at an off-site facility, 
transfer of ownership of the records in the event the investigator leaves the site. 
8.4.
Audits
To ensure compliance with GCP and all applicable regulatory requirements, GSK may 
conduct a quality assurance audit. Regulatory agencies may also conduct a regulatory 
inspection of this study. Such audits/inspections can occur at any time during or after 
completion of the study. If an audit or inspection occurs, the investigator and institution 
agree to allow the auditor/inspector direct access to all relevant documents and to allocate 
his/her time and the time of his/her staff to the auditor/inspector to discuss findings and 
any relevant issues.
8.5.
Posting of information on public registers
Study information from this protocol will be posted on public registers (e.g. GSK Clinical 
Study Register, clinicaltrials.gov) before enrolment of subjects begins as applicable.
8.6.
Ownership, confidentiality and publication
8.6.1.
Ownership
All information provided by GSK and all data and information generated by the site as 
part of the study (other than a subject’s medical records) are the sole property of GSK.
All rights, title, and interests in any inventions, know-how or other intellectual or 
industrial property rights which are conceived or reduced to practice by site staff during 
the course of or as a result of the study are the sole property of GSK, and are hereby 
assigned to GSK.
If a written contract for the conduct of the study which includes ownership provisions 
inconsistent with this statement is executed between GSK and the study site, that 
contract’s ownership provisions shall apply rather than this statement.
8.6.2.
Confidentiality
Documented evidence that a potential investigator is aware and agrees to the confidential 
nature of the information related to the study must be obtained by means of a 
confidentiality agreement.
All information provided by GSK and all data and information generated by the site as 
part of the study (other than a subject’s medical records) will be kept confidential by the 
investigator and other site staff. This information and data will not be used by the 
investigator or other site personnel for any purpose other than conducting the study. 
These restrictions do not apply to: (i) information which becomes publicly available 
through no fault of the investigator or site staff; (ii) information which it is necessary to 
 
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disclose in confidence to an IEC or IRB solely for the evaluation of the study; (iii) 
information which it is necessary to disclose in order to provide appropriate medical care 
to a study subject; or (iv) study results which may be published as described in the next 
paragraph. If a written contract for the conduct of the study which includes 
confidentiality provisions inconsistent with this statement is executed, that contract’s 
confidentiality provisions shall apply rather than this statement.
8.6.3.
Publication
Prior to submitting for publication, presentation, use for instructional purposes, or 
otherwise disclosing the study results generated by the site (collectively, a ‘Publication’), 
the investigator shall provide GSK with a copy of the proposed Publication and allow 
GSK a period to review the proposed Publication (at least twenty-one working days, or at 
least fifteen working days for abstracts/posters/presentations). Proposed Publications 
shall not include either GSK confidential information other than the study results or 
personal data on any subject, such as name or initials.
At GSK’s request, the submission or other disclosure of a proposed Publication will be 
delayed a sufficient time to allow GSK to seek patent or similar protection of any 
inventions, know-how or other intellectual or industrial property rights disclosed in the 
proposed Publication.
If a written contract for the conduct of the study, which includes publication provisions 
inconsistent with this statement is executed, that contract’s publication provisions shall 
apply rather than this statement.
8.6.4.
Provision of study results to investigators, posting to the clinical 
trials registers and publication
Where required by applicable regulatory requirements, an investigator signatory will be 
identified for the approval of the study report. The investigator will be provided 
reasonable access to statistical tables, figures, and relevant reports and will have the 
opportunity to review the complete study results at a GSK site or other mutually-
agreeable location.
A manuscript will be progressed for publication in the scientific literature if the results 
provide important scientific or medical knowledge.
9.
COUNTRY SPECIFIC REQUIREMENTS
Not applicable.
 
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10.
REFERENCES
Brennan S, Gangell C, Wainwright C, Sly PD. Disease surveillance using 
bronchoalveolar lavage. Paediatric Respiratory Reviews, 2008; 9: 151–159.
de Blic J, Midulla F, Barbato A et al. Bronchoalveolar lavage in children. Eur Respir J,
2000; 15: 217-231.
Clinical and Laboratory Standards Institute (CLSI). M100-S25 Performance 
Standards for Antimicrobial Susceptibility Testing. January 2015. 
De Schutter I, De Wachter E, Crokaert F, et al. Microbiology of Bronchoalveolar Lavage 
Fluid in Children With Acute Nonresponding or Recurrent Community-Acquired 
Pneumonia: Identification of Nontypeable Haemophilus influenzae as a Major Pathogen. 
Clinical Infectious Diseases, 2011; 52 (12): 1437–1444.
Hare KM, Grimwood K, Leach AJ, et al. Respiratory Bacterial Pathogens in the 
Nasopharynx and Lower Airways of Australian Indigenous Children with Bronchiectasis. 
The Journal of Pediatrics, 2010; 157: 1001-1005.
Hausdorff WP and Dagan R. Serotypes and pathogens in paediatric pneumonia. Vaccine, 
2008; 26S: B19–B23.
Le Bourgeois M, Goncalves M, Le Clainche L, et al. Bronchoalveolar Cells in Children 
<3 Years Old With Severe Recurrent Wheezing. Chest, 2002; 122: 791–797.
Marchant JM, Masters B, Taylor SM, et al. Evaluation and Outcome of Young Children 
With Chronic Cough. Chest, 2006; 129:1132–1141.
Marguet C, Jouen-Boedes F, Dean TP, et al. Bronchoalveolar Cell Profiles in Children 
with Asthma, Infantile Wheeze, Chronic Cough, or Cystic Fibrosis. Am J Respir Crit 
Care Med, 1999; 159: 1533–1540.
Mccracken GH. Etiology and treatment of pneumonia. Pediatr Infect Dis J, 2000; 19: 
373–377.
Mulholland K. Childhood pneumonia mortality- a permanent global emergency. Lancet, 
2007; 370: 285–289.
Obaro SK and Madhi SA. Bacterial pneumonia vaccines and childhood pneumonia: are 
we winning, refining, or redefining? Lancet Infect Dis, 2006; 6: 150–161.
Rudan I, Tomaskovic L, Boschi-Pinto C, et al. Global estimate of the incidence of 
clinical pneumonia among children under five years of age. Bulletin of the World Health 
Organization, 2004; 82 (12): 895–903.
Saito J, Harris WT, Gelfond J, et al. Physiologic, Bronchoscopic, and Bronchoalveolar 
Lavage Fluid Findings in Young Children With Recurrent Wheeze and Cough. Pediatric 
Pulmonology, 2006; 41: 709–719.dEA
 
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Schellhase DE, Fawcett DD, Schutze GE, et al. Clinical utility of flexible bronchoscopy 
and bronchoalveolar lavage in young children with recurrent wheezing. J Pediatr, 1998; 
132: 312–318.
Shann F. Haemophilus influenzae pneumonia: type b or non-type b? The Lancet, 1999; 
354: 1488–1490.
Woodhead M, Blasi F, Ewig S, et al. Guidelines for the management of adult lower 
respiratory tract infections. Eur Respir J, 2005; 26: 1138–1180.
 
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APPENDIX A
AMENDMENTS AND ADMINISTRATIVE 
CHANGES TO THE PROTOCOL
GlaxoSmithKline Biologicals
Clinical Research & Development
Protocol Amendment 1
eTrack study number 
and Abbreviated Title
115813 (EPI-STREP-064 BOD ES)
Amendment number:
Amendment 1
Amendment date:
Amended: 08 March 2013
Co-ordinating author:
, Scientific Writer
Rationale/background for changes:
It was initially intended to ship the collected bronchoalveolar lavage (BAL) fluid and 
nasopharyngeal swabs to the GSK designated laboratory (Instituto Valenciano de 
Microbiologia [IVAMI], Spain) within 4-6 hours of collection. For some sites, this is 
not feasible from the operational point of view due to conflicting hospital schedules 
and limited availability of daily flights between the two provinces participating in this 
study. One of the proposals was to bring in a technician from IVAMI for plating of 
samples within 4-6 hours, which again would not be cost-effective and may also create 
differences in test results between the centres, making it difficult to compare the results 
at the end of the study.
An alternative approach that has been decided is immediate freezing of the collected 
BAL fluid and nasopharyngeal swab samples between -70∃to -80∃C. As a result, the 
tertiary objective of comparing the bacterial load of the samples plated within 4-6 hours 
with delayed plating (22-26 hours and 46-50 hours) cannot be met.
Amended text has been included in bold italics in the following sections:
1.
Title page: List of contributing authors:
#
, Study Data Manager
#
, Laboratory Study Manager, Business and Decision for 
GSK Biologicals
#
, Global Study Manager (Consultant, Harrison Clinical 
Research)
#
, Local Study Coordinator
2.
Sponsor signatory page
, Director, Life cycle Management, Epidemiology, GlaxoSmithKline 
Biologicals
, GVD-Director, Pediatric Vaccines-Epidemiology, GlaxoSmithKline 
Biologicals
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3.
Synopsis-Rationale for study and Section 1.2 (Rationale for the study):
The study also provides an opportunity to understand more technical aspects of the BAL 
procedure, which might be useful for future studies. For instance, the effect of delayed 
plating of BAL fluid samples for bacterial culture has not been documented. Since the 
maximum time recommended before BAL sample processing is 4-6 hours, comparing 
bacterial load in BAL samples plated within the standard recommendation of 4-6 hours 
with delayed plating (22-26 hours and 46-50 hours) may provide updated logistical 
recommendations that could be helpful for future multi-centre studies.
4.
Synopsis –Tertiary objective and Section 2.3 (Tertiary objective) has been 
deleted.
To compare the bacterial load detected by quantitative culture in a subset of up to 50% of 
the collected BAL samples plated within standard recommended 4-6 hours with delayed 
plating (22-26 hours and 46-50 hours). 
Refer to Section 7.1.3 for the definition of the tertiary endpoint.
5.
Synopsis-Tertiary Endpoint and Section 7.1.3 (Tertiary Endpoint) has been 
deleted.
Occurrence of bacteria as determined by quantitative culture in a subset of up to 50% of 
the collected BAL samples plated within standard recommended 4-6 hours and with 
delayed plating (22-26 hours and 46-50 hours).
6.
Section 5.6.2 – Biological Samples
Table 4
Biological samples
Sample type
Quantity
Time point
BAL
Maximum possible At least 2mL
Visit 1
Day 0
Nasopharyngeal swab
1 swab
 
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7.
Section 5.6.3-Laboratory assays
Table 5
Laboratory assays
Pathogen/ 
Marker
Method
Scale
Plating timelines
Sample type
Qualitative
Quantitative
BAL
NP
Identification and/ or quantification of bacteria 
S. pneumoniae 
Culture
X
X
Within 4-6h
after collection
(Culture)
X
X
Molecular 
techniques (PCR)
X
X
Culture
X
X
at 22-26h
at 46-50h
after first plating*
X
H. influenzae
Culture
X
X
Within 4-6h
after collection
(Culture)
X
X
Molecular 
techniques (PCR)
X
X
Culture
X
X
at 22-26h
at 46-50h
after first plating*
X
M. catarrhalis
Culture
X
X
Within 4-6h
after collection
(Culture)
X
X
Molecular 
techniques (PCR)
X
X
Culture
X
X
at 22-26h
at 46-50h
after first plating*
X
Other 
pathogens (if 
detected)
Culture
X
Within 4-6h
after collection
X
X
Serotyping of bacterial pathogens
S. pneumoniae
Agglutination assay 
or molecular 
techniques
X
X
X
H. influenzae 
Monovalent antisera 
and
Molecular 
techniques 
(differentiation 
between Hi and 
Haemophilus 
haemolyticus)†
X
X
X
X
Antibiotic sensitivity*: Including penicillin, erythromycin, azithromycin, tetracycline, levofloxacin, trimethoprim/ 
sulfamethoxazole, amoxicillin/ clavulanate; and additionally a beta-lactamase test for H. influenzae and M. catarrhalis
*: Delayed plating should be performed for every second BAL sample that is collected, as applicable
†: PCR differentiation between Hi and H. haemolyticus will be performed on isolates from BAL/ NP swab samples that 
are H. influenzae positive
*: For BAL samples only
8.
Section 7.6
Statistical methods: Section 7.6.3 (Analysis of tertiary 
objective) has been deleted.
The comparison of bacterial load will be done for at least 50% of the enrolled subjects 
using cross-tables for each bacteria and as per the plating time.
 
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GlaxoSmithKline Biologicals
Clinical Research & Development
Protocol Amendment 2
eTrack study number 
and Abbreviated Title
115813 (EPI-STREP-064 BOD ES)
Amendment number:
Amendment 2
Amendment date:
05 October 2015
Co-ordinating author:
, Scientific Writer, Keyrus Biopharma for 
GSK Biologicals
Rationale/background for changes:
The EPI-STREP-064 BOD ES protocol has been updated for the following reasons:
∀
It has been clarified that: 
#
the antibiotic susceptibility profile (including penicillin, erythromycin, 
azithromycin, tetracycline, levofloxacin, trimethoprim/ sulfamethoxazole, 
amoxicillin/ clavulanate) will be determined for Haemophilus influenzae (H. 
influenzae), Streptococcus pneumoniae (S. pneumoniae) and Moraxella 
catarrhalis (M. catarrhalis) identified not only from bronchoalveolar lavage 
(BAL) fluid but also from nasopharyngeal swab samples;
#
the beta-lactamase test will be performed for H. influenzae and M. catarrhalis
identified not only from BAL fluid but also from nasopharyngeal swab 
samples;
#
ampicillin resistance testing will be performed for beta-lactamase negative H. 
influenzae from BAL fluid and from nasopharyngeal swab samples.
∀
In addition, inconsistencies between the secondary endpoints in the synopsis and 
the secondary endpoints in the body of the protocol amendment 1 have been 
clarified.
Other changes have been made for simplification, clarification or consistency.
Amended text has been included in bold italics and deleted text in strikethrough in 
the following sections:
Title page - List of contributing authors:
∀
, Senior Manager, Epidemiology
∀
, Study Statistician, freelance for GSK Biologicals
∀
, Study Delivery Lead
∀
, Study Delivery Manager
∀
, Study Data Manager, TCS for GSK Biologicals
∀
, Safety Associate

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∀
, GVCL Project Manager
∀
, GVCL Study Manager
∀
, Local Delivery Lead Local Study Coordinator
Sponsor signatory page:
, Director, Life cycle Management, Head of Global Epidemiology, 
GlaxoSmithKline Biologicals
Synopsis – Rationale for the study:
The present study aims to identify and characterise bacteria present in the lower airways 
of children with suspected chronic LRTIs and for whom bronchoalveolar lavage (BAL)
is indicated by the clinician.
Synopsis – Secondary objectives and Section 2.2. Secondary objectives:
To determine the antibiotic susceptibility profile for H. influenzae, S. pneumoniae and M. 
catarrhalis identified from BAL fluid and nasopharyngeal swab samples.
Synopsis - Secondary endpoints:
In a hospital setting and among subjects with suspected chronic LRTIs:
∀
To describe the bacterial load of H. influenzae, S. pneumoniae and M. catarrhalis
detected by quantitative culture and by molecular techniques (PCR) in the BAL 
fluid. 
∀
To describe the presence of other bacterial pathogens detected by qualitative culture 
in the BAL fluid.
∀
To describe the bacterial load of H. influenzae, S. pneumoniae and M. catarrhalis
detected by quantitative culture and by molecular techniques (PCR) in the 
nasopharyngeal swab samples.
∀
To describe the presence of other bacterial pathogens detected by qualitative culture 
in the nasopharyngeal swab samples.
∀
To describe colonisation of the upper airways by characterising H. influenzae, S. 
pneumoniae, M. catarrhalis and other pathogens in nasopharyngeal swab samples.
∀
To determine the serotypes of H. influenzae and S. pneumoniae identified from BAL 
fluid samples.
∀
To determine the serotypes of H. influenzae and S. pneumoniae identified from 
nasopharyngeal swab samples.
∀
To determine the antibiotic susceptibility profile for H. influenzae, S. pneumoniae
and M. catarrhalis identified from BAL fluid samples.

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
To describe, according to microbiological results observed:

Age and gender

Clinical symptoms and radiological evidence

Pneumococcal conjugate vaccine, H. influenzae type b vaccine and influenza 
vaccine status

Medical history and co-morbidities

Information on feeding and day care practice, environmental exposure and 
smoking environment

History of antibiotic use in the past six months as well as other treatments for 
chronic lower respiratory disease

Laboratory results [including white blood cell counts, C-Reactive Protein (CRP) 
level and erythrocyte sedimentation rate, if available].

Bacterial load of H. influenzae, S. pneumoniae and M. catarrhalis in the BAL 
fluid as determined by quantitative culture and PCR. 

Description of other bacterial pathogens in the BAL fluid as determined by 
qualitative culture.

Bacterial load of H. influenzae, S. pneumoniae and M. catarrhalis in 
nasopharyngeal swab samples as determined by quantitative culture and PCR. 

Description of other bacterial pathogens in nasopharyngeal swab samples as 
determined by qualitative culture.

Occurrence of H. influenzae, S. pneumoniae, M. catarrhalis and other pathogens 
detected by culture in the nasopharyngeal swab samples. 

Occurrence of H. influenzae and S. pneumoniae serotypes identified from BAL 
fluid samples and nasopharyngeal swab samples. 

Occurrence of H. influenzae, S. pneumoniae and M. catarrhalis minimum 
inhibitory concentration (MIC) values identified from BAL fluid and 
nasopharyngeal swab samples.

Demographic characteristics (age, gender, etc), clinical characteristics (including 
medical history, LRTI symptoms, treatment history, vaccination status, etc), 
radiological evidence and laboratory results (including white blood cell counts, 
CRP level and erythrocyte sedimentation rate, if available).
 
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Section 1.1. Background:
Table 1 Bacterial aetiology of BAL fluid culture from children with respiratory 
symptoms
Reference
Population
Number of 
samples
H. influenzae S. pneumoniae M. catarrhalis
[De Schutter, 
2011]
Acute nonresponsive CAP (cut off 
≥ 104 10^4 CFU/mL)
127
26%
6.3%
8.7%
Recurrent CAP (cut off ≥ 104 
10^4 CFU/mL)
123
51.2%
7.3%
21.1%
[Hare, 2010]
Bronchiectasis (cut off > 104 10^4
CFU/mL)
45
47%
18%
20%
[Marchant, 
2006]
Chronic cough of > 3 weeks 
duration of unknown etiology
20
47%
35%
26%
[Saito, 2006]
Persistent bacterial bronchitis 
(persistent, wet cough for >1month 
that resolves with appropriate 
antibiotic treatment)
19
26.3%
21%
42.1%
[Le Bourgeois, 
2002]
Severe recurrent wheezy 
bronchitis unresponsive to inhaled
steroids
30
30%
13.33%
10%
[Marguet, 1999]
5 groups: 
asthma
chronic cough
infantile wheeze
cystic fibrosis
control
72 (total)
12
9
23
10
10
8.33%
33.33%
30.43%
20%
30%
8.33%
22.22%
0.04%
10%
-
16.67%
11.11%
13.04%
-
-
[Schellhase, 
1998]
Recurrent wheezing
27
3.7%
-
7.4%
CAP = community-acquired pneumonia
 
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Section 5.6.3. Laboratory assays:
Table 5 Laboratory analysis
Pathogen/ 
Marker
Method
Scale
Sample type
Qualitative
Quantitative
BAL
NP
Identification and/ or quantification of bacteria
S. 
pneumoniae 
Culture
X
X
X
Molecular 
techniques (PCR)
X
H. influenzae
Culture
X
X
X
Molecular 
techniques (PCR)
(differentiation 
between Hi and 
Haemophilus 
haemolyticus)†
X
M. catarrhalis
Culture
X
X
X
Molecular 
techniques (PCR)
X
Other 
pathogens (if 
detected)
Culture
X
X
X
Serotyping of bacterial pathogens
S. 
pneumoniae
Agglutination assay 
or molecular 
techniques
X
X
X
H. influenzae
Monovalent antisera 
and
Molecular 
techniques 
(differentiation 
between Hi and 
Haemophilus 
haemolyticus)†
X
X
X
Antibiotic sensitivity :
Including penicillin, erythromycin, azithromycin, tetracycline, levofloxacin, trimethoprim/ sulfamethoxazole, 
amoxicillin/ clavulanate susceptibility testing for H. influenzae, S. pneumoniae and M. catarrhalis isolated 
from BAL and NP swab samples;
and additionally a beta-lactamase test for H. influenzae and M. catarrhalis isolated from BAL and NP swab 
samples;
and ampicillin resistance testing by broth microdilution for beta-lactamase negative H. influenzae isolated 
from BAL and NP swab samples.
†: PCR differentiation between Hi and H. haemolyticus will be performed on isolates from BAL/ NP swab samples that 
are H. influenzae positive
*: For BAL samples only
 
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Section 7.1.2. Secondary endpoints:

Occurrence of H. influenzae, S. pneumoniae and M. catarrhalis minimum inhibitory 
concentration (MIC) values identified from BAL fluid and nasopharyngeal swab
samples.
Section 7.6.3. Analysis of secondary objectives:
The serotype distribution (number and percentage) of H. influenzae and S. pneumoniae
and other bacterial isolates detected in BAL fluid will be presented in frequency tables. 
For the antibiotic susceptibility, H. influenzae, and S. pneumoniae and M. catarrhalis 
isolates (detected by culture of BAL fluid and nasopharyngeal swab), classified as 
susceptible, intermediate or resistant (following Clinical and Laboratory Standards 
Institute CLSI guidelines [CLSI, 2015]) will be tabulated by bacterial pathogen and 
antimicrobial agent.
Section 10. References:
Clinical and Laboratory Standards Institute (CLSI). M100-S25 Performance 
Standards for Antimicrobial Susceptibility Testing. January 2015.
 
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PPD