A Cohort Study to Assess the Burden of Dengue Illness in Household Members From Selected Communities in Southeast Asia and Latin America
Phase: NA
Conditions: Dengue, Dengue Vaccines
Sponsor: GlaxoSmithKline

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Study Protocol
Sponsor:
GlaxoSmithKline Biologicals
14200 Shady Grove Road
Rockville, MD 20850 USA
eTrack study number and 
Abbreviated Title
200318 (EPI-DENGUE-007 BOD)
Date of protocol
Final Version 2: 17 April 2015
Date of protocol amendment
Amendment 1 Final: 28 January 2016
Amendment 2 Final: 11 May 2017
Amendment 3 Final: 24 July 2017
Title
A cohort study to assess the burden of dengue illness 
in household members from selected communities in 
Southeast Asia and Latin America.
Detailed Title
A prospective, multi-centre, cohort study to assess 
the burden of dengue illness in household members 
(aged between 6 months and 50 years) from selected 
communities in Southeast Asia and Latin America.
Co-ordinating author
, Scientific Writer
Contributing authors
(Amended: 24 July 2017)
!
, Senior Manager, 
Epidemiology
!
, Director, Epidemiology
!
, 
, 
 
, Study Delivery Leads
!
, Clinical Development and 
Research Lead
!
, Specialist, Biostatistics
!
, Statistician
!
, Lead Statistician
!
, Study Data Manager, for GSK 
Biologicals, from Tata Consultancy Services
!
, Study Data Manger, for GSK 
Biologicals for Tata Consultancy Services
!
,
, Clinical Safety 
representatives
!
, Clinical read-out Lead 
(CRTL), CLS
!
, Lab study manager
!
, Director, Regional Epidemiology 
and Health Outcomes, Asia-Pacific
!
, Senior Manager, 
Epidemiologist, LATAM
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 14.1.1
Copyright 2015-2017 of the GlaxoSmithKline group of companies. All rights reserved. 
Unauthorised copying or use of this information is prohibited.

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Protocol Amendment 3 Sponsor Signatory Approval
eTrack study number and 
Abbreviated Title
200318 (EPI-DENGUE-007 BOD)
Date of protocol Amendment
Amendment 3 Final: 24 July 2017
Detailed Title
A prospective, multi-centre, cohort study to assess 
the burden of dengue illness in household members 
(aged between 6 months and 50 years) from 
selected communities in Southeast Asia and Latin 
America.
Sponsor signatory
Robert Paris
US RDC
Clinical & Epidemiology Project Lead
GlaxoSmithKline Biologicals
Signature
Date
 
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Protocol Amendment 3 Rationale
Amendment number:
Amendment 3
Rationale/background for changes:
This amendment was developed to clarify that a blood sample will be collected for 
testing related to tertiary endpoints even when dengue is ruled out by the study 
physician during clinical examination.
 
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Protocol Amendment 3 Investigator Agreement
I agree:

To conduct the study in compliance with this protocol, any mutually agreed future 
protocol amendments or protocol administrative changes, with the terms of the study 
agreement and with any other study conduct procedures and/or study conduct 
documents provided by GlaxoSmithKline (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 (including serum 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 acceptable 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
200318 (EPI-DENGUE-007 BOD)
Date of protocol amendment
Amendment 3 Final: 24 July 2017
Detailed Title
A prospective, multi-centre, cohort study to assess 
the burden of dengue illness in household members 
(aged between 6 months and 50 years) from selected 
communities in Southeast Asia and Latin America.
Investigator name
Signature
Date
 
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Sponsor Information
1.
Sponsor
GlaxoSmithKline Biologicals
14200 Shady Grove Road
Rockville, MD 20850 USA
2.
Sponsor Medical Expert for the Study
Refer to the local study contact information document.
3.
Sponsor Study Monitor
Refer to the local study contact information document.
4.
Study Contact for Reporting of a Serious Adverse Event (SAE)
GSK Biologicals Central Back-up Study Contact for Reporting SAEs: refer to
protocol Section 8.3.2.
 
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SYNOPSIS
Detailed Title
A prospective, multi-centre, cohort study to assess the burden 
of dengue illness in household members (aged between 6 
months and 50 years) from selected communities in Southeast 
Asia and Latin America.
Rationale for the 
study 
The purpose of this study is to describe the burden of DENV 
illness among household members aged 6 months to 50 years 
of selected communities in Latin America and Southeast Asia. 
The study will describe the immune profile of study 
participants against DENV and possibly other flaviviruses at 
enrolment.
This study is also seeking to maintain/set up febrile illness 
surveillance and operational capacity in sites in countries of 
Latin America and Southeast Asia for the targeted study 
population (6 months to 50 years) with the perspective of 
preparing these sites for future dengue vaccine efficacy 
studies.
Objectives
Primary

To estimate the overall incidence rate of reverse-
transcriptase quantitative polymerase chain reaction (RT-
qPCR)-confirmed symptomatic DENV infection in a 
multi-centre cohort of household members aged 6 months 
to 50 years.
Secondary

To estimate the incidence rate of virologically confirmed
(through RT-qPCR or NS1) and probable (based on 
serological evidence) symptomatic DENV cases, 
separately and combined - by age, gender, site, serotype 
(if applicable) and anti-DENV IgG serological status at 
enrolment.

To estimate the prevalence of anti-DENV 
immunoglobulin G (IgG) antibodies against dengue in the 
study population, overall and by age and site (at 
enrolment), for all participants and among dengue cases
(confirmed and probable).

To describe clinical presentations of dengue cases
(confirmed and probable).
 
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Tertiary

To describe the spatial and temporal distribution of 
dengue cases (confirmed and probable) among cohort 
participants in the study areas and analyse determinants 
of spatio-temporal transmission (e.g. environmental, 
entomological, socio-demographic or ecological factors).

To describe the DENV, and other flaviviruses (i.e. 
Japanese Encephalitis Virus [JEV; mostly relevant in 
Southeast Asia]) or West Nile Virus [WNV; mostly 
relevant in Latin America]) neutralising antibody profile 
in a subset of subjects.

To explore other infectious aetiologies than dengue in 
subjects with episodes of febrile illness referred to as 
“suspected dengue case” (chikungunya, Zika, influenza, 
leptospirosis).

To explore determinants of past dengue infection and 
dengue illness.

To further characterise the immune status at enrolment 
(e.g. antibody avidity, etc.).
Study design

Type of design: Prospective, multicentre, household-
based, cohort study/surveillance study.

Study population: Subjects aged between 6 months and 
50 years at the time of enrolment living in 
geographically-defined communities in Latin America 
and Southeast Asia. The study population will comprise 
household members. The study population should include 
between 30% and 50% of adults (aged 18 years or above) 
per site.

Recruitment: The appropriate recruitment strategy will be 
selected by each participating site. Two approaches may 
be considered: a school-based approach and a 
community-based approach without school involvement.
A school-based approach: Recruitment will be initiated 
in primary and/or secondary schools. The 
parent(s)/legally acceptable representatives [LAR(s)] of 
the student attending participating schools will be asked 
to allow voluntary participation of their child/ward. The 
parent(s)/LAR(s) of the student or those living in the 
same household will also be invited to enrol 
himself/herself/themselves (if 50 years) and their 
younger/older child who may not be attending the study 
school(s). Other household members (if 50 years) will 
also be invited to participate in the study.
 
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A community-based approach without school 
involvement: Recruitment will be organised by study 
staff at the household level and/or other participating 
sites. Study staff could also conduct home visits for 
recruitment but collection of blood sample will be done 
preferably at a designated facility.

Duration of longitudinal follow-up and number of visits:
Each subject will have 3 scheduled visits at enrolment, at 
Month 12 and a conclusion visit at Month 24. The Month 
12 and Month 24 visits may occur via a telephone contact 
for the Mexican site. The study duration will be 24 
months for each participating site. In countries with 
marked seasonality, the recruitment period would 
preferably occur outside the period of peak incidence of 
dengue, based on the local epidemiology of dengue in the 
past years and preferentially outside the holiday period (if 
the investigator believes that families are more likely to 
leave the study area during this period). These periods 
will be described for each site and may be modified upon
mutual agreement between the investigator and the 
central study team based on available epidemiological 
information.

Type of study: self-contained.

Data collection: Electronic Case Report Form (eCRF).

Duration of the study: Because of differences in local 
regulations, dengue seasonality and holiday period 
between countries, the recruitment period will be 
staggered. However, to facilitate site activities, sites will 
target to have all the subjects recruited in a 3-month 
period. For each subject, the study duration will be 24
months.

Epoch 001: Prospective data collection starting at Visit 1 
(Day 0) and ending at the conclusion visit (Month 24).
Synopsis Table 1
Study groups and epochs foreseen in the study
Study Groups
Number of 
subjects
Age (Min/Max)
Epochs
Epoch 001
Prospective
1750
6 months to 50 years 
x
Discussion of study 
design
A prospective cohort study design has been selected as it 
allows estimation of the incidence of infection in a defined 
population (with a known denominator), describing the 
spectrum of clinical outcomes and year-to-year variation. 
The study population will be composed of individuals aged 6 
 
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months to 50 years. Households represent a platform of 
research to understand the transmission dynamics and 
spectrum of disease among closely-related individuals. 
The study will include multiple sites, mainly because it will 
contribute to developing or maintaining the field infrastructure 
required for future vaccine phase III efficacy studies. In this 
perspective, multiple sites in different countries in Asia and 
Latin America will need to be involved to capture significant 
number of dengue cases caused by all four dengue serotypes. 
The study may allow for the detection of differences across 
sites in the transmission pattern.
The age range of the study population is large (6 months-50 
years), including infants, young children, adolescents and 
adults. DENV infections affect human population of all age 
groups worldwide [WHO, 2012b]. Due to potential reduction 
of vaccine efficacy by maternal antibodies in infants and a 
reduced memory response [van Panhuis, 2011], infants aged < 
6 months are currently not targeted for vaccination and thus, 
will not be included in this epidemiological study. 
Number of subjects
Approximately, 1750 subjects are expected to be enrolled into 
this study with about 300-500 subjects expected per study site. 
Subjects aged between 6 months and 50 years at the time of 
enrolment living in geographically-defined communities in 
Latin America and Southeast Asia will be part of this study.
Endpoints
Primary

RT-qPCR confirmed symptomatic DENV infection (all 
DENV types) during the study period. 
Secondary

DENV-type specific confirmed symptomatic DENV 
infection.

Virologically confirmed (by RT-qPCR or NS1) and 
probable (based on serological evidence) symptomatic 
DENV infection. 

Status for anti-DENV IgG at enrolment (indicative of past 
DENV infection).

Symptoms and severity of symptomatic dengue. 
 
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Tertiary
Exploratory objectives may be assessed based on, but not 
limited to the following endpoints:

Spatial coordinates of the houses of participating 
households and of dengue cases and date of symptoms 
onset (applicable only for symptomatic cases).

Neutralising antibodies titres against DENV 1-4.

Neutralising antibody titres against other flaviviruses 
(such as JEV and WNV).

Other infectious aetiology than dengue in subjects with 
episodes of febrile illness referred to as “suspected 
dengue case” (chikungunya, Zika, influenza, 
leptospirosis)

Determinants of past dengue infection and dengue illness.

Characteristics of immune status against DENV infection
(e.g.: antibody avidity, etc.).
 
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TABLE OF CONTENTS
PAGE
SPONSOR INFORMATION ............................................................................................6
SYNOPSIS......................................................................................................................7
LIST OF ABBREVIATIONS...........................................................................................19
GLOSSARY OF TERMS ...............................................................................................21
1.
INTRODUCTION....................................................................................................24
1.1.
Background ................................................................................................24
1.2.
Rationale for the study................................................................................25
2.
BENEFIT: RISK ASSESSMENT.............................................................................26
2.1.
Risk Assessment ........................................................................................26
2.2.
Benefit Assessment....................................................................................26
2.3.
Overall Benefit: Risk Conclusion.................................................................27
3.
OBJECTIVES.........................................................................................................27
3.1.
Primary objective ........................................................................................27
3.2.
Secondary objectives..................................................................................27
3.3.
Tertiary objectives.......................................................................................27
4.
STUDY DESIGN OVERVIEW ................................................................................28
4.1.
Discussion of study design .........................................................................29
4.1.1.
Rationale for study design ...........................................................29
5.
CASE DEFINITIONS..............................................................................................30
5.1.
Suspected symptomatic dengue case (SDC)..............................................30
5.2.
Laboratory confirmation ..............................................................................31
5.3.
Classification of severity of dengue.............................................................31
5.3.1.
The 1997 WHO classification [WHO, 1997] .................................32
5.3.2.
The 2009 WHO classification [WHO, 2009, Gutiérrez, 
2013; Macedo, 2014] ...................................................................32
6.
STUDY POPULATION...........................................................................................34
6.1.
Number of subjects/ centres .......................................................................34
6.2.
Overview of recruitment plan ......................................................................34
6.2.1.
Recruitment of study centres .......................................................35
6.3.
Inclusion criteria for subject enrolment........................................................35
6.4.
Exclusion criteria for subject enrolment.......................................................36
7.
CONDUCT OF THE STUDY ..................................................................................36
7.1.
Regulatory and ethical considerations, including the informed 
consent process..........................................................................................36
7.2.
Subject and household number attribution..................................................38
7.3.
General study aspects ................................................................................38
7.4.
Outline of study procedures ........................................................................39
7.5.
Detailed description of study procedures ....................................................42
 
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7.5.1.
Procedures at scheduled visit 1 (enrolment) ................................42
7.5.1.1.
Informed consent and assent .....................................43
7.5.1.2.
Check inclusion and exclusion criteria ........................43
7.5.1.3.
Check evidence of subject being member of 
the household.............................................................43
7.5.1.4.
Attribute subject number and household 
number.......................................................................43
7.5.1.5.
Collect spatial coordinates of the households.............43
7.5.1.6.
Record socio-demographic and household 
information .................................................................43
7.5.1.7.
Physical examination, medical history and 
JE/YF/dengue vaccination history...............................44
7.5.1.8.
Distribute subject ID card and instruction kit ...............44
7.5.1.9.
Collect blood sample (Amended: 24 July 2017)..........44
7.5.1.10.
Instruct/ remind subjects/subject’s 
parent(s)/LAR(s) or designate on assessment 
procedures in case of acute febrile illness ..................44
7.5.1.11.
Issue diary logs to subject/subject’s 
parent(s)/LAR(s) or designate and train them 
on how it is to be filled out in the event of an 
acute febrile illness.....................................................44
7.5.1.12.
Report Serious Adverse Events (SAEs) related 
to study procedures....................................................44
7.5.2.
Procedures at Visit 2 and conclusion visit (Visit 3) 
(Amended: 24 July 2017).............................................................45
7.5.3.
Dengue surveillance ....................................................................45
7.5.4.
Procedures at unscheduled visits and management of 
SDC (Amended: 24 July 2017) ....................................................46
7.6.
Biological sample handling and analysis.....................................................47
7.6.1.
Use of specified study materials ..................................................48
7.6.2.
Biological samples (Amended: 24 July 2017)...............................49
7.6.3.
Laboratory assays for primary and secondary endpoints .............49
7.6.4.
Laboratory assays to be performed for tertiary endpoints 
(Amended: 24 July 2017).............................................................51
7.6.5.
Biological samples evaluation......................................................52
8.
SAFETY (AMENDED: 24 JULY 2017)....................................................................53
8.1.
Safety definitions ........................................................................................54
8.1.1.
Definition of a serious adverse event ...........................................54
8.1.1.1.
Clinical laboratory parameters and other 
abnormal assessments qualifying as SAEs ................55
8.2.
Detecting and recording SAEs....................................................................55
8.2.1.
Time periods for detecting and recording SAEs ...........................55
8.2.2.
Evaluation of SAEs......................................................................56
8.2.2.1.
Active questioning to detect SAEs..............................56
8.2.2.2.
Assessment of the intensity of SAEs ..........................57
8.2.2.3.
Assessment of causality .............................................57
8.2.2.4.
Assessment of outcomes............................................58
8.3.
Reporting of SAEs ......................................................................................58
8.3.1.
Prompt reporting of SAEs related to study participation to 
GSK.............................................................................................58
8.3.2.
Contact information for reporting SAEs to GSK............................59
 
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8.3.3.
Completion and transmission of SAEs reports related to 
study participation to GSK ...........................................................59
8.3.3.1.
Back-up system in case the electronic 
reporting system does not work..................................59
8.3.4.
Updating of SAE information after freezing of the subject’s 
eCRF...........................................................................................59
8.3.5.
Regulatory reporting requirements for SAEs................................60
8.4.
Follow-up of SAEs ......................................................................................60
8.4.1.
Follow-up during the study...........................................................60
8.4.2.
Follow-up after the subject is discharged from the study..............60
9.
SUBJECT COMPLETION AND WITHDRAWAL.....................................................61
9.1.
Subject completion .....................................................................................61
9.2.
Subject withdrawal......................................................................................61
10. STATISTICAL METHODS......................................................................................62
10.1.
Endpoints....................................................................................................62
10.1.1.
Primary endpoint..........................................................................62
10.1.2.
Secondary endpoints ...................................................................62
10.1.3.
Tertiary endpoints........................................................................62
10.2.
Determination of sample size......................................................................62
10.3.
Cohorts for Analyses ..................................................................................65
10.3.1.
Total cohort..................................................................................65
10.3.2.
According-To-Protocol (ATP) cohort ............................................65
10.4.
Derived and transformed data.....................................................................65
10.5.
Analysis of demographics...........................................................................65
10.6.
Descriptive analyses of laboratory results...................................................65
10.7.
Analysis of primary objective.......................................................................66
10.8.
Analysis of secondary objectives ................................................................66
10.9.
Other analyses ...........................................................................................67
10.10. Interpretation of analyses............................................................................68
10.11. Conduct of analyses ...................................................................................68
10.11.1. Sequence of analyses..................................................................68
10.11.2. Statistical considerations for interim analyses..............................68
11. ADMINISTRATIVE MATTERS ...............................................................................68
11.1.
Electronic Case Report Form instructions...................................................68
11.2.
Study monitoring by GSK Biologicals..........................................................69
11.3.
Record retention .........................................................................................70
11.4.
Quality assurance.......................................................................................70
11.5.
Posting of information on publicly available registers and publication 
policy ..........................................................................................................70
11.6.
Provision of study results to investigators ...................................................71
11.7.
Collection, analysis, storage of spatial data ................................................71
12. COUNTRY SPECIFIC REQUIREMENTS...............................................................71
12.1.
Thailand specific information and requirements ..........................................71
12.1.1.
List name, affiliation and contact details of all investigators .........71
12.1.2.
Definition of burden on the study protocol (Amended: 24 
July 2017)....................................................................................74
 
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12.1.3.
Duration of the study, site address and number of 
participating subjects in Thailand (Amended: 24 July 
2017) ...........................................................................................74
12.1.4.
Study population groups and Calculation of study 
population groups in Thailand (Amended: 24 July 2017)..............74
12.1.5.
Overview of the Subject Recruitment Plan for Thailand ...............75
12.1.6.
Adding Secondary Objective Explanations...................................75
12.1.7.
Informed Consent and Consent for Thailand................................75
12.1.8.
Compiling Area Coordinates of Households in Thailand ..............76
12.1.9.
Managing Biological Samples in Thailand....................................77
12.1.10. Potential Risks and Inconveniences, and Prevention 
Measures.....................................................................................77
12.1.11. Benefits, Compensation, Reimbursement, Treatment and 
Solution in the Event of Complications for the Investigator...........77
12.1.12. Compensation and Reimbursement (Amended: 24 July 
2017) ...........................................................................................77
12.1.13. Other Ethical Issues.....................................................................78
12.1.14. Signature of all investigators........................................................79
13. REFERENCES.......................................................................................................80
 
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LIST OF TABLES
PAGE
Table 1
Study groups and epochs foreseen in the study.....................................29
Table 2
List of study procedures .........................................................................39
Table 3
Procedures in case of urgent care visit or hospitalisation or at 
any non-study health care facility for acute febrile illness and 
SDC detected through active surveillance..............................................42
Table 4
Intervals between study visits/contacts...................................................42
Table 5
Volume of whole blood for study related testing (Amended: 24 
July 2017) ..............................................................................................49
Table 6
Possible laboratory assays on study blood samples for primary 
and secondary endpoints .......................................................................49
Table 7
Humoral immunity (DENV antibody determination) for primary 
and secondary endpoints (Amended: 24 July 2017)...............................50
Table 8
Virology for primary and secondary endpoints........................................50
Table 9
Possible laboratory assays on study blood samples for tertiary 
endpoints (Amended: 24 July 2017).......................................................51
Table 10
Humoral immunity for tertiary objectives.................................................52
Table 11
Virology for tertiary endpoints.................................................................52
Table 12
Possible assays for primary and secondary endpoints for blood 
samples collected at scheduled visits.....................................................52
Table 13
Aliquot preparation for blood samples collected at visits for 
suspected dengue and follow-up contact ...............................................53
Table 14
Reporting periods for SAEs....................................................................55
Table 15
Timeframes for submitting SAEs related to study participation to 
GSK .......................................................................................................58
Table 16
Expected 95% confidence intervals for the incidence rate of 
virologically confirmed dengue cases.....................................................64
Table 17
Outsourced laboratories.........................................................................85
 
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LIST OF FIGURES
PAGE
Figure 1
Schematic representation of the study design........................................28
Figure 2
Active tracking algorithm ........................................................................46
 
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LIST OF APPENDICES
PAGE
APPENDIX A LABORATORY ASSAYS .......................................................................83
APPENDIX B CLINICAL LABORATORIES ..................................................................85
APPENDIX C AMENDMENTS AND ADMINISTRATIVE CHANGES TO THE 
PROTOCOL...........................................................................................86
 
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LIST OF ABBREVIATIONS
AE
Adverse Event
ATP
According-To-Protocol
CBC
Complete Blood Count
DENV
Dengue virus
DHF
Dengue Hemorrhagic Fever
DSS
Dengue Shock Syndrome
eCRF
electronic Case Report Form
ELISA
Enzyme-linked Immunosorbant Assay
GCP
Good Clinical Practice
GIS
Geographic Information System
GPP
Good Pharmacoepidemiology Practices
GPS
Global Positioning System
GSK
GlaxoSmithKline
HCT
Haematocrit
ICF
Informed Consent Form
ICH
International Conference on Harmonisation
IEC
Independent Ethics Committee
IgG
Immunoglobulin G
IgM
Immunoglobulin M
IRB
Institutional Review Board
JE
Japanese Encephalitis
JEV
Japanese Encephalitis Virus
LAR
Legally Acceptable Representative
NA
Not applicable
 
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NS1
Non-structural protein 1
RNA
Ribonucleic acid
RT-qPCR
Reverse-transcriptase quantitative polymerase chain reaction
SAE
Serious Adverse Event
SAP
Statistical Analysis Plan
SDC
Suspected Dengue Cases
SPM
Study Procedures Manual
WHO
World Health Organisation
WNV
West Nile Virus
YF
Yellow Fever
 
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GLOSSARY OF TERMS
Adverse event:
Any untoward medical occurrence in a subject, 
temporally associated with the use of a medicinal product, 
whether or not considered related to the medicinal 
product, or temporally associated with a study procedure.
An AE can therefore be any unfavourable and unintended 
sign (including an abnormal laboratory finding), 
symptom, or disease (new or exacerbated) temporally 
associated with the use of a medicinal product. For 
marketed medicinal products, this also includes failure to 
produce expected benefits (i.e., lack of efficacy), abuse or 
misuse.
Anonymised data:
Information about an individual that GSK or a third party 
cannot reasonably attribute to the individual, or could 
only attribute to the individual by expending a 
disproportionate amount of time, effort or expense (e.g. 
de-identified or aggregated information). For the purpose 
of this policy, Key-Coded personally identifiable 
information shall not be considered Anonymised 
Information
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.
Cohort study:
A form of epidemiological study where subjects in a 
study population are classified according to their 
exposure status/disease and followed over time 
(prospective/ retrospective) to ascertain the outcome(s). 
Dengue naïve subject:
A subject who has no detectable anti-DENV antibodies 
(tested negative for anti-DENV IgG antibodies)
 
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Designate:
A designate is defined as a person who helps the 
subject/subject’s parent(s)/LAR(s) with performing some 
of the study procedures if the subject/subject’s 
parent(s)/LAR(s) has difficulties to perform them alone 
(such as completion of diary log, receiving phone calls 
and planning of the study visits), e.g. a relative of the 
subject, a field worker who is linked to this study. 
Designates are appointed by the subject or his/ her LAR 
for help with the study procedures solely and cannot 
make decisions on behalf of the subject.
Eligible:
Qualified for enrolment into the study based upon strict 
adherence to inclusion/exclusion criteria.
Epidemiological study:
An observational or interventional study without 
administration of medicinal product(s) as described in a 
research protocol.
Epoch:
An epoch is a self-contained set of consecutive time 
points or a single time point from a single protocol. Self-
contained means that data collected for all subjects at all 
time points within that epoch allows to draw a complete 
conclusion. Typical examples of epochs are retrospective 
data collection and prospective data collection, etc.
eTrack:
GSK Biologicals’ tracking tool for clinical/ 
epidemiological 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 10.3 for details on criteria for evaluability).
Holiday period:
Public holidays or school holidays e.g. long school 
holidays or traditional holiday time for family vacations.
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 objectives.
Protocol amendment:
The International Conference on Harmonisation (ICH) 
defines a protocol amendment as: ‘A written description 
of a change(s) to or formal clarification of a protocol.’ 
GSK Biologicals further details this to include a change 
to an approved protocol that affects the safety of subjects, 
scope of the investigation, study design, or scientific 
integrity of the study.
 
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Research protocol:
A document that describes the objective(s), design, 
methodology, statistical considerations, and organisation 
of a study. The protocol usually also gives the 
background and rationale for the study, but these could be 
provided in other protocol referenced documents.
Self-contained study:
Study with objectives not linked to the data of another 
study.
Site Monitor:
An individual assigned by the sponsor who is responsible 
for assuring the proper conduct of epidemiological studies 
at one or more investigational sites.
Source Documents
Original documents, data, and records (e.g., hospital 
records, clinical and office charts, laboratory notes, 
memoranda, subjects' diaries or evaluation checklists, 
pharmacy dispensing records, recorded data from 
automated instruments, copies or transcriptions certified 
after verification as being accurate copies, microfiches, 
photographic negatives, microfilm or magnetic media, x-
rays, subject files, and records kept at the pharmacy, at 
the laboratories and at medico-technical departments 
involved in the epidemiological trial).
Study population:
Sample of population of interest.
Sub-cohort:
A subgroup of the total cohort of study subjects for whom 
the planned study procedures are different from those 
planned for the other study subjects.
Subject:
Term used throughout the protocol to denote an 
individual who has been contacted in order to participate 
or participates in the epidemiological study or a person 
about whom some medical information has been recorded 
in a database.
Subject number:
A unique number identifying a subject, assigned to each 
subject consenting to participate in the study.
Surveillance:
The ongoing systematic collection, collation, analysis, 
and interpretation of descriptive epidemiological health 
data on a specific disease. Surveillance can monitor 
incidence and/or prevalence, and/or inform about when 
and where health problems are occurring and who is 
affected.
 
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1.
INTRODUCTION
1.1.
Background
Dengue is a mosquito-borne disease endemic in large parts of Southeast Asia, Central and 
South America and in the Caribbean [Letson, 2010; Thomas, 2011]. Autochthonous 
transmission is also found in areas of Africa and Middle East. Dengue is caused by 4 
flaviviruses, dengue virus (DENV) types 1-4, and results in acute febrile illness 
[Edelman, 2008; Thomas 2011].
Dengue fever, commonly of duration 2-7 days is accompanied by severe headache, 
muscle pain, joint pain, bone pain, and pain behind the eyes as well as gastrointestinal 
symptoms [Edelman 2008; WHO, 2012a]. In infants with dengue, high fever usually lasts 
2-7 days as seen in older children. Upper respiratory tract symptoms like cough, nasal 
congestion, runny nose and dyspnoea, gastrointestinal symptoms and febrile convulsion 
are more common in infants than in older children [Capeding, 2010; Hung, 2004;
Kalayanarooj, 2003]. Severe dengue is potentially fatal as plasma leakage may lead to 
shock and/or fluid accumulation with respiratory distress. Severe haemorrhage and severe 
organ impairment may develop. The critical period for complications occur 3–7 days 
after the first symptoms, typically coinciding with fever abatement (below 38°C/ 100°F) 
and can include warning signs such as severe abdominal pain, persistent vomiting,
mucosal bleeding, clinical fluid accumulation, lethargy or restlessness [WHO, 2012b].
Transmission of dengue virus is variable: the intensity of transmission and distribution of 
serotypes typically vary significantly from one year to another. The four viruses act 
independently and immunity to one type only provides short-lived immunity to the other 
3 viruses estimated to range from 4-6 months [Letson, 2010] to 1.6 years, as suggested by 
recent analysis of cohort studies [Endy, 2014]. Individuals who have recovered from a 
primary DENV infection are then fully susceptible to infection and disease by subsequent 
infection with heterologous dengue virus types [Endy, 2004]. Re-infection with the same 
serotype has been documented but rarely leads to disease. Neutralising antibodies elicited 
by a primary infection are predominantly serotype specific whereas cross-reactive 
responses are seen following secondary infection. Secondary DENV infection has been 
identified as an epidemiological risk factor for severe dengue. Third or even fourth 
infections are thought to be clinically mild, although the risk of developing severe dengue 
remains [Whitehorn, 2011].
Dengue transmission has been shown to be very focal in several settings [Mammen, 
2008; Yoon, 2012; Honório, 2009]. In cluster sampling studies investigating infections in 
the peridomestic area of index cases, different levels of transmission intensity have been 
observed [Martínez-Vega, 2012]. Analysis of spatial dengue transmission is a key area of 
research to understand the disease and plan for control measures [Salje, 2012; Vazquez-
Prokopec, 2010].
 
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1.2.
Rationale for the study
The purpose of this study is to describe the burden of DENV illness among household 
members aged 6 months to 50 years of selected communities in Latin America and 
Southeast Asia. The results will inform the design of future clinical trials, supporting 
decision making on target age groups and stratification, sample size estimation, 
recruitment method. The study will describe the population pre-existing immune status to 
dengue and other relevant flaviviruses reflecting past exposure. It will explore spatial and 
temporal characteristics of dengue transmission in the selected communities. The data 
generated may support modelling work to assess the impact of heterogeneity 
(heterogeneity of exposure, heterogeneity of individual response influenced by the pre-
existing immune status) on the outcomes of an efficacy trial. 
In addition, the study may shed light of other infectious aetiologies than dengue in 
patients with febrile illness episodes.
This study is also seeking to maintain/set up febrile illness surveillance and operational 
capacity in sites in countries of Latin America and Southeast Asia for the targeted study 
population (6 months to 50 years) with the perspective of preparing these sites for future 
dengue vaccine efficacy and effectiveness studies.
 
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2.
BENEFIT: RISK ASSESSMENT
The following section outlines the risk assessment and mitigation strategy for this study 
protocol:
2.1.
Risk Assessment
Risks associated with blood collection (such as pain at blood sampling site, haematoma 
or thrombus, vasovagal reaction, syncope or fainting) can be reduced by following best 
practices listed in the [WHO guidelines] for drawing blood (2010). Some examples are 
provided in the table below.
Important 
Potential/Identified Risk
Data/Rationale for 
Risk
Mitigation Strategy
Study procedure: Blood 
sample collection.
Pain at blood 
sampling site

Well-trained person should take 
the blood sample

Use needle of smaller gauge 
than the vein
Haematoma or 
thrombus

Enter vessel at an angle of 30 
degrees or less

Use gauge of needle smaller 
than the vein

Apply pressure to a straight arm 
for 3–5 minutes after drawing 
blood
Vasovagal reaction
Syncope, fainting

Hydrate patient, take postural 
blood pressure if
dehydrated

Reduce anxiety

Have patient lie down if the 
person expresses concern

Provide audio-visual distraction
Please refer to [WHO guidelines] for drawing blood (2010): best practices in 
phlebotomy, in particular section 8.5.3 on risk assessment and risk reduction strategies.
2.2.
Benefit Assessment
The subjects may not receive any other direct benefit than facilitated access to healthcare 
in case of febrile illness during study conduct. Appropriate care will be provided to the 
subjects diagnosed with dengue or other diseases.
 
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2.3.
Overall Benefit: Risk Conclusion
Taking into account the measures taken to minimise risk to subjects participating in this 
study, the potential or identified risks identified are justified by the potential benefits that 
may be afforded to subjects.
3.
OBJECTIVES
3.1.
Primary objective

To estimate the overall incidence rate of reverse-transcriptase quantitative 
polymerase chain reaction (RT-qPCR)-confirmed symptomatic DENV infection in a 
multi-centre cohort of household members aged 6 months to 50 years.
Refer to Section 10.1.1 for the definition of the primary endpoint.
3.2.
Secondary objectives

To estimate the incidence rate of virologically confirmed (through RT-qPCR or NS1) 
and probable (based on serological evidence) symptomatic DENV cases, separately 
and combined - by age, gender, site, serotype (if applicable) and anti-DENV IgG 
serological status at enrolment.

To estimate the prevalence of anti-DENV immunoglobulin G (IgG) antibodies 
against dengue in the study population, overall and by age and site (at enrolment), 
for all participants and among dengue cases (confirmed and probable).

To describe clinical presentations of dengue cases (confirmed and probable).
Refer to Section 10.1.2 for the definition of the secondary endpoints.
3.3.
Tertiary objectives

To describe the spatial and temporal distribution of dengue cases (confirmed and 
probable) among cohort participants in the study areas and analyse determinants of 
spatio-temporal transmission (e.g. environmental, entomological, socio-demographic 
or ecological factors).

To describe the DENV, and other flaviviruses (i.e. Japanese Encephalitis Virus 
[JEV; mostly relevant in Southeast Asia]) or West Nile Virus [WNV; mostly 
relevant in Latin America]) neutralising antibody profile in a subset of subjects.

To explore other infectious aetiologies than dengue in subjects with episodes of 
febrile illness referred to as “suspected dengue case” (chikungunya, Zika, influenza, 
leptospirosis).

To explore determinants of past dengue infection and dengue illness.

To further characterise the immune status at enrolment (e.g. antibody avidity, etc.).
Refer to Section 10.1.3 for the definition of the tertiary endpoints.
 
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4.
STUDY DESIGN OVERVIEW
Protocol waivers or exemptions are not allowed with the exception of immediate safety 
concerns. Therefore, adherence to the study design requirements, including those 
specified in the outline of study procedures (Section 7.4), are essential and required for 
study conduct. 
Figure 1 presents a schematic representation of the study design.
Figure 1
Schematic representation of the study design
*The Month 12 and Month 24 visits may occur via a telephone contact for the Mexican site and there will be no 
blood sample collection and physical examination at these visits for subjects in Mexico.

Type of design: Prospective, multi-country, multi-centre, household-based, cohort 
study/surveillance study.

Study population: Subjects aged between 6 months and 50 years at the time of 
enrolment living in geographically-defined communities in Latin America and 
Southeast Asia. The study population will comprise household members. The study 
population should include between 30% and 50% of adults (aged 18 years or above) 
per site.

Recruitment: The appropriate recruitment strategy will be selected by each 
participating site. Two approaches may be considered: a school-based approach and 
a community-based approach without school involvement (refer to Section 6.2).

Duration of longitudinal follow-up and number of visits: The study duration will be 
24 months for each participating site. Subjects will have a scheduled visit at 
 
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enrolment, one visit at Month 12 and a conclusion visit at Month 24. The Month 12 
and Month 24 visits may occur via a telephone contact for the Mexican site. The 
number of visits in case of febrile illness, referred as “suspected symptomatic dengue 
case”, will vary by subject.

Type of study: self-contained.

Data collection: Electronic Case Report Form (eCRF ).

Duration of the study: Because of differences in local regulations, dengue seasonality 
and holiday period between countries, the recruitment period will be staggered. 
However, to facilitate site activities, sites will target to have all the subjects recruited 
in a 3-month period. For each subject, the study duration will be approximately 24
months.

Epoch 001: Prospective data collection starting at Visit 1 (Day 0) and ending at 
the conclusion visit (Month 24).
Table 1
Study groups and epochs foreseen in the study
Study Groups
Number of subjects
Age (Min/Max)
Epochs
Epoch 001
Prospective
1750
6 months to 50 years
x
4.1.
Discussion of study design
4.1.1.
Rationale for study design
A prospective cohort study design has been selected as it allows estimation of the 
incidence of infection in a defined population (with a known denominator), describing 
the spectrum of clinical outcomes and year-to-year variation. This design is suited to 
study the natural infection history and identify marker(s) associated with protection 
against future symptomatic dengue infection.
The study population will be composed of individuals aged 6 months to 50 years. 
Households represent a platform of research to understand the transmission dynamics and 
spectrum of disease among closely-related individuals. Dengue cases tend to cluster 
within households, which could be the result of a single or very few infected mosquitoes 
biting different household members during a short period of time [Dussart, 2012]. 
The study will include multiple sites, mainly because it will contribute to developing or 
maintaining the field infrastructure required for future vaccine phase III efficacy studies. 
In this perspective, several sites in different countries in Asia and Latin America will 
need to be involved to capture significant number of dengue cases caused by all four 
dengue serotypes. The study may allow for the detection of differences across sites in the 
transmission pattern.
 
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The age range of the study population is large (6 months-50 years), including infants, 
young children, adolescents and adults. Dengue virus infections affect human population 
of all age groups worldwide [WHO, 2012b]. The clinical burden of dengue (in particular 
severe dengue) in much of Southeast Asia primarily falls on children/adolescents aged 
less than 15 years. However, dengue also affects adults throughout the world including
several Southeast Asian countries that include Malaysia, Sri Lanka, and Singapore 
[Cuong, 2011]. In a recent study in Hanoi, Vietnam, 85% of notified dengue cases were 
aged over 15 years [Cuong, 2011]. In several countries in Latin America, incidence of 
dengue was highest in teenagers and young adults [San Martín, 2010]. In Mexico, the 
incidence of dengue peaks in the age range of 10 to 19 years [Epidemiological Panorama 
Dengue Fever and Hemorrhagic Fever, 2014]. Due to potential reduction of vaccine 
efficacy by maternal antibodies in infants and a reduced memory response [Van Panhuis, 
2011], infants aged < 6 months are currently not targeted for vaccination and thus, will 
not be included in this epidemiological study. Because dengue is endemic in the countries 
where study sites are located, the study population should have a mixed anti-DENV 
immunological background, providing an opportunity to describe different response 
patterns to new infections.
5.
CASE DEFINITIONS
The following case definitions will be used in this study.
5.1.
Suspected symptomatic dengue case (SDC)
Acute febrile illness measured as 38.0°C with a thermometer by any route or recent 
history of febrile illness (onset in the past 8 days) reported by the subject/the 
parent(s)/LAR(s)/ designate (if applicable) of the subject for at least two consecutive days 
(a duration of approximately 36-48 hours) and of less than 7 days duration, which may be 
accompanied by other dengue symptoms or signs and does not have a defined focus or an 
obvious reason unrelated to dengue (based on physician judgement).
Note: Besides fever, other dengue associated signs and symptoms include but are not 
limited to: upper respiratory tract symptoms like cough, nasal congestion, runny nose and 
dyspnoea, gastrointestinal symptoms and febrile convulsion in infants/older children and
fatigue, headache, pain behind the eyes, abdominal pain, nausea, vomiting, muscle ache, 
joint ache, diffuse rash on the trunk, itching of skin (pruritis) in adults/adolescents.
A number of infectious and non-infectious diseases mimic dengue and severe dengue. 
Physicians should be careful not to rule out dengue diagnosis too quickly, when the 
differential diagnosis cannot be accurately made. Early symptoms of dengue may include 
gastrointestinal and upper respiratory symptoms and may be misdiagnosed as diarrhoeal 
diseases or other diseases with flu-like symptoms. The WHO handbook for clinical 
management of dengue provides a list of conditions that mimic the febrile phase and the 
critical phase of dengue infection [WHO, 2012b].
 
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A SDC presenting at the health care facility within 5 days following the onset of 
symptoms will be defined as an ‘early presenter’. A SDC presenting at the health care 
facility 6 days or more after the onset of symptoms will be defined as ‘late presenter’. 
Both early and late presenters will have a scheduled visit at the study healthcare centre
including blood sampling for laboratory diagnosis.
Seven consecutive calendar days without fever (body temperature 38.0°C), in the 
absence of antipyretic medication, are required to separate two episodes of SDC.
5.2.
Laboratory confirmation

RT-qPCR confirmed symptomatic DENV infection 
A SDC confirmed by RT-qPCR

A virologically confirmed symptomatic DENV infection
A SDC confirmed by RT-qPCR or NS1.

Probable dengue case 
A SDC with:

DENV RT-qPCR negative or not performed (late presenter)
and

DENV Non-structural protein 1 (NS1) negative or undetermined (early or late 
presenter)
and

Anti-DENV Immunoglobulin M (IgM) positive with a rapid 
immunochromatographic (ICT) assay or an ELISA assay)
Or 

Anti-DENV Ig G positive (rapid ICT assay or ‘capture ELISA’ assay)
5.3.
Classification of severity of dengue
The clinical and biological signs at presentation of SDC, potential return visits and during 
hospitalisation will be recorded. The physician will record in the eCRF the occurrence of 
warning signs (2009 WHO definition) during the course of illness.
When the episode is considered terminated (which may be at the follow-up contact for 
subjects with ambulatory management or at hospital discharged for hospitalized 
subjects), the physician will record in the eCRF whether the patient met criteria for 
dengue haemorrhagic fever or dengue shock syndrome according to the 1997 WHO 
definition and for severe dengue according to the 2009 WHO definition.
The classification of severity for research purpose within the study should not interfere 
with patient management, which will be conducted as per local guidelines.
 
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5.3.1.
The 1997 WHO classification [WHO, 1997]
Criteria for dengue hemorrhagic fever (DHF): The following criteria must all be 
present:

Fever, or history of acute fever, lasting 2–7 days, occasionally biphasic,

Haemorrhagic- tendencies, evidenced by at least one of the following 

A positive tourniquet test,

Petechiae, ecchymoses or purpura,

Bleeding from the mucosa, gastrointestinal tract, injection sites or other 
locations,

Haematemesis or melaena,

Thrombocytopenia (100,000 cells per mm3 or less),

Evidence of plasma leakage due to increased vascular permeability, manifested by at 
least one of the following:

A rise in the haematocrit (HCT) equal to or greater than 20% above average for 
age, sex and population,

A drop in the HCT following volume-replacement treatment equal to or greater 
than 20% of baseline,

Signs of plasma leakage such as pleural effusion, ascites and hypoproteinaemia,
Criteria for dengue shock syndrome (DSS): The criteria mentioned for DHF must 
all be present, plus evidence of circulatory failure manifested by:

Rapid and weak pulse, and

Narrow pulse pressure [<20mmHg (2.7 kPa)]
OR

Hypotension for age (this is defined as systolic pressure < 80 mmHg for those aged 
less than 5 years, or < 90 mmHg for those ages 5 years and older), and

Cold, clammy skin and restlessness.
5.3.2.
The 2009 WHO classification [WHO, 2009, Gutiérrez, 2013; 
Macedo, 2014]
Criteria for dengue with warning signs: At least one of the following should be 
present:

Abdominal pain or tenderness, 

Persistent vomiting (three or more emesis in a period of one hour, or five or more in 
a period of six hours), 
 
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
Clinical fluid accumulation (peri-orbital, facial or lower limb oedema as reported by 
the study physician, or pleural effusion, ascites or gall-bladder wall thickening ≥3 
mm as observed via X-ray radiography or ultrasonography), 

Mucosal bleed (any of the following: hemoptysis, epistaxis, gingival bleeding, 
melena, hematemesis, hematuria, menorrhagia, vaginal bleeding, or subconjunctival 
hemorrhage as observed by a study physician or reported by the patient), 

Liver enlargement (liver enlarged >2 cm below the edge of the ribs as palpated by a 
study physician), 

Increase in HCT concurrent with rapid decrease in platelet count (interpreted as any 
HCT >20% over baseline with platelet 50,000/mm3),

Lethargy (Glasgow coma scale score <15 for children aged 5 years or more or 
Blantyre coma scale <5 for children under 5, as evaluated by a study physician) and 
restlessness.
Criteria for severe dengue: dengue with at least one of the following criteria:

Severe Plasma Leakage leading to Shock (DSS),1
Fluid accumulation with respiratory distress2,

Severe Bleeding3,

Severe organ involvement;
Liver: AST or ALT ≥ 1000 IU/L,
Central nervous system: impaired consciousness4 ,
Failure of heart and other organs5.
                                                
1 A clinical state of reduced perfusion to end-organs (such as the skin), defined as presence of a weak pulse, 
and/or narrowing of pulse pressure, and/or hypotension for age and one of the following: 1) Cold, clammy 
skin, 2) Increased capillary refill time, 3) Peripheral cyanosis, 4) Skin mottling
2 defined as respiratory discomfort, dyspnea, respiratory failure, or increased respiratory rate of >60 
breaths/min for ages <2 months; >50 breaths/min for ages 2 months to 1 year; >40 breaths/min for ages 1 to 
5 years; >30 breaths/min for ages 5 to 8 years; and >20 breaths/min for those older than 8 years);
3 As evaluated by clinician WHO Grade 2 or above: haematemesis, melena, menorrhagia or clinical drop in 
haemoglobin requiring whole blood or packed red cell transfusion;
4 Any reduction in coma score that may be accompanied by convulsions and/or meningism and/or abnormal 
neurological signs
5 For renal impairment: Stage 2 Acute Kidney Injury defined as serum creatinine increase of 100% over 
baseline or calculated norm for age/gender/race)
 
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6.
STUDY POPULATION
The study will be conducted in countries of Latin America and Southeast Asia. 
6.1.
Number of subjects/ centres
Approximately, 1750 subjects are expected to be enrolled into this study with about 300 
to 500 subjects expected per study site. Subjects aged between 6 months and 50 years at 
the time of enrolment living in geographically-defined communities in Latin America and 
Southeast Asia will be part of this study. The number of households will depend on the 
number of subjects enrolled per household. For a site enrolling 500 subjects, the number
of households would likely range between 125 (assuming 4 subjects on average per 
household) and 250 (assuming 2 subjects on average per household).
6.2.
Overview of recruitment plan
The recruitment strategy will be selected by each participating site. One of the two 
approaches may be considered:

A school-based approach
Recruitment will be initiated in primary and/or secondary schools. The parent(s)/ LAR(s) 
of the student attending participating schools will be asked to allow voluntarily 
participation of their child/ward. The parent(s)/LAR(s) of these students will also be 
invited to enrol himself/herself/themselves (if 50 years) and their younger/older child 
who may not be attending the study school(s). Other household members will also be 
invited to participate in the study (if 50 years).
Recruitment will be organised by study staff, preferably at participating schools (primary 
and/or secondary) or designated facility (e.g. healthcare centre, local community centre, 
etc.). If any member of a given household refuses to participate in the study, it will not 
preclude inclusion of other household members. Study staff could also conduct home 
visits for recruitment but collection of blood sample will be done preferably at a 
designated facility. 

A community-based approach (without school involvement)
Recruitment will be organised by study staff at the household level and/or at other 
designated facilities (e.g. healthcare centre, local community centre, etc.), using various 
modes of communication (e.g.: public announcements or community meetings to inform 
about the study). Study staff could also conduct home visits for recruitment but collection 
of blood sample will be done preferably at a designated facility.
Whichever approach is chosen, the various modes of communication used for recruitment 
will be reviewed and approved by the Institutional Review Board (IRB)/Independent 
Ethics Committee (IEC) according to the local legislation (for e.g.: public announcements 
or community meetings to inform about the study).
 
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Note: For the first 3 months of the study after overall study start (in each centre), the 
recruitment target will be fixed for every centre. At month 3 of enrolment at each centre, 
the recruitment per country will be evaluated. If at the time of evaluation, recruitment at 
one or more countries is below the expected target, the number of subjects to be recruited 
will be re-evaluated.
Recruitment period
In countries with marked seasonality, the recruitment period should preferably occur 
outside the period of peak incidence of dengue, based on the local epidemiology of 
dengue in the past years. It should also preferably occur outside the holiday period if the 
investigator believes that families are more likely to leave the study area during this 
period. 
These periods will be described for each site and may be modified upon mutual 
agreement between the investigator and the central study team based on available 
epidemiological information.
6.2.1.
Recruitment of study centres
Selection of communities and schools
The following criteria will be considered as guidance for selecting the communities.

Areas which are known to be endemic for dengue with high rates of transmission in 
most recent years.

Easy access for the study population to a healthcare centre that can evaluate SDC 
(fast track) and manage the enrolled subjects.

Safe access by study personnel.
In addition, the following criteria will be considered as guidance for selecting schools, in 
case the school based approach is selected for recruitment:

Of sufficient size to allow the enrolment target of 300-500 subjects (children and 
adults) per site in one geographically-defined community.
6.3.
Inclusion criteria for subject enrolment
Deviations from inclusion criteria are not allowed because they can potentially jeopardise 
the scientific integrity of the study, regulatory acceptability or subject safety. Therefore, 
adherence to the criteria as specified in the protocol is essential.
All subjects must satisfy ALL the following criteria at study entry:

Written and signed informed consent (and assent if the subject is below the legal age 
of consent) obtained from the subject/from subject’s parent(s)/LAR(s). For a subject 
below the legal age of consent is, his/her signature will be obtained on the informed 
assent form, if applicable.
 
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
A male or female between, and including 6 months and 50 years of age at the time of 
enrolment (Subjects become ineligible on their 51st birthday).

Subject and/or the subject’s parent(s)/LAR(s) who the investigator believes can 
comply with the requirements of the protocol (e.g., willingness to go to the 
hospital/healthcare centre for visit(s) in case of acute febrile illness, able to observe 
the signs of dengue and to understand how to take and report body temperature, etc).

Subject who plans, at the time of enrolment, to remain at same residence/study area 
during the one or two year study period (as applicable).

Household should be reachable by phone (residence phone or mobile phone).
Note: Pregnant or lactating female or female planning to become pregnant can be 
recruited into the study.
6.4.
Exclusion criteria for subject enrolment
Deviations from exclusion criteria are not allowed because they can potentially 
jeopardise the scientific integrity of the study, regulatory acceptability or subject safety. 
Therefore, adherence to the criteria as specified in the protocol is essential.
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:

Child in care.
Please refer to the glossary of terms for the definition of child in care. 

Participation (current or planned) in another epidemiological study or in a clinical 
trial that would conflict with the current study, based on investigator’s judgement.

Terminal illness or severe mental incapacity.
7.
CONDUCT OF THE STUDY
7.1.
Regulatory and ethical considerations, including the 
informed consent process
The study will be conducted in accordance with the ICH Guideline for Good Clinical 
Practice (GCP), Guidelines for Good Pharmacoepidemiology Practices (GPP) 
[International Society for Pharmacoepidemiology (ISPE), 2007], 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 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.
 
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Conduct of the study includes, but is not limited to, the following:

IRB/ IEC review and favourable opinion/approval of study protocol and any 
subsequent amendments.

Subject/ subject’s parent(s)/LAR(s) informed consent and subject informed assent, as 
appropriate

Investigator reporting requirements as stated in the protocol.
GSK Biologicals will provide full details of the above procedures to the investigator, 
either verbally, in writing, or both. 
Freely given and written informed consent must be obtained from each subject and/or 
each subject’s parent(s)/LAR(s) or the impartial witness and subject informed assent, 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, GPP, 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. 
In accordance with the ICH Harmonised Tripartite Guidelines for GCP, a subject who 
can only be enrolled in the study with the consent of his/her parent(s) or LAR(s) (e.g., 
minors/ subjects below the legal age for consent), should be informed about the study to 
the extent compatible with the subject’s understanding and, if capable, the subject should 
sign and personally date a written informed assent form (IAF). It is required that the 
assent be signed by each subject, if capable, in addition to the informed consent that is to 
be signed by his/her parent or legal representative. It should be assessed whether an 
assent is required depending on the age of the study population and the local 
requirements.
GSK Biologicals strongly recommends that if the subject reaches the age of consent 
during the study they will be asked to provide consent at the next study visit (if 
applicable). This procedure should be applied according to local laws and regulations.
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.
 
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7.2.
Subject and household number attribution
Subject numbers will be assigned sequentially to subjects consenting to participate/to be 
included in the study, according to the range of subject numbers allocated to each study 
centre. Household numbers will also be assigned sequentially at each site to eligible 
household.
7.3.
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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7.4.
Outline of study procedures
Table 2 presents the list of study procedures.
Table 2
List of study procedures
Age
6 months to 50 Years
Epoch
Epoch 001 
Data Collection
Prospective data Collection
Visit
Scheduled visits
Unscheduled visits 
(for acute febrile illness and suspected dengue 
at hospital/healthcare centre)
Time points
Visit 1
Day 0
Regular 
Surveillance 
contact between 
visitsa
Visit 2
Month 12 g 
Conclusion visit 3
Month 24g
First visit
Return visit*
Follow-up 
contact**
Informed consent and assent as applicable
●
Check inclusion/exclusion criteria
●
Check evidence of subject being member of the 
household (family book or other administrative 
document)
O
Attribute subject number and household number 
attribution
●
Collect spatial coordinates of householdse 
●e
Record/ update household and socio-demographic 
information
●
●
●
Medical history and Japanese encephalitis 
(JE)/Yellow fever (YF)/ Dengue (if applicable) 
vaccination history or updates
●
●
●
Distribute subject ID card and instruction kitd
O
Collect blood sample
●
  ●f
●f
Instruct/ remind subjects/subjects’ parent(s)/LAR(s) 
or designate (if applicable) on assessment 
procedures in case of acute febrile illness
●
O
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Age
6 months to 50 Years
Epoch
Epoch 001 
Data Collection
Prospective data Collection
Visit
Scheduled visits
Unscheduled visits 
(for acute febrile illness and suspected dengue 
at hospital/healthcare centre)
Time points
Visit 1
Day 0
Regular 
Surveillance 
contact between 
visitsa
Visit 2
Month 12 g 
Conclusion visit 3
Month 24g
First visit
Return visit*
Follow-up 
contact**
Issue diary logs to subject/subject’s parent(s)/LAR(s)/ 
designate and train them on how it is to be filled out 
in the event of an acute febrile illness†
O
O
O
Contact the subject/subject’s parent(s)/LAR(s)/ 
designate regarding any acute febrile illness and 
remind him/her/them of the procedures
O
Physical examination and medical history (see 
Section 7.5.4)
●
●f
●f
●
●
Collect or verify diary logs if applicableb
O
O
Record if subject meets the definition of a SDC; if 
dengue is discarded, record the alternative diagnosis
●
Record laboratory results prescribed as per local 
guidelines by the treating physician (may include 
CBC, liver enzymes, bilirubin)
●
●
Record patient management c
●
●
Collect blood sample for diagnosis#
●
Outcome of dengue episode
●
Report Serious Adverse Events (SAEs) related to 
study procedures‡
 ●
●
●
●
●
●
Study conclusion
●
● is used to indicate a study procedure that requires documentation in the individual eCRF.
O is used to indicate a study procedure that does not require documentation in the individual eCRF.
* A return visit is a visit linked to the first visit for acute febrile illness, occurring if the subject’s physical condition necessitates medical evaluation, according to local medical practice. 
The investigator will determine whether this return visit is necessary and whether it is considered as part of the same episode. See Section 7.5.2 for details.
** A follow-up contact will be done to record the outcome of the dengue episode
aRefer to section 7.5.3 for the more details on regular contact between study visits.
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bA diary log given at visit 1 and potentially subsequently (via surveillance contacts or mail) are only to be filled out in the event that the subject has acute febrile illness or suspected 
dengue symptoms. See Section 7.5.2for details.
cDepending on the clinical manifestations and other circumstances, patients may be either be sent home, be referred for in-hospital management for close observation or require 
emergency treatment and urgent referral. In case of in hospital/emergency room management, data on clinical evolution and results of biological and paraclinical examinations will be 
collected in the eCRF.
dThe instruction kit includes a thermometer, study contact information (phone numbers) and an instruction card with information about dengue symptoms and steps to take if acute 
febrile illness occurs.
eCollection of spatial coordinates can be done at any time during the recruitment period and not necessarily at Visit 1.
fThe blood sample collection, physical examination and collection of medical history procedures at Month 12 and Month 24 were included as part of the protocol amendment 2 and will 
not be applicable for Mexico.
gThe Month 12 and Month 24 visits may occur via a telephone contact for the Mexican site.
#Testing includes RT-qPCR and NS1/IgG capture/ IgM rapid ICT or ELISA. One purpose is to provide rapid results to the physician and the subject.
‡SAE = Serious adverse event- see Section 7.1.1for definition.
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Table 3 presents the procedures involved in case of hospitalisation or urgent care visit at 
any non-study health care facility for acute febrile illness and SDC detected through 
active surveillance.
Table 3
Procedures in case of urgent care visit or hospitalisation or at any 
non-study health care facility for acute febrile illness and SDC 
detected through active surveillance
Procedures involved
Time point/Data to be collected
Documentation
Timeframe to record the event
As soon as possible after detection of event
○
Contact with hospital/health care 
facility visited by the patient
Phone call/visit, as appropriate
○
Data to be collected
Date of visit/entry, main symptoms and severity, 
diagnosis, date of discharge (if applicable)
●
Verification of diagnoses 
By phone contact/visit with treating physician/written 
report by treating physician/hospital discharge report
○
●is used to indicate a study procedure that requires documentation in the individual CRF/eCRF.
○is used to indicate a study procedure that does not require documentation in the individual CRF/eCRF.
Table 4 presents the intervals between study visits/contacts.
Table 4
Intervals between study visits/contacts
Interval
Optimal length of interval*
Allowed interval
Visit 1 (Day 28) Visit 2 (Month12)
12 months
28 days
Visit 2 (Month 12) Conclusion visit 
(Month 24)
12 months
28 days
Interval between First visit for a SDC and 
the final follow-up contact for a case
21 days
7 days
*Whenever possible the investigator should arrange study visits/contacts within this interval.
7.5.
Detailed description of study procedures
There will be one scheduled visit (Visit 1) at enrolment, a second visit at Month 12 (Visit 
2) and a final conclusion visit at Month 24 (Visit 3). The Month 12 and Month 24 visits 
may occur via a telephone contact for the Mexican site. During the study, detection of 
febrile illness in the cohort will be performed using active surveillance and enhanced 
passive surveillance. Subjects with febrile illness will have a medical visit; if the subject 
meets the case definition of SDC, a blood sample will be collected for laboratory 
diagnosis.
7.5.1.
Procedures at scheduled visit 1 (enrolment)
The visit 1 (enrolment) will take place at a designated facility, as determined by each 
participating site. Home visits may also be organised, at the convenience of the study 
team.
 
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7.5.1.1.
Informed consent and assent
The informed consent/assent will be obtained from all eligible subjects of the household, 
participating in the study.
The signed/witnessed/thumb printed informed consent of the subject/subject’s 
parent(s)/LAR(s) must be obtained before study participation. The signed informed 
assent of a subject below the age of consent (i.e., minor) should be obtained in addition to 
the signed informed consent by his/her parent(s)/LAR(s) according to local rules and 
regulations. Refer to Section 7.1 for the requirements on how to obtain informed consent 
and assent, as appropriate.
7.5.1.2.
Check inclusion and exclusion criteria
Check all applicable inclusion and exclusion criteria as described in Sections 6.3 and 6.4
before enrolment.
7.5.1.3.
Check evidence of subject being member of the household
The evidence of the subjects being members of the same household (e.g. family book or 
other administrative documents) will be checked by the site and then documented in the 
source notes.
7.5.1.4.
Attribute subject number and household number 
Refer to Section 7.2.
7.5.1.5.
Collect spatial coordinates of the households
The participating households may be geo-referenced with GPS (Global Positioning 
System) devices. This method has been used in other studies characterising spatial 
transmission and using households or individual dengue cases as the primary unit of 
analysis [Martínez-Vega, 2012; Rabaa, 2013; Siqueira-Junior, 2008; Paz-Soldan, 2014]. 
A geographic masking technique may be applied (see Section 11.7) to remove the 
possibility of re-identification of participants.
Collection of spatial coordinates may be done at any time during the recruitment period 
and not necessarily at Visit 1.
7.5.1.6.
Record socio-demographic and household information
Socio-demographic and household information such as age, gender, type of dwelling and 
parental relationship with other participants in the house will be collected and recorded in 
the eCRF at each scheduled visit.
 
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7.5.1.7.
Physical examination, medical history and JE/YF/dengue vaccination 
history
A detailed clinical examination will be carried out to assess the subject’s general 
condition, cardiac and respiratory rates, blood pressure, dengue associated clinical 
signs/symptoms.
Information on subject’s medical history and JE, YF and dengue vaccination history (if 
applicable, should a vaccine be licensed and available) will be collected and recorded in 
the eCRF.
7.5.1.8.
Distribute subject ID card and instruction kit
Each household will be given a instruction kit which includes a thermometer, study 
contact information (phone numbers) and an instruction card with information about 
dengue symptoms and steps to take if acute febrile illness occurs.
7.5.1.9.
Collect blood sample (Amended: 24 July 2017)
At Visit 1, a blood sample of 3.5 mL will be collected from subjects aged between 6
months and 2 years (all sites), and a blood sample of 10 mL will be collected from 
subjects aged between 2 and 50 years (all sites).
7.5.1.10.
Instruct/ remind subjects/subject’s parent(s)/LAR(s) or designate on 
assessment procedures in case of acute febrile illness
Study personnel will train subjects/subject’s parent(s)/LAR(s) or designate (if applicable)
to recognise acute febrile illness, and will instruct them to contact the study personnel or 
come to a designated study healthcare centre/ hospital for medical evaluation as soon as 
possible, preferably within 5 days of the occurrence of acute febrile illness.
7.5.1.11.
Issue diary logs to subject/subject’s parent(s)/LAR(s) or designate and 
train them on how it is to be filled out in the event of an acute febrile 
illness
The subjects’/subjects’ parent(s)/LAR(s) or designate (if applicable) will be instructed to 
record temperature and main symptoms in a diary log in case of suspected acute febrile 
illness until the medical appointment. The diary log will complement the patient 
interview and will facilitate the recording of symptoms in the eCRF. Please refer to the 
SPM for more details on the dengue kit and diary log. If a subject/subject’s 
parent(s)/LAR(s) is illiterate and is unable to complete the diary log on his/her own, 
he/she may be helped by a designate (refer to the GLOSSARY OF TERMS for the 
definition of designate).
7.5.1.12.
Report Serious Adverse Events (SAEs) related to study procedures
All SAEs related to study procedures (blood collection) will be recorded in the eCRF.
 
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7.5.2.
Procedures at Visit 2 and conclusion visit (Visit 3) (Amended: 24 
July 2017)
The Visit 2 and Visit 3 will take place at a designated facility, as determined by each 
participating site. Home visits may also be organised, at the convenience of the study 
team. The socio-demographic information will be updated in the eCRF (e.g. change of 
household during the conduct of the study). The Japanese encephalitis (JE) /Yellow fever 
(YF)/Dengue (if applicable) vaccination history will be updated.
A detailed clinical examination will be carried out to assess the subject’s general 
condition, cardiac and respiratory rates, blood pressure, dengue associated clinical 
signs/symptoms.
A blood sample of 3.5 mL will be collected from subjects aged between 6 months and 2 
years (all sites), and a blood sample of 10mL will be collected from subjects aged 
between 2 and 50 years (all sites). The Month 12 and Month 24 visits may occur via a 
telephone contact for the Mexican site and there will be no physical examination and 
blood sample collection at these visits for subjects in Mexico.
At study conclusion (Visit 3) for each subject, the investigator will:

Review all the data collected to ensure accuracy and completeness 

Complete the study conclusion screen in the eCRF.
7.5.3.
Dengue surveillance
Between scheduled visits, detection of SDC in the cohort will be performed using active
surveillance of acute febrile illness and enhanced passive surveillance.

Active surveillance: Households will be contacted regularly to enquire about the 
occurrence of febrile illness among the study subjects. Preferably, the frequency of 
contacts will be once per week. In sites with marked dengue seasonality, the 
frequency of contact may be twice per month during periods of low transmission of 
dengue. These periods may be modified upon decision of the central study team 
based on available epidemiological information. The mode of contact may include 
phone calls and/or house visits. In addition to the regular phone contact, some sites 
may opt to check the school absenteeism log and contact the household of the 
children who are absent.

If febrile illness is identified during active surveillance, an appointment will be 
arranged at the designated study healthcare centre/ hospital as soon as possible 
(preferably within 5 days following the onset of symptoms).
A household visit may be planned if the subject cannot come to the designated healthcare 
centre/hospital. This situation should, however, remain exceptional.
 
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
The surveillance operations will be tracked through an active tracking algorithm. In 
sites performing surveillance through phone contacts in first intention, there should 
be at least three documented attempts to contact the household before a home visit is 
planned (refer Figure 2). In sites where surveillance is performed routinely by other 
means (e.g. home visits), there should also be three documented attempts to contact 
the household.
Figure 2
Active tracking algorithm

Enhanced passive surveillance (spontaneous referral): This form of surveillance 
will be key in maximising the likelihood to detect SDC early, during the course of 
illness. Study personnel will instruct subjects/subject’s parent(s)/LAR(s)/ designate 
(if applicable) to contact the study personnel or come to a designated study 
healthcare centre/hospital for medical evaluation in case of febrile illness (preferably 
within 5 days of onset of febrile illness).
7.5.4.
Procedures at unscheduled visits and management of SDC
(Amended: 24 July 2017)

Suspected cases in the cohort may arise from three sources: spontaneous referral 
(enhanced passive surveillance), referral by study personnel during scheduled study 
visits, or as a result of active surveillance.

All subjects with acute febrile illness should be seen at a designated study healthcare 
centre/hospital by a study physician. If dengue is ruled out by the study physician 
during clinical examination because of another obvious alternative diagnosis 
(identified focus of fever), the physician will document his alternative diagnosis, and 
collect a blood sample for testing related to tertiary endpoints. 
Examples of obvious alternative diagnosis are abscess with cellulitis, dental/ gingival 
problems with cellulitis, skin infections with cellulitis, eye infections with periorbital 
cellulitis, otitis media with pain, pneumonia, meningitis, bacillary dysentery, 
purulent tonsillitis, urinary tract infections/pyelonephritis, cholecystitis, peritonitis, 
appendicitis, moderate to severe trauma from accidents, post operation fever.
 
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
Subjects who, for any reason, seek medical assistance at any non-study health care 
facility may be identified, for instance by active surveillance, and if feasible, clinical 
data will be retrospectively collected on the eCRF from those non-study healthcare 
facilities. A study physician will be responsible for collecting the retrospective data 
and informing the local study coordinator for appropriate follow-up. Refer to Table 3
for further details.

The first visit to the study healthcare centre/hospital will include a detailed clinical 
examination to assess the subject’s general condition, cardiac and respiratory rates, 
blood pressure, dengue associated clinical signs/symptoms and other relevant clinical 
signs/symptoms or paraclinical examinations (for example detection of clinical fluid 
accumulation with ultrasonography). A blood sample should be collected by the 
healthcare centre/hospital for confirmation of dengue by RT-qPCR technique for
early presenters. Rapid dengue diagnostic tests (NS1, IgM, and IgG) will be 
performed as well because a result can be provided rapidly to the physician and 
subject and support the diagnosis. These tests will also provide indications on the 
diagnosis for late presenters, in which RT-qPCR will not be performed. If other tests 
(such as CBC, liver enzymes, bilirubin) are prescribed by the treating physician as 
part of routine assessment, the results will be recorded in the eCRF.

Record patient management:
Depending on the clinical manifestations and other circumstances (co-morbidities, 
etc.), patients may either be sent home, be referred for in-hospital management for 
close observation or require emergency treatment and urgent referral. In case of 
hospital/emergency room management, data on clinical evolution and main results of 
biological (platelet counts, evidence of hemoconcentration, etc.) and paraclinical 
examinations (e.g. chest X-ray, ultrasonography, etc.) will be collected in the eCRF. 
The duration of hospitalisation, potential management in the intensive care unit and 
the treatment administered (fluid therapy/ blood product transfusion) will also be 
collected in the eCRF.

Subsequent visits (‘return visits’) may be needed and will be conducted as directed 
by national guidelines by the study physician. Medical data related to any SDC will 
be collected for hospitalised subjects and for subjects in outpatient settings at return 
visits. Treatment will be given according to local standard routines, following 
national guidelines.

The physician will classify the severity of the episode in the eCRF at the time of 
follow-up contact or hospital discharge, if applicable.
7.6.
Biological sample handling and analysis
Please refer to the SPM and any document provided e.g.: Investigator manual, 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). 
 
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
Collected samples will be used for protocol mandated research. In addition, these 
samples may be used to perform research related to the improvement, development 
and quality assurance of the laboratory tests described in this protocol. This may 
include the management of the quality of these tests, the maintenance or 
improvement of these tests, the development of new test methods, as well as making 
sure that new tests are comparable to previous methods and work reliably.

It is also possible that future findings may make it desirable to use the samples 
acquired in this study for future research, not described in this protocol. Therefore, 
all subjects in countries where this is allowed will be invited to give another specific 
consent to allow GSK or a contracted partner use the samples for future research 
including development of tests and their quality assurance. Future research will be 
subject to the laws and regulations in the respective countries and will only be 
performed once an independent Ethics Committee or Review Board has approved 
this research.
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/subject’s parent(s)/LAR(s). 
Refer also to the Investigator Agreement, where it is noted that the investigator cannot 
perform any other biological assays on the samples collected for the study except those 
described in the protocol or its amendment(s). Other biological assays prescribed as part 
of routine care or local guidelines should be performed on separate biological samples.
Collected samples will be stored for a maximum of 20 years (counting from when the last 
subject performed the last study contact), 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.
7.6.1.
Use of specified study materials
When materials are provided by GSK Biologicals, it is MANDATORY that all samples 
(including serum 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 10.3 for the definition of study cohorts/data 
sets to be analysed). 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.
 
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7.6.2.
Biological samples (Amended: 24 July 2017)
Table 5 presents the volume of blood that will be collected from subjects for study-
related testing during each visit.
Table 5
Volume of whole blood for study related testing (Amended: 24 July 
2017)
Age group
Type of Visit
Visits 1, 2 
and 3
SDC – first visit
Cases where dengue 
is ruled out
Early presenter
Late Presenter
6 months to <2 years
3.5 ml 
3.5 ml
3.5 ml
3.5 mL
2-50 years
10 ml
10 ml
10 ml
9 mL
If blood collection is not feasible, a finger prick test may alternatively be performed for 
testing with a IgM/IgG/NS1 ICT.
7.6.3.
Laboratory assays for primary and secondary endpoints
Please refer to APPENDIX A for a detailed description of the assays performed in the 
study. Please refer to APPENDIX C for the address of the clinical laboratories used for 
sample analysis.
Table 6 presents the possible laboratory assays for primary and secondary endpoints on 
the blood sample that will be drawn from the subject.
Table 6
Possible laboratory assays on study blood samples for primary and 
secondary endpoints
Time point
Possible parameters/assays measured
Scheduled visit 1, 2 and 3*
- Anti-DENV indirect IgG Enzyme Linked Immunosorbant Assay 
(ELISA)
Unscheduled Visit: SDC, early presenter (≤ 5 
days following onset of symptoms)
- DENV RT-qPCR
- DENV isolation for sequencing purpose
- DENV sequence
- DENV NS1 rapid test (ICT) or ELISA
- IgM/Ig G rapid test (ICT) or SD BIOLINE Dengue Duo
(Dengue NS1 Ag + IgG/IgM)A
*Blood sample will not be collected at Visit 2 and Visit 3 for subjects in Mexico
Assays for the determination of DENV types will be performed using standardised and 
qualified or validated procedures (refer to Table 7 and Table 8).
Diagnostic tests (NS1, IgG and IgM) will be available at each site. The purpose of these 
diagnostic tests is to provide prompt laboratory results to the physician and the subject. 
Rapid ICT (point of care) will be used. Providing rapid results to the subject may be 
perceived as a benefit of study participation and may help in retaining subjects.
Table 7 presents humoral immunity to DENV.
 
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Table 7
Humoral immunity (DENV antibody determination) for primary and 
secondary endpoints (Amended: 24 July 2017)
System
Component
Method
Kit / Manufacturer
Laboratory
Serum
IgM/IgG to 
DENV
Rapid ICT assay or 
Dengue NS1 Ag + IgM/IgG
Standard Diagnostics 
(ICT) or equivalent/ SD 
BIOLINE Dengue 
Duo(Dengue NS1 Ag 
+IgG/IgM)
or equivalent
Local
IgG to DENV
(for scheduled 
visit 1, 2 and 3*)
ELISA
Indirect IgG Panbio or 
equivalent
Central or local GSK 
designated laboratory 
DENV types
1-4 neutralising 
antibodies
Dengue neutralisation
assay
In-house
GSK designated 
laboratory
*Blood sample will not be collected at Visit 2 and Visit 3 for subjects in Mexico.
Additional potential exploratory tests including neutralising antibodies against JEV and 
other flaviviruses may be performed. Table 8 presents virology.
Table 8
Virology for primary and secondary endpoints
System
Component
Method
Laboratory
Serum
DENV RNA
RT-qPCR
Central or local GSK designated laboratory
Note: Viral isolation may be conducted if needed.
The GSK Biologicals’ clinical laboratories have established a Quality System supported 
by procedures. The activities of GSK Biologicals’ clinical laboratories are audited 
regularly for quality assessment by an internal (sponsor-dependent) but laboratory-
independent Quality Department.
 
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7.6.4.
Laboratory assays to be performed for tertiary endpoints
(Amended: 24 July 2017)
Table 9 presents the possible laboratory assays for tertiary endpoints on the blood sample 
that will be drawn from the subject.
Table 9
Possible laboratory assays on study blood samples for tertiary 
endpoints (Amended: 24 July 2017)
Time point
Possible parameters/assays measured
Scheduled visit 1, 2 and 3*
- DENV neutralising antibody assay
- JEV neutralising antibody assay
- WNV neutralising antibody assay
- Tertiary exploratory assays to characterize DENV immune 
status (e.g. antibody avidity assay, etc.)
- Chikungunya serology (IgM/IgG detection) and neutralizing 
antibodies
- Zika serology (IgM/IgG detection) and neutralizing antibodies
Unscheduled Visit: SDC, early presenter (≤ 5 
days following onset of symptoms)
-  PCR/serology for other infectious agents causing febrile 
illness (chikungunya, Zika, influenza, leptospirosis)
Unscheduled Visit: SDC, late presenter (> 6 
days following onset of symptoms)
- DENV NS1 rapid test 
- IgM/IgG rapid test or IgM/IgG capture ELISA
- PCR/serology for other infectious agents causing febrile 
illness (chikungunya, Zika, influenza, leptospirosis)
Unscheduled Visit: Cases where dengue is 
ruled out
-PCR/serology for other infectious agents causing febrile 
illness (chikungunya, Zika, influenza, leptospirosis)
*Blood sample will not be collected at Visit 2 and Visit 3 for subjects in Mexico.
Laboratory assays related to tertiary endpoints will only be performed after the primary 
and secondary endpoints have been ensured, and the local epidemiology of dengue virus 
disease is better understood. 
The results of the primary and secondary endpoints, public health needs, and availability 
of biological samples will guide:

The subset of subjects selected for which assays related to tertiary endpoints will be 
conducted

Which pathogens should be tested (JEV, WNV, chikungunya, Zika, influenza, 
leptospirosis)

The assays and tests to be performed.
If testing for tertiary endpoints will be conducted, these parameters and rationale will be 
provided to the local ethical committees and relevant health authorities for approval. 
Testing may include, but are not limited to the following tests for humoral immunity and 
virology.
 
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Table 10
Humoral immunity for tertiary objectives
System
Component
Method
Kit / Manufacturer
Laboratory
Serum
Antibodies against Zika 
virus
To be determined
To be determined
GSK designated lab
Serum
Zika virus neutralizing 
antibody
Zika neutralization assay
To be determined
GSK designated 
laboratory
Serum
Antibodies against 
chikungunya virus
To be determined
To be determined
To be determined
Serum
YF virus neutralizing 
antibody
YF neutralization assay
To be determined
GSK designated 
laboratory
Serum
JEV neutralizing antibody
JEV neutralization assay
To be determined
GSK designated 
laboratory
Serum
WNV neutralizing 
antibody
WNV neutralization assay
To be determined
GSK designated 
laboratory
Table 11
Virology for tertiary endpoints
System
Component
Method
Kit/Manufacturer
Laboratory
Serum
Zika RNA
RT-PCR
To be determined
GSK designated laboratory
Serum
Chikungunya RNA
RT-PCR
To be determined
GSK designated laboratory
Serum
JEV RNA
RT-PCR
To be determined
GSK designated laboratory
Serum
WNV RNA
RT-PCR
To be determined
GSK designated laboratory
7.6.5.
Biological samples evaluation
Table 12 presents the aliquoting of blood samples collected at scheduled visits.
Table 12
Possible assays for primary and secondary endpoints for blood
samples collected at scheduled visits
Blood sampling time point
Possible assays
Estimated 
number of 
subjects
Serum 
aliquot
Components 
priority rank
Type of contact/ 
Sampling 
time point
Scheduled Visit 1, 
Visit 2 and 
Visit 3
Day 0
Month 12
Month 24
Anti-DENV IgG indirect 
ELISA
All
200 l
1
DENV virus 1-4 neutralising 
antibody assay
Subset
1x 1 ml or 
remaining 
volume
2
Blood sample will not be collected at Visit 2 and Visit 3 for subjects in Mexico
 
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Table 13 presents the possible assays and aliquot preparation for blood samples collected 
at first unscheduled visit for suspected dengue (for early and late presenters).
Table 13
Aliquot preparation for blood samples collected at visits for 
suspected dengue and follow-up contact
Blood sampling 
time point
Serum aliquot 
volume
Possible Assays
Estimated 
number of 
subjects
Components 
priority rank
First visit –
presentation within 
5 days following 
onset of symptoms
(early presenter)
750 l
DENV RT-qPCR
Unknown
1
Approximately 
500 l
DENV NS1/IgM/IgG rapid test
(ICT) 
Unknown
2
1 ml or remaining 
volume
PCR/serology of other 
infectious aetiologies 
(chikungunya, Zika, influenza, 
leptospirosis)
Unknown
3
Viral isolation and sequencing
First visit –
presentation 6 days 
or more following
onset of symptoms 
(late presenter)
Approximately 
500 l
DENV NS1/IgM/IgG rapid test
(ICT) 
Unknown
1
1 ml or remaining 
volume
PCR/serology of other 
infectious aetiologies 
(chikungunya, Zika, influenza, 
leptospirosis)
Unknown
2
Collected samples may 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.
8.
SAFETY (AMENDED: 24 JULY 2017)
In this prospective cohort study no test product/vaccine will be given. However, blood 
samples will be collected at the enrolment visit (Visit 1), Visit 2, conclusion visit (Visit 
3), for suspected dengue and for subjects where dengue is ruled out. SAEs related to any 
study procedure 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/ subject’s parent(s)/ LAR(s) or designate (if applicable) will be instructed to 
contact the investigator immediately should they/the subject manifest any signs or 
symptoms they perceive as serious/ of concern or indicating a change in their health 
status.
 
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8.1.
Safety definitions
8.1.1.
Definition of a serious adverse event
An SAE is any untoward medical occurrence 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 an 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 
ankle) which may interfere or prevent everyday life functions but do not constitute a 
substantial disruption.
or
e.
Is a congenital anomaly/birth defect in the offspring of a study subject.
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.
 
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8.1.1.1.
Clinical laboratory parameters and other abnormal assessments 
qualifying as SAEs
In absence of diagnosis, 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 (refer to Section 8.1.1). Clinically significant abnormal laboratory 
findings or other abnormal assessments that are present at baseline and significantly 
worsen following the start of the study will also 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.
8.2.
Detecting and recording SAEs
8.2.1.
Time periods for detecting and recording SAEs
SAEs related to study participation (blood sample collection) will be collected and 
recorded from the time of the first study visit until the subject is discharged from the 
study, by the investigator or his/ her designate on the SAE form, within 24 hours of being 
aware of the SAE, irrespective of intensity.
All reported SAEs related to the study procedure will be followed-up by the investigator 
until they have resolved, subsided, stabilised, disappeared, or until the event is otherwise 
explained, or the subject is lost to follow-up.
See Section 8.3 for instructions on reporting of SAEs.
An overview of the protocol-required reporting periods for SAEs is given in Table 14.
Table 14
Reporting periods for SAEs
Study activity
Visit 1
Visit 2
Visit 3
Conclusion visit/
Follow-up contact for 
unscheduled visits 
(whichever occurs last)
Report SAEs related to study procedures
 
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8.2.2.
Evaluation of SAEs
8.2.2.1.
Active questioning to detect SAEs
Each subject/ subject’s parents/ LAR(s) / designate (if applicable) will be instructed to 
contact the investigator immediately should the subject manifest any signs and symptoms 
(s)he perceives/ they perceive as serious.
All SAEs either observed by the investigator or his/ her staff or reported by the subject/ 
subject’s parents/ LAR(s) / designate (if applicable) spontaneously or in response to a 
direct question will be evaluated by the investigator. The nature of each event, date and 
time of onset, outcome, intensity and possible 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 the SAE 
in the eCRF. The investigator is not allowed to send photocopies of the subject’s medical 
records to GSK Biologicals instead of appropriately completing 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.
 
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8.2.2.2.
Assessment of the intensity of SAEs
The investigator will assess the maximum intensity that occurred over the duration of the 
event for all SAEs recorded during the study. The assessment will be based on the 
investigator’s clinical judgement.
The intensity should be assigned to one of the following categories:
1 (mild)
=
An SAE which is easily tolerated by the subject, causing minimal 
discomfort and not interfering with everyday activities.
2 (moderate)
=
An SAE which is sufficiently discomforting to interfere with 
normal everyday activities.
3 (severe)
=
An SAE which prevents normal, everyday activities (in a young 
child, such an SAE would, for example, prevent attendance at 
school/ kindergarten/ a day-care centre and would cause the 
parent(s)/ LAR(s) to seek medical advice. In adults/ adolescents, 
such an SAE would, for example, prevent attendance at work/ 
school and would necessitate the administration of corrective 
therapy.)
An event is defined as ‘serious’ when it meets one of the pre-defined outcomes as 
described in Section 8.1.1.
8.2.2.3.
Assessment of causality
The investigator should assess the causality of each SAE. The investigator will use 
clinical judgement to determine the relationship between the SAEs and study 
participation. Alternative causes, such as natural history of the underlying diseases, other 
concomitant therapy and other risk factors will be considered and investigated.
There may be situations when a 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 report to GSK Biologicals. The investigator may change 
his/her opinion of causality in light of follow-up information and update the SAE 
information accordingly. 
Is there a reasonable possibility that the SAE may have been caused by the study 
procedure (blood sample collection)?
YES
:
There is a reasonable possibility that the study procedure 
contributed to the SAE. 
NO
:
There is no reasonable possibility that the SAE is causally related 
to the study procedure.
 
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If an event meets the criteria to be considered as ‘serious’ (see section 8.1.1), additional 
examinations/tests will be performed by the investigator in order to determine ALL 
possibly contributing factors to each SAE.
Possibly contributing factors include:

Medical history.

Concomitant medication.

Protocol required procedure.

Other procedure not required by the protocol.
8.2.2.4.
Assessment of outcomes
The investigator will assess the outcome of all SAEs recorded during the study as:

Recovered/ resolved.

Recovering/ resolving.

Not recovered/ not resolved.

Recovered with sequelae/ resolved with sequelae.

Fatal (SAEs only).
8.3.
Reporting of SAEs
8.3.1.
Prompt reporting of SAEs related to study participation to GSK 
SAEs that occur in the time period defined in Section 8.2.1 will be reported promptly to 
GSK within the timeframes described in Table 15 once the investigator determines that 
the event meets the protocol definition of an SAE.
Table 15
Timeframes for submitting SAEs related to study participation to 
GSK
Type of event
Initial reports
Follow-up of relevant information
on a previous report
Timeframe
Documents
Timeframe
Documents
SAEs related to study participation‡
24 hours*
SAE screen
24 hours*
SAE screen
* Timeframe allowed after receipt or awareness of the information.
‡ The investigator will be required to confirm review of the SAE causality by ticking the ‘reviewed’ box in the electronic 
Expedited Adverse Events Report within 72 hours of submission of the SAE.
 
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8.3.2.
Contact information for reporting SAEs to GSK 
Back-up Study Contact for Reporting SAEs
24/24 hour and 7/7 day availability:
GSK Biologicals Clinical Safety & Pharmacovigilance
Outside US & Canada sites: 
Fax: 
 or 
 
Email address: 
8.3.3.
Completion and transmission of SAEs reports related to study 
participation to GSK
Once an investigator becomes aware that an SAE has occurred in a study subject, the 
investigator (or designee) must complete the information in the electronic Expedited 
Adverse Event Report WITHIN 24 HOURS. The report 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 report should still be completed within 24 
hours. Once additional information is received, the report should be updated WITHIN 24 
HOURS.
The investigator will always provide an assessment of causality at the time of the initial 
report. The investigator will be required to confirm the review of the SAE causality by 
ticking the ‘reviewed’ box in the electronic Expedited Adverse Events Report within 72 
hours of submission of the SAE.
8.3.3.1.
Back-up system in case the electronic reporting system does not work
If the electronic reporting system does not work, the investigator (or designee) must 
complete, then date and sign a paper Expedited Adverse Event Report 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 reporting system is not working 
and NOT if the system is slow. As soon as the electronic reporting system is working 
again, the investigator (or designee) must complete the electronic Expedited Adverse 
Event Report within 24 hours. The final valid information for regulatory reporting will be 
the information reported through the electronic reporting system. 
8.3.4.
Updating of SAE information after freezing of the subject’s eCRF
When additional SAE information is received after freezing of the subject’s eCRF, new 
or updated information should be recorded on a paper Expedited Adverse Event Report, 
with all changes signed and dated by the investigator. The updated report should be faxed 
to the GSK Biologicals Clinical Safety and Pharmacovigilance department or to the 
Study Contact for Reporting SAEs (see the Sponsor Information) within the designated 
reporting time frames specified in Table 15.

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8.3.5.
Regulatory reporting requirements for SAEs
The investigator will promptly report all SAEs to GSK Biologicals in accordance with 
the procedures detailed in Section 8.3.1. GSK Biologicals has a legal responsibility to 
promptly notify, as appropriate, both the local regulatory authority and other regulatory 
agencies about the safety of a product under epidemiological investigation. Prompt 
notification of SAEs by the investigator to the Study Contact for Reporting SAEs is 
essential so that legal obligations and ethical responsibilities towards the safety of other 
subjects are met.
8.4.
Follow-up of SAEs
8.4.1.
Follow-up during the study
After the initial SAE report, the investigator is required to proactively follow each subject 
and provide further relevant information on the subject’s condition to GSK Biologicals 
(within 24 hours for SAEs, refer to Table 15).
All SAEs documented at a previous visit/ contact and recorded as not recovered/ not 
resolved or recovering/ resolving will be reviewed at subsequent visits/ contacts until the 
end of the study.
8.4.2.
Follow-up after the subject is discharged from the study
The investigator 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.
If the investigator receives additional relevant information on a previously reported SAE, 
he/ she will provide this information to GSK Biologicals using a paper Expedited 
Adverse Event Report.
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 any available post-mortem findings, including 
histopathology.
 
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9.
SUBJECT COMPLETION AND WITHDRAWAL
9.1.
Subject completion
A subject who is available for the concluding visit foreseen in the protocol is considered 
to have completed the study.
9.2.
Subject withdrawal
Subjects who are withdrawn because of SAEs related to study participation must be 
clearly distinguished from subjects who are withdrawn for other reasons. The investigator 
will follow subjects who are withdrawn as the result of an SAE until resolution of the 
event (see Section 8.4.2).
Withdrawals will not be replaced.
From an analysis perspective, a ‘withdrawal’ from the study refers to any subject who 
was not available for the concluding visit foreseen in the protocol.
All data collected until the date of withdrawal/last visit of the subject will be used for the 
analysis.
A subject is considered a ‘withdrawal’ from the study when no study procedure has 
occurred, no follow-up has been performed and no further information has been collected 
for this subject from the date of withdrawal/last contact. The withdrawal of one subject 
does not have impact on the participation of other subjects from the same household.
Investigators will make an attempt to contact those subjects who do not return for 
scheduled visits or follow-up. 
Information relative to the withdrawal will be documented in the eCRF. The investigator 
will document whether the decision to withdraw a subject from the study was made by 
the subject himself/herself, by the subject’s parent(s) or LAR(s), or by the investigator, as 
well as which of the following possible reasons was responsible for withdrawal:

SAE.

Protocol violation.

Consent withdrawal, not due to an SAE*

Moved from the study area.

Lost to follow-up.

Others
*In case a subject is withdrawn from the study because he/she/the subject’s parent(s)/ 
LAR(s) has withdrawn consent, the investigator will document the reason for withdrawal 
of consent, if specified by the subject/ the subject’s parent(s)/LAR(s), in the eCRF.
 
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Subjects who are withdrawn from the study because of SAEs must be clearly 
distinguished from subjects who are withdrawn for other reasons. Investigators will 
follow subjects who are withdrawn from the study as result of an SAE until resolution of 
the event (see Section 8.4.2).
10.
STATISTICAL METHODS
10.1.
Endpoints
10.1.1.
Primary endpoint

RT-qPCR confirmed symptomatic DENV infection (all DENV types) during the 
study period.
10.1.2.
Secondary endpoints

DENV-type specific confirmed symptomatic DENV infection.

Virologically confirmed (by RT-qPCR or NS1) and probable (based on serological 
evidence) symptomatic DENV infection.

Status for anti-DENV IgG at enrolment (indicative of past DENV infection).

Symptoms and severity of symptomatic dengue.
10.1.3.
Tertiary endpoints
Exploratory objectives may be assessed based on, but not limited to the following 
endpoints:

Spatial coordinates of the houses of participating households and of dengue cases 
and date of symptoms onset (applicable only for symptomatic cases).

Neutralising antibodies titres against DENV 1-4.

Neutralising antibody titres against other flaviviruses (such as JEV and WNV).

Other infectious aetiology than dengue in subjects with episodes of febrile illness 
referred to as “suspected dengue cases” (chikungunya, Zika, influenza, 
leptospirosis).

Determinants of past dengue infection and dengue illness.

Characteristics of immune status against DENV infection.
10.2.
Determination of sample size
The target population to be enrolled is estimated at 1750 subjects (approximately 300 to
500 per site). A dropout rate of 5% per year is estimated, which would lead to 
approximately 1662 subjects completing 1 year of follow-up and approximately 1579
completing 2 years of follow-up, accumulating 3327 patient-years.
 
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The incidence of dengue is likely to vary by site and by age group. The study population 
should include between 30% and 50% of adults, but the distribution is not further 
specified as it would make operational feasibility more complex. 
Supposing that the study population is composed of 70% of children with an expected 
incidence of 8 RT-qPCR-confirmed dengue case per 1000 person-years and 30% of 
adults with an expected incidence of 5 RT-qPCR confirmed dengue cases per 1000 
person-years, the study would detect about 19 cases in children and 5 cases in adults (12
cases in year one with a cohort of 1750 subjects and 12 cases in year 2 with a cohort of 
about 1662 subjects). 
The enrolment will be done by household. Each household can be considered as a cluster 
and this induces a design effect to account for the between-cluster variability when 
estimating the confidence intervals (CI) of the incidence rates. The design effect 
measures the increase in the standard error of the incidence rate estimate due to the 
sampling design used and is given by: D = 1 + (b – 1) rho, where rho is the intra-cluster 
correlation (a measure of rate of homogeneity within clusters) and b is the average 
number of subjects sampled per household. Here, we assume b is assumed to be 3. 
Although in theory ‘rho’ can have a value up to 1, in practice values higher than 0.4 are 
uncommon. We have used a conservative estimate of 0.4 for this study [Bennett, 1991]. 
The design effect is then estimated to 1.8.
Table 16 shows the 95% CI for a range of scenarios in term of cohort size, dropout rates 
and incidence rates. With an overall sample size of a minimum of about 1750 evaluable 
subjects, a follow-up period of two years and the expected incidences of 8 cases per 1000 
person-years in children and 5 cases per 1000 person-years in adults, the overall 
incidence rate would be 7.1 with an exact Poisson 95% CI of [4.5; 10.6], and with a CI 
based on the normal approximation and accounting for the design effect of [3.3; 10.9].
 
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Table 16
Expected 95% confidence intervals for the incidence rate of virologically confirmed dengue cases
Cohort 
size
Proportion 
of adults
Dropout 
rate (per 
year)
Incidence rates
Person-
years
Number 
of cases
Overall 
incidence 
(1000 p-y)
Confidence intervals (1000 p-y)
Children (p-y)*
Adults (p-y)
Exact (Poisson)
Normal approx + DE**
Lower
Upper
Lower
Upper
1400
30%
5%
0.008
0.003
2662
17
6.5
3.8
10.4
2.4
10.6
1575
30%
5%
0.008
0.003
2994
19
6.5
3.9
10.1
2.6
10.4
1750
30%
5%
0.008
0.003
3327
22
6.5
4.1
9.9
2.8
10.2
1400
30%
5%
0.008
0.005
2662
19
7.1
4.2
11.1
2.8
11.4
1575
30%
5%
0.008
0.005
2994
21
7.1
4.4
10.8
3.1
11.1
1750
30%
5%
0.008
0.005
3327
24
7.1
4.5
10.6
3.3
10.9
1400
30%
5%
0.01
0.003
2662
21
7.9
4.9
12.1
3.4
12.4
1575
30%
5%
0.01
0.003
2994
24
7.9
5.0
11.8
3.6
12.2
1750
30%
5%
0.01
0.003
3327
26
7.9
5.2
11.5
3.8
12.0
1400
30%
5%
0.01
0.005
2662
23
8.5
5.4
12.8
3.8
13.2
1575
30%
5%
0.01
0.005
2994
25
8.5
5.5
12.5
4.1
12.9
1750
30%
5%
0.01
0.005
3327
28
8.5
5.7
12.3
4.3
12.7
*p-y: Person-years.
**: Confidence intervals based on the normal approximation and accounting for a design effect of 1.8.
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10.3.
Cohorts for Analyses
10.3.1.
Total cohort
The Total cohort will include all subjects enrolled in the study.
10.3.2.
According-To-Protocol (ATP) cohort
The ATP cohort will include all evaluable subjects, that is, subjects who meet all 
eligibility criteria, complying with the procedures defined in the protocol and for whom 
data of at least one follow-up contact are available. The surveillance period for one 
subject will be defined as the duration from the date of enrolment until the date of the last 
follow-up contact for this subject. Reasons for elimination from ATP analyses will be 
established at the time of data cleaning and documented.
10.4.
Derived and transformed data
RT-qPCR-confirmed symptomatic DENV cases and probable symptomatic DENV cases 
will be detected on the basis of the definitions presented in Section 5.
The severity of the dengue symptomatic infections will be derived as described in Section 
5.3.
10.5.
Analysis of demographics
Demographic characteristics (age at study entry and gender), medical history and 
vaccination history for JE and YF will be summarised by group using descriptive 
statistics:

Frequency tables will be generated for categorical variable such as centre.

Mean, median, standard error will be provided for continuous data such as age.
10.6.
Descriptive analyses of laboratory results
The primary analysis will be based on the ATP cohort. If the percentage of enrolled 
subjects with serological results excluded from the ATP cohort is 5% or more, a second 
analysis based on the Total cohort will be performed to complement the ATP analysis. 
Descriptive analyses will be performed at enrolment (Visit 1) for each laboratory test 
available, by site and overall:

The seroprevalence of each DENV serotype, and other flaviviruses (i.e. JEV; 
(Southeast Asia only) or WNV; (Latin America only) will be calculated with 95% 
CI.

If applicable, GMT with 95% CI will be tabulated for antibodies against each virus.
 
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
If applicable, the range and distribution of antibody concentrations for each virus
will be tabulated.
Descriptive analyses will be performed at visits for suspected dengue for each laboratory 
test available, by site and overall: 

The seroprevalence of each DENV serotype, and other flaviviruses (i.e. JEV; 
(Southeast Asia only) or WNV; (Latin America only) will be calculated with 95%CI. 
The denominator will be the number of visits.

If applicable, GMT with 95% CI will be calculated for antibodies against each virus.

If applicable, the range and distribution of neutralizing antibody titres for each virus
will be tabulated.
95% CI will account for clustering of observations within households for the analyses by 
centre and also the stratification by centres for the overall analyses. Details of the 
methods will be described in the statistical analysis plan (SAP).
10.7.
Analysis of primary objective
The primary analysis will be based on the ATP cohort. If the percentage of enrolled 
subjects with serological results excluded from the ATP cohort is 5% or more, a second 
analysis based on the Total cohort will be performed to complement the ATP analysis.
The following analyses will be performed:
Incidence rate of RT-qPCR confirmed symptomatic dengue infection with 95% CI: the 
numerator will be the number of subjects with RT-qPCR confirmed symptomatic dengue 
infection during the study period. The denominator will be the total person-years at risk, 
i.e., from enrolment until the first RT-qPCR confirmed symptomatic dengue infection 
during the entire study period.
CIs for incidence rates and prevalence will account for clustering of observations within 
households for the analyses by centre and also the stratification by centres for the overall 
analyses. Details of the methods will be described in the statistical analysis plan (SAP).
10.8.
Analysis of secondary objectives
The following analyses will be performed within each centre and overall, and for each 
year/season and overall:

The analyses planned for the primary objective will be repeated by age class, gender, 
serotype, and dengue serological status at the beginning of the analysis period (visit 
1 for the first season and overall, or the last visit for suspected dengue cases prior to 
season for the second season). The analysis period for the second season will be 
determined according to the peak of the season.
 
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
Incidence rates of probable symptomatic DENV infection with 95% CI by age class, 
gender, dengue serological status at the beginning of the analysis period, and overall: 
the numerator will be the number of subjects with probable dengue infection during 
the analysis period in each subset. The denominator will be the total person-years at 
risk in each subset, i.e. from the beginning of the analysis period until the first 
probable symptomatic dengue infection.

Incidence rate of RT-qPCR confirmed and probable symptomatic DENV infection
combined with 95% CI by age class, gender, dengue serological status at the 
beginning of the analysis period, and overall: the numerator will be the number of 
subjects with RT-qPCR confirmed symptomatic and probable dengue infection 
during the analysis period in each subset. The denominator will be the total person-
years at risk in each subset, i.e. from the beginning of the analysis period until the 
first RT-qPCR confirmed symptomatic or probable symptomatic dengue infection.
The following analyses will be performed within each centre and overall:

The (crude) seroprevalence of DENV infection with 95% CI at enrolment (visit 1), 
by age class and overall: the proportion of dengue IgG positive subjects among all 
subjects and among dengue cases (confirmed and probable) with available results. 

The clinical characteristics of symptomatic DENV infection (symptoms, 
hospitalisations, severity) will be presented as overall and per sub-group (type 
(DENV 1-4), gender and age class). 
CIs for incidence rates and proportions will account for clustering of observations within 
households for the analyses by centre and also the stratification by centres for the overall 
analyses. Details of the methods will be described in the SAP.
10.9.
Other analyses
All analyses will be performed on the ATP cohort.
The following analyses will be performed within each centre and overall, and for each 
year/season and overall:

The attack rate within households with an original RT-qPCR-confirmed symptomatic 
DENV infection. All types of DENV infections will be considered. The attack rate 
will be the proportion of subjects with symptomatic DENV infection among subjects 
with an original RT-qPCR-confirmed symptomatic DENV infection within their 
household.

Analysis of spatio-temporal transmission
The proportion of dengue infections among study participants living in circles of 
various radius (less than 20 metres, 20-40 metres, 60-80 metres, 80-100 metres and 
up to 200 metres) in the neighbourhood of laboratory confirmed dengue cases 
(positive index case) and participants without evidence of dengue infection (negative 
index case) will be estimated. 
 
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The time distribution of laboratory-confirmed symptomatic dengue infections will be 
described. The cumulative distribution of the cases every month will be presented in 
graphs for each season.
The spatial distribution of participants will support in making a model to simulate the 
conduct of an efficacy trial and assess potential sources of biases (heterogeneity of 
spatial transmission, baseline immune status, etc.)
The analyses for the other exploratory objectives will be described in detail in the SAP.
10.10.
Interpretation of analyses
There is no hypothesis testing in this study. 95% CI will be computed for incidence rates 
and proportions. CIs for incidence rates and proportions will account for clustering of 
observations within households for the analyses by centre and also the stratification by 
centres for the overall analyses. Details of the methods will be described in the SAP.
10.11.
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.
10.11.1.
Sequence of analyses
The final analysis will be performed when all data have been collected and cleaned.
10.11.2.
Statistical considerations for interim analyses
No interim analysis is planned for this study.
11.
ADMINISTRATIVE MATTERS
To comply with ICH GCP, GPP, administrative obligations relating to data collection, 
monitoring, archiving data, audits, confidentiality, ownership and publications must be 
met.
11.1.
Electronic Case Report Form instructions
A validated GSK defined electronic data collection tool 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.
 
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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 designee. In all cases, the 
investigator remains accountable for the study data. 
Once the database is archived and the clinical study report is complete and approved by 
all parties, each participating investigator will be provided with a CD-ROM of the final 
version of the data generated at his/her investigational site.
11.2.
Study monitoring by GSK Biologicals
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, any other 
study agreements, GCP and all applicable regulatory requirements. 
The subject information will be anonymised (see GLOSSARY OF TERMS) using a code 
number to assure confidentiality of the subjects’ data. No GSK Biologicals personnel will 
have the ability to link data to an identifiable individual. The investigator and the head of 
the medical institution (where applicable) agrees to allow the monitor direct access to all 
relevant documents.
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 a eCRF review and a Source Document 
Verification (SDV). By SDV we understand verifying eCRF 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 eCRF. 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 eCRF will serve as the source must be identified, agreed and 
documented in the source documentation agreement form.
For eCRF, the monitor will mark completed and approved screens at each visit. 
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.
 
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11.3.
Record retention
Following closure of the study, the investigator must maintain all site study records 
(except for those required by local regulations to be maintained elsewhere) in a safe and 
secure location. The records must be easily accessible, when needed (e.g., audit or 
inspection), and must be available for review 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); however, caution needs to be 
exercised before such action is taken. The investigator must ensure 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 that an acceptable back-up of the reproductions exists and that 
there is an acceptable quality control procedure in place 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 a particular site, as dictated by ICH GCP or other applicable 
guidelines, any institutional requirements or applicable laws or regulations, or GSK 
standards/procedures; otherwise, the minimum retention period will default to 15 years. 
The investigator/institution must notify GSK of any changes in the archival 
arrangements, including, but not limited to, archival at an off-site facility and transfer of 
ownership of the records in the event the investigator leaves the site. 
11.4.
Quality assurance
To ensure compliance with GCP or other applicable guidelines 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.
11.5.
Posting of information on publicly available registers and 
publication policy
Study information from this protocol will be posted on public registers before enrolment 
of subjects begins.
Observational studies that do not evaluate vaccines/products are progressed for 
publication in the scientific literature when the results provide important scientific or 
medical knowledge.
 
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11.6.
Provision of study results to investigators
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 Biologicals site or other 
mutually-agreed location.
GSK Biologicals will also provide the investigator with the full summary of the study 
results. The investigator is encouraged to share the summary results with the study 
subjects, as appropriate.
11.7.
Collection, analysis, storage of spatial data
Access to spatial data will be restricted to the site having collected the data and to study 
team members performing the spatial and temporal analysis described in Section 10.9. 
Spatial coordinates will be modified using an algorithm respecting the relative position of 
all subjects, but removing the link with the original location of the household. Only 
modified coordinates will be archived. 
12.
COUNTRY SPECIFIC REQUIREMENTS
12.1.
Thailand specific information and requirements
12.1.1.
List name, affiliation and contact details of all investigators
Name and Address of Principal Investigator
Principal Investigator
Assoc. Prof. Dr. Pornthep Chanthavanich
Department of Tropical Pediatrics,
Faculty of Tropical Medicine, Mahidol University
, 
,
, 
, Thailand 10400
Tel: 
, 
, Mobile: 
Fax: 
Email: 

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PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
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Consultant
Dr. 
, .FRCST
Deputy  Nakhonpathom Provincial  Chief  Medical  Officer,
Expert Public Health Physician (Preventive Medicine)
Nakhonpathom Provincial Health Office
, 
 
, Nakhon Pathom 73000
Tel: 
, Mobile: 
, 
Fax: 
Email: 
Sub-Investigator
Assoc. Prof. Dr. Chukiat Sirivichayakul
Department of Tropical Pediatrics,
Faculty of Tropical Medicine, Mahidol University
, 
,
, 
, Thailand 10400
Tel: 
, 
, Mobile: 
Fax: 
Email: 
Assist. Prof. Dr. Kriengsak Limkittikul
Department of Tropical Pediatrics,
Faculty of Tropical Medicine, Mahidol University
, 
,
, 
, Thailand 10400
Tel: 
, 
, Mobile: 
Fax: 
Email: 

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PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD

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Assist. Prof. Watcharee Chokejindachai
Department of Tropical Pediatrics,
Faculty of Tropical Medicine, Mahidol University
, 
,
, 
, Thailand 10400
Tel: 
, 
, Mobile: 
Fax: 
Email: 
Dr. Weerawan Hattasingh
Department of Tropical Pediatrics,
Faculty of Tropical Medicine, Mahidol University
, 
,
, 
, Thailand 10400
Tel: 
, 
, Mobile: 
Fax: 
Email: 
Assist. Prof. Wijitr Fungladda (Retired)
Department of Social and Environmental Medicine,
Faculty of Tropical Medicine, Mahidol University
, 
,
, 
, Thailand 10400
Tel: 
 Mobile: 
Fax: 
Email: 
Dr. Supawat Chatchen
Department of Tropical Pediatrics,
Faculty of Tropical Medicine, Mahidol University
, 
,
, 
, Thailand 10400
Tel: 
, 
, Mobile: 
Fax: 
Email: 

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PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
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12.1.2.
Definition of burden on the study protocol (Amended: 24 July 
2017)
Burden in the study protocol means incidence and severity of Dengue infection and 
hospital admission of volunteer/subjects.
12.1.3.
Duration of the study, site address and number of participating 
subjects in Thailand (Amended: 24 July 2017)
Trial Site in Thailand
There is 1 trial site participating in Thailand at Sam Kwai Phuek Health Promoting 
Hospital, Muang District, Nakhon Pathom Province. The site is planning to enrol 500 
subjects in this study. Study team members are from Department of Pediatric Tropical 
Medicine, Faculty of Tropical Medicine, Mahidol University, and Deputy Nakhonpathom 
Provincial Chief Medical Officer, Nakornpathom provincial Health Office, consultant of 
the study.
This study is an international multicentre (International) cohort study that will be 
conducted in 4 countries. Sample sizes are listed below:

Thailand is planning to enrol 500 subjects

Malaysia is planning to enrol 400 subjects

Philippines is planning to enrol 500 subjects

Mexico is planning to enrol 350 subjects
Duration of the study in Thailand
The study duration for Thailand is 2.0 years (24 months).
Planning of recruitment period is October 2017-June 2018.
Follow up visit period of the study is October 2017- October 2020.
Planning of the study closure is October 2020.
For each subject, the study duration is approximately 2 years (24 months)
12.1.4.
Study population groups and Calculation of study population 
groups in Thailand (Amended: 24 July 2017)
The size of Thailand study population is derived from the average yearly incidence of 
dengue fever in Sam Khwai Phuak Sub-district from 2009 to 2013 of 283 per 100,000 
people. If a lower than actual incidence is assumed, according to population data of Sam 
Khwai Phuak Sub-District of approximately 10,900 people, a sample size of 500 people 
will be able to detect the incidence of dengue fever in Sam Khwai Phuak Sub-district of 
close to 600 per 100,000 people, with a confidence level of 80 percent and an estimated 
data deficiency of no more than 15 percent.
 
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In Thailand, a total of approximately 500 subjects will be recruited and there will be a 
total of 1 study site participating in this study, which is Sam Khwai Phuek Health 
Promotion Hospital, Muang District, Nakhon Pathom Province.
Department of Tropical Pediatrics, Faculty of Tropical Medicine, Mahidol University, is 
the main site for administration with no recruitment. Investigator and study team are 
affiliated with Department of Tropical Pediatrics, Faculty of Tropical Medicine, Mahidol 
University.
Deputy Nakhonpathom Provincial Chief Medical Officer [Expert Public Health Physician 
(Preventive Medicine)] from Nakhon Pathom Province, a local person, is the project 
consultant.
The roles of study team at the Sam Kwai Phuek Health Promotion Hospital is:
To be the local person coordinating public utility payments at the study site for which the 
research project is the party responsible for, and coordinating with community leaders 
and the community in the area. 
12.1.5.
Overview of the Subject Recruitment Plan for Thailand
The method of finding/recruiting appropriate subjects will be selected by each research 
site participating in the study. One of the following 2 methods may be selected:

School recruitment method

Community recruitment method (without schools being involved)
For Thailand, however, subjects will be recruited using the community recruitment 
method (without schools being involved).
12.1.6.
Adding Secondary Objective Explanations
To describe the clinical symptoms of patients infected with the dengue virus (which have 
been confirmed and which are within the scope of suspected infection)*
*This includes studying clinical symptoms and the severity of the dengue virus infection. 
The severity of the dengue virus infection in this study will be monitored with the subject 
by taking their physical examination history, and tracking the progress of the disease and 
hospitalization during the follow-up contact after 21 days of the first visit. 
12.1.7.
Informed Consent and Consent for Thailand
Obtaining consent from subjects in this study means requesting consent from individual 
subjects.
Subjects/guardians of subjects will be given an explanation of various details, including 
both the natures and risks involved with the study. The above process will be carried out 
by the head of protocol or protocol participants prior to obtaining signatures of consent 
from 
the 
subjects/imprinting 
the 
fingerprints 
of 
the 
subjects 
and/or 
the 
father/mother/legally acceptable representative of the subjects prior to their participation 
 
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in the study, including the signature of the person providing protocol details 
(investigator) and relevant witnesses. The person providing protocol details must allow 
sufficient time for the subjects to decide whether or not to participate in the study, with 
the signing of the Subject Information Sheet and Informed Consent Form for adults, and 
Subject Information Sheet and Informed Assent Form for children, or Subject 
Information Sheet and Informed Consent Form for the father/mother/legally acceptable 
representative of the subjects to the amount of 2 copies of each. The first copy will be 
kept at the study site and the second copy will be kept by the subject. In addition, if the 
subjects or the father/mother/legally acceptable representative of the subjects would like 
to appoint a designee* to help with certain parts of the study procedures, including to 
help in monitoring and checking the body temperature of the subjects in the event they 
have a fever, completing daily records, answering calls from study staff who will ask 
whether or not the participant has a fever, and reminding the subjects of the study 
procedure, and help planning study visits, this designee is not able to make any decision 
on behalf of the subjects, or the father/mother/legally acceptable representative of the 
subjects. Moreover, the above designee must sign their name to receive consent from the 
subjects or the father/mother/legally acceptable representative of the subjects, or the 
father/mother/legally acceptable representative of the subjects together with the signature 
of the person providing protocol details (investigator) and the witnesses in the designee 
information document and letter of consent to participate in the study, to the amount of 2 
copies. The first copy will be kept at the research site and the second copy will be kept by 
the subject.
The Subject Information Sheet and Informed Consent Form for this study are as follows:

Subject Information Sheet for research subject that are between 20 – 50 years old

Informed Consent Form for subjects that are between 20 – 50 years old

Subject Information Sheet for the father/mother/legally acceptable representative of 
subject from 6 months old to less than 20 years old

Informed Consent Form for the father/mother/legally acceptable representative of 
subject from 6 months old to less than 20 years old

Subject Information Sheet for subject from 7 years to less than 13 years old

Assent Form for subject from 7 years to less than 13 years old.

Subject Information Sheet for subject from 13 years to less than 20 years old. 

Assent Form for subject from 13 years to less than 20 years old. 

Subject Information Sheet for designees.

Informed Consent Form for designees.
12.1.8.
Compiling Area Coordinates of Households in Thailand
This study involves the compilation of the geographical area coordinates of households. 
The compilation of the area coordinates of subjects participating in the protocol with a 
Global Positioning System (GPS) device will deliver only subject number data and their 
area coordinates. It will not specify the name, surname, home address, or any data that 
could affect the privacy of the subjects. 
 
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12.1.9.
Managing Biological Samples in Thailand
For Thailand, samples of subjects will be stored for a period of 5 years after the study is 
completed (counting from the date of the last subject's last contact in the study). In the 
event that investigator needs to store blood samples for longer than 5 years, the 
investigator and the study team will request approval from the Ethical Review Committee 
for Research in Human Subjects for a further period of 5 years, and the samples 
remaining after the period approved will be destroyed. 
Subject samples will only be used for tests in this study.
12.1.10.
Potential Risks and Inconveniences, and Prevention Measures 
Blood drawing may cause slight pain in the area blood is drawn from. There may be 
swelling or bruising, or dizziness. After blood is drawn some people may feel faint, or 
nauseous, and in very rare cases infection may arise in the area blood is drawn. 
Investigator will use sterile blood drawing techniques to prevent infection from blood 
drawing. We will use hospitals with experience in blood drawing, especially for small 
children, and will use a scalp vein of appropriate size. For any side effects that may be 
caused by blood drawing, investigator will offer treatment until full recovery free of 
charge. 
12.1.11.
Benefits, Compensation, Reimbursement, Treatment and 
Solution in the Event of Complications for the Investigator
Benefits, Compensation, Reimbursement, Treatment and Solution in the Event of 
Complications for the Investigator
Subjects may not receive direct benefits from this study. However, knowledge regarding 
dengue fever derived from this study will be of great benefit in the prevention and 
management of dengue fever and administration of vaccines to prevent dengue fever in 
the future. 
Subjects will receive treatment for any fever symptoms from a doctor or healthcare 
professional at the Sam Khwai Phuak Sub-District Health Promotion Hospital in 
accordance with standard medical practice guidelines.
Subjects will be tested for dengue virus diagnoses (NS1, IgG and IgM) as specified in the 
protocol, and will know the results within 15 minutes. This will benefit subject treatment 
at no expense.
12.1.12.
Compensation and Reimbursement (Amended: 24 July 2017)
Compensation
None
Reimbursement
 
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Subjects will receive the amount of 1,000 baht per person per visit as specified in the 
protocol as a reimbursement for time and travel costs, except the case where you have a 
fever when attending a scheduled visit, but the person providing treatment suspects that 
he/she may have dengue fever, in which case a return visit appointment will be made on 
the following day.
12.1.13.
Other Ethical Issues 
Confidentiality
Investigator will keep personal information, study documents, and study results related to 
this study confidential, without disclosing them publicly. The investigator certifies that 
the personnel working in this project and the study sponsor will act in the same way. This 
study will keep subject information confidential and will disclose it in the form of overall 
study results that does not include any names or other data which may be able to identify 
the subjects, by using a numerical code instead of a name. This information, including 
medical records, may be examined by authorized personnel from Ministry of Public 
Health of Thailand or the Ethical Review Committee for Research in Human Subjects, 
only for the benefit of oversight, monitoring, and research evaluation. Regardless, the 
study sponsor and representatives, including those involved in all parts, must act in 
accordance with good clinical practices, and must, to the best of their ability, endeavour 
to keep personal information of subjects confidential. 
Budget Details and Funding Sources
Study sponsor GlaxoSmithKline Biologicals
Budget Details: As per documents showing research budget details.
 
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12.1.14.
Signature of all investigators
Principal Investigator
____________________________________
________________
(Assoc. Prof. Dr. Pornthep Chanthavanich)
Day / Month/ Year
Consultant
____________________________________
________________
(Dr. 
)
Day / Month/ Year
Sub-Investigator
____________________________________
________________
      (Assoc. Prof. Dr. Chukiat Sirivichayakul)
Day / Month/ Year
____________________________________
________________
           (Assist. Dr. Kriengsak Limkittikul)
Day / Month/ Year
____________________________________
________________
      (Assist. Prof. Dr. Watcharee Chokejindachai)
Day / Month/ Year
____________________________________
________________
                (Dr. Weerawan Hattasingh)
Day / Month/ Year
____________________________________
________________
          (Assoc. Prof. Dr. Wijitr Fungladda)
Day / Month/ Year
____________________________________
________________
                (Dr. Supawat Chatchen)
Day / Month/ Year
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13.
REFERENCES
Bennett S, Wood T, Liyanage WM et al. A simplified general method for cluster-sample 
surveys of health in developing countries. World Health Stat Q (1991); 44(3):98-106.
Capeding RZ et al. The incidence, characteristics, and presentation of dengue virus 
infections during infancy. Am J Trop Med Hyg. (2010);82(2):330–6.
Cuong HQ, Hien NT, Duong TN. Quantifying the Emergence of Dengue in Hanoi, 
Vietnam: 1998-2009. PLoS Negl Trop Dis (2011);5(9):1-7.
Dussart P, Baril L, Petit L. Clinical and virological study of dengue cases and the 
members of their households: the multinational DENFRAME Project. PLoS Negl Trop
Dis (2012); 6(1)1482.
Edelman R, Hombach J. “Guidelines for the clinical evaluation of dengue vaccines in 
endemic areas”: Summary of a World Health Organization Technical Consultation. 
Vaccine (2008); 26:4113-4119.
Endy TP, Nisalak A, Chunsuttitwat S, et al. Relationship of pre-existing dengue virus 
(DV) neutralising antibody levels to viremia and severity of disease in a prospective 
cohort study of DV infection in Thailand. J Infect Dis. 2004;189(6):990-1000.
Endy TP. Human immune responses to dengue virus infection: lessons learned from 
prospective cohort studies. Front Immunol. 2014; 5:183.
Epidemiological Panorama Dengue Fever and Hemorrhagic Fever. 
(http://www.epidemiologia.salud.gob.mx/doctos/panodengue/PANORAMAS_2013/Pano
_dengue_sem51_sem2013.pdf) Accessed on March 29th 2014.
Gutiérrez G, Gresh L, Pérez MÁ, Elizondo D, Avilés W, Kuan G, Balmaseda A, Harris 
E. Evaluation of the diagnostic utility of the traditional and revised WHO dengue case 
definitions. PLoS Negl Trop Dis. 2013;7(8):e2385
Honório NA, Nogueira RM, Codeço CT, Carvalho MS, Cruz OG, Magalhães M de A, et 
al. Spatial evaluation and modelling of Dengue seroprevalence and vector density in Rio 
de Janeiro, Brazil. PLoS Negl Trop Dis. 2009;3(11):e545.
Hung NT et al., Dengue hemorrhagic fever in infants: a study of clinical and cytokine 
profiles. J Infect Dis. (2004);189:221–32.
International Society for Pharmacoepidemiology (ISPE). Guidelines for Good 
Pharmacoepidemiology Practices. GPP, 
http://www.pharmacoepi.org/resources/guidelines_08027.cfm; 2007. Accessed 17 April 
2014.
Kalayanarooj S, Nimmannitya S. Clinical presentations of dengue hemorrhagic fever in 
infants compared to children. J Med Assoc Thai. (2003);86(3):S673–80.
 
CONFIDENTIAL
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200318 (EPI-DENGUE-007 BOD)
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Letson GW, Singhasivanon P, Fernandez E et al. Dengue vaccine trial guidelines and role 
of large-scale, post proof-of-concept demonstration projects in bringing a dengue vaccine 
to use in dengue endemic areas. Human Vaccines (2010);6(10):802-809.
Macedo GA, Gonin ML, Pone SM, Cruz OG, Nobre FF, Brasil P. Sensitivity and 
specificity of the World Health Organization dengue classification schemes for severe 
dengue assessment in children in Rio de Janeiro. PLoS One. 2014 28;9(4):e96314.
Mammen MP, Pimgate C, Koenraadt CJ, Rothman AL, Aldstadt J, Nisalak A, et al. 
Spatial and temporal clustering of dengue virus transmission in Thai villages. PLoS Med. 
2008;5(11):e205.
Martínez-Vega RA, Danis-Lozano R, Velasco-Hernández J, Díaz-Quijano FA, González-
Fernández M, Santos R, et al. A prospective cohort study to evaluate peridomestic 
infection as a determinant of dengue transmission: protocol. BMC Public Health. 2012; 
12:262.
Paz-Soldan VA, Reiner RC Jr, Morrison AC, Stoddard ST, Kitron U, Scott TW, et al. 
Strengths and Weaknesses of Global Positioning System (GPS) Data-Loggers and Semi-
structured Interviews for Capturing Fine-scale Human Mobility: Findings from Iquitos, 
Peru. PLoS Negl Trop Dis. 2014;8(6):e2888. 
Rabaa MA, Klungthong C, Yoon IK, Holmes EC, Chinnawirotpisan P, Thaisomboonsuk 
B, et al. Frequent in-migration and highly focal transmission of dengue viruses among 
children in Kamphaeng Phet, Thailand. PLoS Negl Trop Dis. 2013;7(1):e1990. 
Salje H, Lessler J, Endy TP, Curriero FC, Gibbons RV, Nisalak A, N et al. Revealing the 
microscale spatial signature of dengue transmission and immunity in an urban population. 
Proc Natl Acad Sci U S A. 2012;109(24):9535-8.
San Martín JL, Brathwaite O, Zambrano B. The epidemiology of dengue in the Americas 
over the last three decades: A worrisome reality. Am. J. Trop. Med. Hyg
(2010);82(1):128-135.
Siqueira-Junior JB, Maciel IJ, Barcellos C, Souza WV, Carvalho MS, Nascimento NE, 
Oliveira RM, Morais-Neto O, Martelli CM. Spatial point analysis based on dengue 
surveys at household level in central Brazil. BMC Public Health. 2008; 8:361.
Thomas SJ. The necessity and quandaries of dengue vaccine development. Journal of 
Infectious Diseases (2011);203:299-303.
Van Panhuis WG, Luxemburger C, Pengsaa K et al. Decay and persistence of maternal 
dengue antibodies among infants in Bangkok. Am. J. Trop. Med. Hyg. (2011); 
85(2):355-362.
Vazquez-Prokopec GM, Kitron U, Montgomery B, Horne P, Ritchie SA. Quantifying the 
spatial dimension of dengue virus epidemic spread within a tropical urban environment. 
PLoS Negl Trop Dis. 2010;4(12):e920.
 
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Whitehorn J, Simmons CP. The pathogenesis of dengue. Vaccine. 2011; 23;29(42):7221-
8.
WHO. Geneva, Switzerland. Dengue Haemorrhagic Fever: Diagnosis, Treatment, 
Prevention and Control. 1997. Second edition.
WHO. Geneva, Switzerland. Dengue: Guidelines for Diagnosis, Treatment, Prevention 
and Control. 2009.
World Health Organization (WHO). Dengue and severe dengue. Fact sheet no. 117. 
World Health Organization (2012a). URL: 
http://www.who.int/mediacentre/factsheets/fs117/en/.
World Health Organization (WHO). Handbook for clinical management of dengue. 
World Health Organization (2012b): 1-124. URL: 
http://www.who.int/denguecontrol/9789241504713/en/
WHO guidelines on drawing blood. Best practices in phlebotomy. 2010. URL: 
http://apps.who.int/iris/bitstream/10665/44294/1/9789241599221_eng.pdf. Last accessed: 
23 November 2015.
Yoon IK, Rothman AL, Tannitisupawong D, Srikiatkhachorn A, Jarman RG, Aldstadt J, 
et al. Under recognized mildly symptomatic viremic dengue virus infections in rural Thai
schools and villages. J Infect Dis. 2012;206(3):389-98.
 
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APPENDIX A
LABORATORY ASSAYS
SD Bioline Dengue Duo (NS1 Ag + Ab Combo)
The SD Bioline Duo rapid test is commercial CE labelled individual test from Standard 
Diagnostics, Inc. It is an in-vitro immunochromatographic, one step assay designed to 
detect both dengue virus NS1 antigen and differential IgG/IgM antibodies to dengue virus 
in human serum, plasma or whole blood. 
Performance characteristics of the assay are described in the manufacturer package insert.
NS1-antigen ELISA
Platelia™ Dengue NS1 Ag is a one step sandwich format microplate enzyme 
immunoassay for the qualitative or semi-quantitative detection of Dengue virus NS1 
antigen in human serum or plasma.
Performance characteristics of the assay are described in the manufacturer package insert.
Dengue IgG Indirect ELISA
The Panbio Dengue IgG Indirect ELISA is a commercial CE labelled kit for the 
qualitative detection of IgG antibodies to dengue antigen serotypes (1, 2, 3 and 4) in 
serum.
Performance characteristics of the assay are described in the manufacturer package insert.
Dengue IgG Capture ELISA
The Panbio Dengue IgG Capture ELISA is a commercial CE labelled kit for the 
qualitative presumptive detection of elevated IgG antibodies to dengue virus (serotypes 
1-4) in patients with secondary infection.
Performance characteristics of the assay are described in the manufacturer package insert.
Dengue IgM Capture ELISA
The Panbio Dengue IgM Capture ELISA is a commercial CE labelled kit for the 
qualitative detection of IgM antibodies to dengue antigen in serum, as an aid in the 
clinical laboratory diagnosis of patients with clinical symptoms consistent with dengue 
fever.
Performance characteristics of the assay are described in the manufacturer package insert.
Dengue serotype-specific RT-qPCR
The dengue serotype-specific RT-qPCR assay has been developed for the detection of 
DENV serotype RNA in serum samples. Viral RNA is extracted from serum samples and 
reverse-transcribed into cDNA. The resulting cDNA is quantified by real time PCR using 
specific DNA primers targeting the DENV capsid gene. PCR is performed as 2 duplex 
amplification (DENV-1/-3 and DENV-2/-4).
 
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Simplexa TM Dengue REF MOL3100 Rev. C 
A real-time RT-PCR assay for the in vitro detection and typing of dengue virus serotypes 
1, 2, 3 and 4. 
Principles of the procedure: The assay is a real-time RT-PCR that discriminates serotypes 
1 and 4 in one reaction (well), and serotypes 2 and 3 in another reaction (well). The assay 
is composed of two principal steps: (1) extraction of RNA from specimens, and (2) 
amplification of the extracted RNA using bi-functional fluorescent probe-primers and 
reverse primers. The assay amplifies four serotype specific regions: dengue 1 (NS5 gene), 
dengue 2 (NS3 gene), dengue 3 (NS5 gene) and dengue 4 (capsid gene). An RNA 
internal control is used to monitor the extraction process and to detect RT-PCR 
inhibition.
Viral micro-plaque-reduction neutralisation (µPRN) assay
The anti-DENV 1-4 PRN assay is performed in 96-well plates on Vero cells Serial 
serum dilutions are incubated with a fixed target amount of 75 PFU DENV. The serum-
virus mixture is then added to Vero cells and incubated for 2 days in parallel to control 
wells with virus only. The replication of the infectious (non-neutralised) virus is 
monitored by detecting the expression of DENV envelope (gE) protein by infected cells 
with pan-flaviviruses anti-gE mouse monoclonal antibody (4G2, from ATCC, Rockville, 
MD, USA) conjugated to peroxidase. Using a precipitating chromogenic substrate for 
peroxidase, plaques of infected cells are counted. The number of plaques in a given 
serum-virus well is compared to the number of plaques in virus control wells. The 
Neutralisation titre corresponds to the inverse of the serum dilution giving 50 % 
reduction in the number of plaques as compared to the virus control wells.
 
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APPENDIX B
CLINICAL LABORATORIES
Table 17
Outsourced laboratories
Laboratory
Address
Laboratório de Tecnologia Virológica, Bio-
Manguinhos, Fiocruz
Rio de Janeiro, Brazil
Q2 Solutions
26081 Avenue Hall, Suite 150, Valencia Ca, 
91355-1241, United States
 
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APPENDIX C
AMENDMENTS AND ADMINISTRATIVE 
CHANGES TO THE PROTOCOL
GlaxoSmithKline Biologicals
Vaccine Value & Health Science (VVHS)
Protocol Amendment 1
eTrack study number 
and Abbreviated Title
200318 (EPI-DENGUE-007 BOD)
Amendment number:
Amendment 1
Amendment date:
28 January 2016
Co-ordinating author:
, freelance writer for GSK Biologicals
Rationale/background for changes: This amendment was primarily developed 
to incorporate comments from the ethics committees of the countries in which the 
study will be conducted.
!
The mitigation strategy for risks associated with blood sample collection was 
revised based on the WHO guidelines on drawing blood.
!
The amount of blood collected in subjects 2-50 years enrolled in Vietnam was 
reduced from 7 ml to 5 ml at the enrolment visit and at visit for dengue suspicion, 
for early presenters, based on country-specific acceptability requirements.
!
The list of other infectious aetiologies than dengue for which the samples collected 
in subjects with episodes of febrile illness (referred to as “suspected dengue 
cases”) may be tested has been fully defined to include chikungunya, influenza, 
Zika and leptospirosis.
!
Details on anonymisation of subject data were added based on comments from the 
Ethics Committee in Thailand.
!
The study duration for each country was specified under the ‘Country specific 
requirements’ section of the protocol.
!
The name of one of the laboratories performing testing was updated from ‘Quest 
Diagnostics’ to ‘Q2 solutions’.
Amended text has been included in bold italics and deleted text in strikethrough in 
the following sections:
The name of the Global Vaccines Clinical Laboratories (GVCL) department was changed 
to “Clinical Laboratory Sciences (CLS). The name of this function has been updated in 
the title page.
!
, Clinical read-out Lead (CRTL)Project Manager, GVCL CLS
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Synopsis and Section 3.3 Tertiary Objectives

To explore other infectious aetiologies than dengue in subjects with episodes of 
febrile illness referred to as “suspected dengue case” (e.g.: chikungunya, Zika, 
influenza, leptospirosis, etc.).
Section 2.1 Risk Assessment
Risks associated with blood collection (such as pain at blood sampling site, haematoma 
or thrombus, vasovagal reaction, syncope or fainting) can be reduced by following best 
practices listed in the [WHO guidelines] for drawing blood (2010). Some examples are 
provided in the table below.
Important Potential/Identified 
Risk
Data/Rationale for 
Risk
Mitigation Strategy
Study procedures
Study procedure: Blood sample 
collection While giving blood the 
subject may feel faint, or locally 
experience mild pain, bruising, 
irritation or redness.
Spontaneous data Pain 
at blood sampling 
site

Subjects will be 
observed for at least 30 
minutes after blood 
sample collection.

Well-trained person 
should take the blood 
sample

Use needle of smaller 
gauge than the vein
Haematoma or 
thrombus

Enter vessel at an 
angle of 30 degrees or 
less

Use gauge of needle 
smaller than the vein

Apply pressure to a 
straight arm for 3–5 
minutes after drawing 
blood
Vasovagal reaction
Syncope, fainting

Hydrate patient, take 
postural blood 
pressure if
dehydrated

Reduce anxiety

Have patient lie down 
if the person 
expresses concern

Provide audio-visual
distraction
Please refer to [WHO guidelines] for drawing blood (2010): best practices in 
phlebotomy, in particular section 8.5.3 on risk assessment and risk reduction strategies.
 
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Section 2.2 Benefit Assessment
The subjects may not receive any other direct benefit than facilitated access to 
healthcare in case of febrile illness during study conduct.will be followed up for febrile 
illness due to dengue or other infectious aetiologies throughout the study. Appropriate 
care will be provided to the subjects diagnosed with dengue or other diseases.
Section 5.1 Suspected symptomatic dengue case (SDC)
Acute febrile illness measured as 38.0°C with a thermometer by any route or recent 
history of febrile illness (onset in the past 8 days) reported by the subject/the 
parent(s)/LAR(s)/ designate (if applicable) of the subject for at least two consecutive days 
(a duration of approximately 36-48 hours) and of less than 7 days duration, which may be 
accompanied by other dengue symptoms or signs and does not have a defined focus or an 
obvious reason unrelated to dengue (based on physician judgement).
Section 7.5.1.9 Collect blood sample
At Visit 1, a blood sample of 3.5 mL will be collected from subjects aged between 6 
months and 2 years (all sites), and a blood sample of 7 mL will be collected from subjects 
aged between 2 and 50 years (in all sites except Indonesia and Vietnam). 
At Visit 1, a blood sample of 3.5 mL will be collected from subjects enrolled in Indonesia 
aged between 6 months and 50 years. The volume in Indonesia is lower than in other 
countries as testing for DENV neutralising antibodies and other flaviviruses will not be 
performed for these subjects. The lower volume collected for subjects enrolled in 
Indonesia and Vietnam may result occasionally in the impossibility to repeat certain 
assays if needed (for instance in case of invalid results after the first assay run). 
At visit 1, a blood sample of 5 ml will be collected for subjects aged between 2 and 50 
years enrolled in Vietnam to comply with local acceptability requirement. However, 
this lower volume may result occasionally in the impossibility to repeat certain assays if 
needed (for instance in case of invalid results after the first assay run).
Sectin 7.6 Biological sample handling and analysis
Please refer to the SPM and any document provided e.g.: Investigator manual, for 
details of biospecimen management (handling, storage and shipment).
Section 7.6.2 Biological samples
Table 5 presents the volume of blood that will be collected from subjects for study-
related testing during each visit (all sites except Indonesia and Vietnam), Table 6
presents the volume of blood that will be collected from subjects in Vietnam and Table 
7Table 6 presents the volume of blood that will be collected from subjects enrolled in 
Indonesia for study-related testing during each visit. The volume of blood collected for 
subjects enrolled in Indonesia at the enrolment visit will be lower than in other countries 
because neutralising antibodies against DENV and other flaviviruses will not be 
 
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measured. The volume of blood collected for subjects enrolled in Vietnam at the 
enrolment visit and at the initial visit for dengue suspicion will be lower than in other 
countries to comply with local acceptability criteria; however, this may result 
occasionally in the impossibility, due to limited serum volume, to repeat certain assays 
if needed (for instance in case of invalid results after the first assay run).
Table 6
Volume of blood for study related testing in subjects enrolled in 
Vietnam
Age group
Type of Visit
Visit 1 (enrolment)
Day 0
SDC – first visit
Early presenter
Late Presenter
6 months to <2 
years
3.5 ml 
3.5 ml
3.5 ml
2-50 years
5 ml
5 ml
3.5 ml
Section 7.6.3 Laboratory assays
Table 8 Possible laboratory assays on study blood samples
Time point
Possible parameters/assays measured
Scheduled visit 1
- Anti-DENV indirect IgG Enzyme Linked Immunosorbant Assay 
(ELISA)
- DENV neutralising antibody assay
- JEV neutralising antibody assay*
- WNV neutralising antibody assay*
- Tertiary exploratory assays to characterize DENV immune 
status (e.g. antibody avidity assay, etc.)
Unscheduled Visit: SDC, early presenter (≤5 
days following onset of symptoms)
- DENV RT-qPCR
- DENV isolation for sequencing purpose
- DENV sequence
- DENV NS1 rapid test (ICT) or ELISA
- IgM/Ig G rapid test (ICT) or IgM/IgG capture ELISA 
- PCR/serology for other infectious agents causing febrile 
illness (chikungunya, Zika, influenza, leptospirosis) (e.g. 
Chikungunya, etc.)
Unscheduled Visit: SDC, late presenter (> 6 
days following onset of symptoms)
- DENV NS1 rapid test or ELISA
- IgM/IgG rapid test or IgM/IgG capture ELISA
- PCR/serology for other infectious agents causing febrile 
illness (e.g. Chikungunya, etc.) (chikungunya, Zika, influenza, 
leptospirosis) 
*DENV, JEV and WNV neutralising antibody assays will not be done for Indonesia.
 
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Section 7.6.4 Biological samples evaluation
Table 11 Possible assays for blood samples collected at scheduled visits
Blood sampling time point
Possible assays
Estimated 
number of 
subjects
Serum 
aliquot
Components 
priority rank
Type of contact/ 
Sampling 
time point
Scheduled Visit 1
(Day 0)
Anti-DENV IgG indirect 
ELISA
All
200 l
1
DENV virus 1-4 neutralising 
antibody assay, JEV and 
WNV neutralising antibody 
assay, serum bank for
other tertiary exploratory 
assays
Subset**
2 x 1 ml or 
remaining 
volume
2
Table 12 Aliquot preparation for blood samples collected at visits for suspected 
dengue and follow-up contact
Blood sampling 
time point
Serum aliquot 
volume
Possible Assays
Estimated 
number of 
subjects
Components 
priority rank
First visit –
presentation within 
5 days following 
onset of symptoms
(early presenter)
750 l
DENV RT-qPCR
Unknown
1
Approximately 
500 l
DENV NS1/IgM/IgG rapid test
(ICT) or ELISA 
Unknown
2
750 l or 
remaining volume
DENV RT-qPCR
PCR/serology of other 
infectious aetiologies 
(e.g.:chikungunya, Zika, 
influenza, leptospirosis, etc.)
Unknown
3
Viral isolation and sequencing
First visit –
presentation 6 days 
or more following
onset of symptoms 
(late presenter)
Approximately 
500 l
DENV NS1/IgM/IgG rapid test
(ICT) or ELISA 
Unknown
1
750 l or 
remaining volume
PCR/serology of other 
infectious aetiologies (e.g.:
chikungunya, Zika, influenza, 
leptospirosis, etc.)
Unknown
2
Synopsis and Section 10.1.2

Other infectious aetiology than dengue in subjects with episodes of febrile illness 
referred to as “suspected dengue cases” (e.g.: chikungunya, zika, influenza, 
leptospirosis etc.).
Section 11.2 Study monitoring by GSK Biologicals
The subject information will be anonymised (see GLOSSARY OF TERMS) using a 
code number to assure confidentiality of the subjects’ data. No GSK Biologicals 
personnel will have the ability to link data to an identifiable individual. The investigator 
and the head of the medical institution (where applicable) agrees to allow the monitor 
direct access to all relevant documents.
 
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Section 12 Country Specific Requirements
The duration of individual follow-up is 12 month for 2 sites (one site out of 2 in 
Mexico, one site in Indonesia) and 24 months for all the other sites.
Section 13 References
WHO guidelines on drawing blood. Best practices in phlebotomy. 2010. URL: 
http://apps.who.int/iris/bitstream/10665/44294/1/9789241599221_eng.pdf. Last 
accessed: 23 November 2015.
Appendix A Laboratory Assays
Dengue serotype-specific RT-qPCR
The dengue serotype-specific RT-qPCR assay has been developed for the detection of 
DENV serotype RNA in serum samples. Viral RNA is extracted from serum samples and 
reverse-transcribed into cDNA. The resulting cDNA is quantified by real time PCR using 
specific DNA primers targeting the DENV capsid gene. PCR is performed as 2 duplex 
amplification (DENV-1/-3 and DENV-2/-4).
Simplexa TM Dengue REF MOL3100 Rev. C 
A real-time RT-PCR assay for the in vitro detection and typing of dengue virus 
serotypes 1, 2, 3 and 4. 
Principles of the procedure: The assay is a real-time RT-PCR that discriminates 
serotypes 1 and 4 in one reaction (well), and serotypes 2 and 3 in another reaction 
(well). The assay is composed of two principal steps: (1) extraction of RNA from 
specimens, and (2) amplification of the extracted RNA using bi-functional fluorescent 
probe-primers and reverse primers. The assay amplifies four serotype specific regions: 
dengue 1 (NS5 gene), dengue 2 (NS3 gene), dengue 3 (NS5 gene) and dengue 4 (capsid 
gene). An RNA internal control is used to monitor the extraction process and to detect 
RT-PCR inhibition.
Appendix B Clinical Laboratories
The name of one of the laboratories has been changed from Quest Diagnostics to Q2
Solutions.
Table 16
Outsourced laboratories
Laboratory
Address
Quest Diagnostics Q2 Solutions
26081 Avenue Hall, Suite 150, Valencia Ca, 91355-1241, 
United States
Local labs in Indonesia Eijkman Institute for 
Molecular Biology
To be determined Jalan Diponegoro 69, Jakarta 
10430, Indonesia, (within the Cipto Mangunkusumo 
Hospital / RSCM)
 
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GlaxoSmithKline Biologicals
Vaccine Value & Health Science (VVHS)
Protocol Amendment 2
eTrack study number and 
Abbreviated Title
200318 (EPI-DENGUE-007 BOD)
Amendment number:
Amendment 2
Amendment date:
11 May 2017
Co-ordinating author:
, freelance writer for GSK Biologicals
Rationale/background for changes: 
This amendment was primarily developed:
!
To incorporate comments from the Thai Ethics Committee
∃
Provide more detail on the testing for the tertiary endpoints.
!
To facilitate meeting the secondary endpoint (To describe clinical presentations of 
dengue cases (confirmed and probable) and the tertiary endpoint (To explore other 
infectious aetiologies than dengue in subjects with episodes of febrile illness 
referred to as “suspected dengue case” (chikungunya, Zika, influenza, 
leptospirosis)).
∃
Add blood draws at 12 months and 24 months in study sites located in the 
Asia Pacific (Incidence in Guadalajara Mexico, where study enrolment is 
complete at the time of the protocol amendment, is too low to justify to add 
these blood draws for Mexican subjects)
!
To facilitate meeting the tertiary endpoint (To describe the spatial and temporal 
distribution of dengue cases (confirmed and probable) among cohort participants 
in the study areas and analyse determinants of spatio-temporal transmission (e.g. 
environmental, entomological, socio-demographic or ecological factors)).
∃
Update spatial coordinates, household information, etc., during SDC visits, 
where applicable.
!
To account for study sites that will no longer participate in the study (Indonesia, 
Vietnam and Monterrey Mexico)
∃
Adjust sample size
∃
Delete country specific wording that is no longer relevant
!
To clarify
∃
The cut-off time for Late SDC value
!
To remove ELISA kits that are no longer applicable.
!
To decrease the response time to address events of public health significance (such 
as an epidemic of arbovirus aetiology), and to address unforeseen principle 
investigator opinion on the needs of the study (based on GSK experience with 
investigators participating in the EPI-DEN-006 study in Brazil):
∃
Increase the blood draw values in study sites located in the Asia Pacific 
(Incidence in Guadalajara Mexico, where study enrolment is complete at the 
time of the protocol amendment, is too low to justify to increase blood draw 
values for Mexican subjects)
!
To eliminate the option for 12 month study duration (subjects at all sites will have
a study duration of 24 months)
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Amended text has been included in bold italics and deleted text in strikethrough in 
the following sections:
The list of contributing authors was updated:
!
, Director, Epidemiology
!
, 
, Study Delivery Leads
!
, Statistician
!
, Lead Statistician
!
, Study Data Manger, for GSK Biologicals for Tata 
Consultancy Services
!
, 
, Clinical Safety representatives
!
, Lab study manager
The address of the sponsor was updated in the protocol:
GlaxoSmithKline Biologicals
Rue de l'Institut, 89 14200 Shady Grove Road
Rockville, MD 20850 USAB-1330 Rixensart - Belgium
Synopsis and Section 4 Study Design Overview
!
Duration of longitudinal follow-up and number of visits: The study duration will be 
determined 24 months for each participating site (12 or 24 months). Each subject 
will have one 3 scheduled visit at enrolment, at Month 12 and a conclusion visit at 
Month 24. The Month 12 and Month 24 visits may occur via a telephone contact 
for the Mexican site. Overall, asbout 75% of the subjects are expected to enrol in a 
two-year longitudinal follow-up and 25% in a one-year follow-up.
These periods will be described for each site and may be modified upon mutual 
agreement between the investigator and the central study team based on available 
epidemiological information.
In countries with marked seasonality, the recruitment period would preferably occur 
outside the period of peak incidence of dengue, based on the local epidemiology of 
dengue in the past years and preferentially outside the holiday period (if the 
investigator believes that families are more likely to leave the study area during this 
period).
!
Duration of the study: Because of differences in local regulations, dengue seasonality 
and holiday period between countries, the recruitment period will be staggered. 
However, to facilitate site activities, sites will target to have all the subjects recruited 
in a 3-month period. For each subject, the study duration will be either 
approximately 12 or 24 months (determined by site).
!
Epoch 001: Prospective data collection starting at Visit 1 (Day 0) and ending at 
the conclusion visitcontact (Month 12 or 24, as applicable).
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Table 1
Study groups and epochs foreseen in the study
Study Groups
Number of subjects
Age (Min/Max)
Epochs
Epoch 001
Prospective
1750 3000
6 months to 50 years
x
Section 4 Study design overview
Figure 1 Schematic representation of the study design
The Month 12 and 24 time points and the procedures to be carried out at these visits were 
added in the figure and the footnote was modified in the figure.
* The Month 12 and Month 24 visits may occur via a telephone contact for the Mexican site and there will be no 
blood sample collection and physical examination at these visits for subjects in Mexico.
Synopsis and Section 6.1 Number of subjects
Approximately, 1750 3000 subjects are expected to be enrolled into this study with about 
300-500 subjects expected per study site. Subjects aged between 6 months and 50 years 
at the time of enrolment living in geographically-defined communities in Latin America 
and Southeast Asia will be part of this study.
Section 4.1.1 Rationale for study design
Because dengue is endemic in the countries where study sites are located, the study 
population will should have a mixed anti-DENV immunological background, providing 
an opportunity to describe different response patterns to new infections.
 
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Section 6.2 Overview of recruitment plan
Recruitment period
In countries with marked seasonality, the recruitment period should preferably occur 
outside the period of peak incidence of dengue, based on the local epidemiology of 
dengue in the past years.
Section 7.4 Outline of study procedures
Table 2 List of study procedures
Age
6 months to 50 Years
Epoch
Epoch 001 
Data Collection
Prospective data Collection
Visit
Scheduled visits/contacts
Unscheduled visits 
(for acute febrile illness and 
suspected dengue at 
hospital/healthcare centre)
Time points
Visit 1 
Day 0
Regular 
Surveillanc
e contact 
between 
visitsa
Visit 2
Month 
12
Conclusion 
contact Visit
3
Month12 or
24 §
First visit
Return 
visit*
Follow-
up 
contact**
Informed consent and assent as
applicable
●
Check inclusion/exclusion criteria
●
Check evidence of subject being 
member of the household (family 
book or other administrative 
document) 
O
Attribute subject number and 
household number attribution
●
Collect spatial coordinates of 
householdse 
●e
Record/ update household and 
socio-demographic information
●
●
●
Medical history and Japanese 
encephalitis (JE)/Yellow fever 
(YF)/ Dengue (if applicable) 
vaccination history or updates 
●
●
●
Distribute subject ID card and 
instruction kitd
O
Collect blood sample 
●
●f
●f
Instruct/ remind subjects/subjects’ 
parent(s)/LAR(s) or designate (if 
applicable) on assessment 
procedures in case of acute 
febrile illness
●
O
 
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Age
6 months to 50 Years
Epoch
Epoch 001 
Data Collection
Prospective data Collection
Visit
Scheduled visits/contacts
Unscheduled visits 
(for acute febrile illness and 
suspected dengue at 
hospital/healthcare centre)
Time points
Visit 1 
Day 0
Regular 
Surveillanc
e contact 
between 
visitsa
Visit 2
Month 
12
Conclusion 
contact Visit
3
Month12 or
24 §
First visit
Return 
visit*
Follow-
up 
contact**
Issue diary logs to 
subject/subject’s 
parent(s)/LAR(s)/ designate and 
train them on how it is to be filled 
out in the event of an acute febrile 
illness†
O
O 
O
Contact the subject/subject’s 
parent(s)/LAR(s)/ designate 
regarding any acute febrile illness 
and remind him/her/them of the 
procedures 
O
Physical examination and medical 
history (see Section 7.5.4)
●
●f
●f
●
●
Collect or verify diary logs if 
applicableb
O
O
Record if subject meets the 
definition of a SDC; if dengue is 
discarded, record the alternative 
diagnosis 
●
Record laboratory results 
prescribed as per local guidelines 
by the treating physician (may 
include CBC, liver enzymes, 
bilirubin)
●
●
Record patient management c
●
●
Collect blood sample for 
diagnosis#
●
Outcome of dengue episode
●
Report Serious Adverse Events 
(SAEs) related to study 
procedures‡
  ●
●
● 
●
●
●
Study conclusion 
●
● 
§The conclusion contact may be done by phone or by a home visit (at the site’s discretion).
fThese procedures were included as part of the protocol amendment 2 and will not be applicable for Mexico.
Table 4 Intervals between study visits
Interval
Optimal length of interval*
Allowed interval
Visit 1 (Day 28) Conclusion contact
Visit 2 (Month 12 or 2412, as applicable)
12 or 24 12 months
28 days
Visit 2 (Month 12) Conclusion visit 
(Month 24)
12 months
28 days
Interval between First visit for a SDC and 
21 days
7 days
 
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the final follow-up contact for a case
*Whenever possible the investigator should arrange study visits/contacts within this interval.
Section 7.5 Detailed description of study procedures
There will be one scheduled visit (Visit 1) at enrolment, a second visit at Month 12 (Visit 
2) and a final conclusion visit at Month 24 (Visit 3)as well as a conclusion contact which 
may be done by phone or via a home visit, at the discretion of the site. The conclusion 
contact will take place 12 or 24 months (+/- 28 days) after visit 1, depending on the 
duration of follow-up determined for the study site. The Month 12 and Month 24 visits 
may occur via a telephone contact for the Mexican site.
Section 7.5.1.5 Collect spatial coordinates of the households
The participating households will may be geo-referenced with GPS (Global Positioning 
System) devices.
A geographic masking technique will may be applied (see Section 15.7 11.7) to remove 
the possibility of re-identification of participants.
Collection of spatial coordinates can may be done at any time during the recruitment 
period and not necessarily at Visit 1.
Section 7.5.1.7 Physical examination, medical history and JE/YF/dengue vaccination 
history
A detailed clinical examination will be carried out to assess the subject’s general 
condition, cardiac and respiratory rates, blood pressure, dengue associated clinical 
signs/symptoms.
Section 7.5.1.9 Collect blood sample 
At Visit 1, a blood sample of 3.5 - 5 mL will be collected from subjects aged between 6 
months and 2 years (all sites), and a blood sample of 7 10 – 15 mL will be collected from 
subjects aged between 2 and 50 years (in all sites except Indonesia and Vietnam).
At Visit 1, a blood sample of 3.5 mL will be collected from subjects enrolled in Indonesia 
aged between 6 months and 50 years. The volume in Indonesia is lower than in other 
countries as testing for DENV neutralising antibodies and other flaviviruses will not be 
performed for these subjects. The lower volume collected for subjects enrolled in 
Indonesia and Vietnam may result occasionally in the impossibility to repeat certain 
assays if needed (for instance in case of invalid results after the first assay run).   
At visit 1, a blood sample of 5 ml will be collected for subjects aged between 2 and 50 
years enrolled in Vietnam to comply with local acceptability requirement. However, this 
lower volume may result occasionally in the impossibility to repeat certain assays if 
needed (for instance in case of invalid results after the first assay run).
 
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Section 7.5.2 Procedures at Visit 2 and conclusion visit (Visit 3) contact
The Visit 2 and Visit 3 will take place at a designated facility, as determined by each 
participating site. Home visits may also be organised, at the convenience of the study 
team. The conclusion contact will be done by phone contact or by home visit, at the 
convenience of the site.
A detailed clinical examination will be carried out to assess the subject’s general 
condition, cardiac and respiratory rates, blood pressure, dengue associated clinical 
signs/symptoms.
A blood sample of 3.5 - 5mL will be collected from subjects aged between 6 months and 
2 years (all sites), and a blood sample of 10 mL will be collected from subjects aged 
between 2 and 50 years (all sites). The Month 12 and Month 24 visits may occur via a 
telephone contact for the Mexican site and there will be no blood sample collection at 
these visits for subjects in Mexico.
At study conclusion (Visit 3) for each subject, the investigator will:

Review all the data collected to ensure accuracy and completeness 

Complete the study conclusion screen in the eCRF.
Section 7.5.4 Procedures at unscheduled visits and management of SDC
Rapid dengue diagnostic tests (NS1, IgM, and IgG) or ELISA tests (depending on the 
country local standards) will be performed as well because a result can be provided
rapidly to the physician and subject and support the diagnosis.
Section 7.6.2 Biological samples
Table 5 presents the volume of blood that will be collected from subjects for study-
related testing during each visit   (all sites except Indonesia and Vietnam), Table 6
presents the volume of blood that will be collected from subjects in Vietnam and Table 7
presents the volume of blood that will be collected from subjects enrolled in Indonesia for 
study-related testing during each visit. The volume of blood collected for subjects 
enrolled in Indonesia at the enrolment visit will be lower than in other countries because 
neutralising antibodies against DENV and other flaviviruses will not be measured. The 
volume of blood collected for subjects enrolled in Vietnam at the enrolment visit and at 
the initial visit for dengue suspicion will be lower than in other countries to comply with 
local acceptability criteria; however, this may result occasionally in the impossibility, due 
to limited serum volume, to repeat certain assays if needed (for instance in case of invalid 
results after the first assay run)
 
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Table 5
Volume of whole blood for study related testing related to sites with 
the exception of Indonesia and Vietnam
Age group
Type of Visit
Visit 1, 2 and 3 (enrolment)
Day 0
SDC – first visit
Early presenter
Late Presenter
6 months to <2 years
3.5 ml -5ml
3.5 ml -5ml
3.5 ml-5ml
2-50 years
7 10 – 15 ml
7 10 ml
3.5 10 ml
Table 6
Volume of blood for study related testing in subjects enrolled in 
Vietnam 
Age group
Type of Visit
Visit 1 (enrolment)
Day 0
SDC – first visit
Early presenter
Late Presenter
6 months to <2 years
3.5 ml 
3.5 ml
3.5 ml
2-50 years
5 ml
5 ml
3.5 ml
Table 7
Volume of blood for study related testing in subjects enrolled in 
Indonesia
Age group
Type of Visit
Visit 1 (enrolment)
Day 0
SDC – first visit
Early presenter
Late Presenter
6 months to <2 years
3.5 ml 
3.5 ml
3.5 ml
2-50 years
3.5 ml
7 ml
3.5 ml
If blood collection is not feasible in a young child with SDC, a finger prick test may 
alternatively be performed for testing with a IgM/IgG/NS1 ICT.
Section 7.6.3 Laboratory assays for primary and secondary endpoints
Table 8 Table 6 presents the possible laboratory assays for primary and secondary 
endpoints on the blood sample that will be drawn from the subject.
 
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Table 8 Table 6 Possible laboratory assays on study blood samples for primary and 
secondary endpoints
Time point
Possible parameters/assays measured
Scheduled visit 1, 2 and 3*
- Anti-DENV indirect IgG Enzyme Linked Immunosorbant Assay 
(ELISA)
- DENV neutralising antibody assay
- JEV neutralising antibody assay
- WNV neutralising antibody assay*
- Tertiary exploratory assays to characterize DENV immune 
status (e.g. antibody avidity assay, etc.)
Unscheduled Visit: SDC, early presenter (≤ 5 
days following onset of symptoms)
- DENV RT-qPCR
- DENV isolation for sequencing purpose
- DENV sequence
- DENV NS1 rapid test (ICT) or ELISA
- IgM/Ig G rapid test (ICT) or SD BIOLINE Dengue Duo 
(Dengue NS1 Ag + IgG/IgM) IgM/IgG capture ELISA
- PCR/serology for other infectious agents causing febrile 
illness (chikungunya, zika, influenza, leptospirosis)
Unscheduled Visit: SDC, late presenter (> 6 
days following onset of symptoms)
- DENV NS1 rapid test or ELISA
- IgM/IgG rapid test or IgM/IgG capture ELISA
- PCR/serology for other infectious agents causing febrile 
illness (chikungunya, zika, influenza, leptospirosis)
* Blood sample will not be collected at Visit 2 and Visit 3 for subjects in Mexico
*DENV, JEV and WNV neutralising antibody assays will not be done for Indonesia.
The subset of subjects for which assays related to tertiary endpoints (e.g. neutralising 
antibodies, tests to further characterise the immune status to dengue, PCR/serology to 
identify other infectious agents causing febrile illness) will be performed will be 
determined based on preliminary results of the study (identification of laboratory 
confirmed and probable symptomatic dengue cases during the surveillance, medical 
history/vaccination history for other flaviviruses, etc.).
The purpose of these diagnostic tests is to provide prompt laboratory results to the 
physician and the subject. Rapid ICT (point of care) will be used, unless local guidelines 
require the use of ELISA tests.
 
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Table 9 Table 7 presents humoral immunity to DENV.
Table 9
Table 7 Humoral immunity (DENV antibody determination) for 
primary and secondary endpoints 
System
Component
Method
Kit / Manufacturer
Laboratory
Serum
IgM/IgG to 
DENV
Rapid ICT assay or 
Dengue NS1 Ag + 
IgM/IgG  IgM/Ig G capture 
ELISA
Standard Diagnostics 
(ICT) or equivalent/ 
PanBio (Capture ELISA)
SD BIOLINE Dengue 
Duo(Dengue NS1 Ag 
+IgG/IgM) or equivalent
Local
IgG to DENV
(for scheduled 
visit 1)
ELISA
Indirect IgG Panbio or 
equivalent
Central or local GSK 
designated laboratory 
DENV types
1-4 neutralising 
antibodies
Dengue neutralisation
assay*
In-house
GSK designated 
laboratory
*Dengue Neutralisation assay will not be done for Indonesia.
Table 10 Table 8
Virology for primary and secondary endpoints
System
Component
Method
Laboratory
Serum
DENV RNA
RT-qPCR
Central or local GSK designated 
laboratory
NS1 antigen
Rapid test (ICT) or ELISA
Local
Note: Viral isolation may be conducted if needed.
Section 7.6.4 Laboratory assays to be performed for tertiary endpoints
Table 9 presents the possible laboratory assays for primary and secondary endpoints on 
the blood sample that will be drawn from the subject.
Table 9 Possible laboratory assays on study blood samples for tertiary endpoints 
Time point
Possible parameters/assays measured
Scheduled visit 1, 2 and 3*
- DENV neutralising antibody assay
- JEV neutralising antibody assay
- WNV neutralising antibody assay
- Tertiary exploratory assays to characterize DENV immune 
status (e.g. antibody avidity assay, etc.)
- Chikungunya serology (IgM/IgG detection) and neutralizing 
antibodies
- Zika serology (IgM/IgG detection) and neutralizing antibodies
Unscheduled Visit: SDC, early presenter 
(≤ 5 days following onset of symptoms)
- - PCR/serology for other infectious agents causing febrile 
illness (chikungunya, Zika, influenza, leptospirosis)
Unscheduled Visit: SDC, late presenter 
(> 6 days following onset of symptoms)
- DENV NS1 rapid test 
- IgM/IgG rapid test or IgM/IgG capture ELISA
- PCR/serology for other infectious agents causing febrile 
illness (chikungunya, Zika, influenza, leptospirosis)
* Blood sample will not be collected at Visit 2 and Visit 3 for subjects in Mexico.
 
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Laboratory assays related to tertiary endpoints will only be performed after the primary 
and secondary endpoints have been ensured, and the local epidemiology of dengue 
virus disease is better understood. 
The results of the primary and secondary endpoints, public health needs, and 
availability of biological samples will guide:

The subset of subjects selected for which assays related to tertiary endpoints will be 
conducted

Which pathogens should be tested (JEV, WNV, chikungunya, Zika, influenza, 
leptospirosis)

The assays and tests to be performed.
If testing for tertiary endpoints will be conducted, these parameters and rationale will 
be provided to the local ethical committees and relevant health authorities for approval. 
Testing may include, but are not limited to the following tests for humoral immunity 
and virology.
Table 10
Humoral immunity for tertiary objectives
System
Component
Method
Kit / Manufacturer
Laboratory
Serum
Antibodies against Zika 
virus
To be determined
To be determined
GSK designates lab
Serum
Zika virus neutralizing 
antibody
Zika neutralization assay
To be determined
GSK designated 
laboratory
Serum
Antibodies against 
chikungunya virus
To be determined
To be determined
To be determined
Serum
YF virus neutralizing 
antibody
YF neutralization assay
To be determined
GSK designated 
laboratory
Serum
JEV neutralizing 
antibody
JEV neutralization assay
To be determined
GSK designated 
laboratory
Serum
WNV neutralizing 
antibody
WNV neutralization assay
To be determined
GSK designated 
laboratory
Table 11
Virology for tertiary endpoints
System
Component
Method
Kit/Manufacturer
Laboratory
Serum
Zika RNA
RT-PCR
To be determined
GSK designated laboratory
Serum
Chikungunya RNA
RT-PCR
To be determined
GSK designated laboratory
Serum
JEV RNA
RT-PCR
To be determined
GSK designated laboratory
Serum
WNV RNA
RT-PCR
To be determined
GSK designated laboratory
Section 7.6.4 11.1.1 Biological samples evaluation
 
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Table 9 Table 12 presents the aliquoting of blood samples collected at scheduled visits.
Table 9 Table 12
Possible assays for primary and secondary endpoints for blood 
samples collected at scheduled visits
Blood sampling time point
Possible assays
Estimated 
number of 
subjects
Serum 
aliquot
Components 
priority rank
Type of contact/ 
Sampling 
time point
Scheduled Visit 1, 
Visit 2 and 
Visit 3*
Day 0
Month 12
Month 24
Anti-DENV IgG indirect 
ELISA
All
200 l
1
DENV virus 1-4 neutralising 
antibody assay, JEV and 
WNV neutralising antibody 
assay, serum bank for 
other tertiary exploratory 
assays
Subset
1x 1 ml or 
remaining 
volume
2
*Blood sample will not be collected at Visit 2 and Visit 3 for subjects in Mexico **The DENV neutralising assay 
and JEV, WNV and tertiary exploratory assays will not be done for Indonesia.
Table 13 Aliquot preparation for blood samples collected at visits for suspected 
dengue and follow-up contact
Blood sampling 
time point
Serum aliquot 
volume
Possible Assays
Estimated 
number of 
subjects
Components 
priority rank
First visit –
presentation within 
5 days following 
onset of symptoms
(early presenter)
750 l
DENV RT-qPCR
Unknown
1
Approximately 
500 l
DENV NS1/IgM/IgG rapid test
(ICT) or ELISA
Unknown
2
1 ml 750 l or 
remaining volume
DENV RT-qPCR
PCR/serology of other 
infectious aetiologies 
(chikungunya, Zika, influenza, 
leptospirosis)
Unknown
3
Viral isolation and sequencing
First visit –
presentation 6 days 
or more following
onset of symptoms 
(late presenter)
Approximately 
500 l
DENV NS1/IgM/IgG rapid test
(ICT) or ELISA
Unknown
1
1 ml 750 l or 
remaining volume
PCR/serology of other 
infectious aetiologies 
(chikungunya, Zika, influenza, 
leptospirosis)
Unknown
2
Section 13 Subject completion and withdrawal
Section 13.1 Subject completion
A subject who is available for the concluding contact visit foreseen in the protocol is 
considered to have completed the study.
Subject 13.2 Subject withdrawal
From an analysis perspective, a ‘withdrawal’ from the study refers to any subject who 
was not available for the concluding contact visit foreseen in the protocol.
 
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All data collected until the date of withdrawal/last visit contact of the subject will be used 
for the analysis.
Section 10.2 14.2 Determination of Sample size
The target population to be enrolled is estimated at 3000 1750 subjects (approximately 
300 to 500 per site) , with at least 75% expected to enrol for a 2-year longitudinal follow-
up and 25% expected to enrol for a 1-year follow-up. A dropout rate of 5% per year is 
estimated, which would lead to approximately 2850 1662 subjects completing 1 year of 
follow-up and approximately 2031 1579 completing 2 years of follow-up, accumulating 
5008 3327patient-years.
The incidence of dengue is likely to vary by site and by age group. The study population 
should include between 30% and 50% of adults, but the distribution is not further 
specified as it would make operational feasibility more complex. 
Supposing that the study population is composed of 70% of children with an expected 
incidence of 8 RT-qPCR-confirmed dengue case per 1000 person-years and 30% of 
adults with an expected incidence of 5 RT-qPCR confirmed dengue cases per 1000 
person-years, the study would detect about 28 19 cases in children and 8 5 cases in adults 
(2112 cases in year one with a cohort of 3000 1750 subjects and 15 12 cases in year 2 
with a cohort of about 2250 1662vsubjects).
The enrolment will be done by household. Each household can be considered as a cluster 
and this induces a design effect to account for the between-cluster variability when 
estimating the confidence intervals (CI) of the incidence rates. The design effect 
measures the increase in the standard error of the incidence rate estimate due to the 
sampling design used and is given by: D = 1 + (b – 1) rho, where rho is the intra-cluster 
correlation (a measure of rate of homogeneity within clusters) and b is the average 
number of subjects sampled per household. Here, we assume b is assumed to be 3. 
Although in theory ‘rho’ can have a value up to 1, in practice values higher than 0.4 are 
uncommon. We have used a conservative estimate of 0.4 for this study [Bennett, 1991]. 
The design effect is then estimated to 1.8.
Table 15 shows the 95% CI for a range of scenarios in term of cohort size, dropout rates 
and incidence rates. With an overall sample size of a minimum of about 3000 1750
evaluable subjects, a follow-up period of two years and the expected incidences of 8 
cases per 1000 person-years in children and 5 cases per 1000 person-years in adults, the 
overall incidence rate would be 7.0 7.1 with an exact Poisson 95% CI of [5.0, 9.5 4.5; 
10.6], and with a CI based on the normal approximation and accounting for the design 
effect of [4.1, 9.93.3;10.9].
 
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Table 16
Expected 95% confidence intervals for the incidence rate of virologically confirmed dengue cases
Coho
rt 
size
Proportion of 
adults
Dropout rate (per 
year)
Incidence rates
Person-
years
Number of 
cases
Overall incidence (1000 
p-y)
Confidence intervals (1000 p-y)
Children (p-
y)*
Adults (p-
y)
Exact 
(Poisson)
Normal approx + 
DE**
Low
er
Uppe
r
Lower
Uppe
r
1400
30%
5%
0.008
0.003
2662
17
6.5
3.8
10.4
2.4
10.6
1575
30%
5%
0.008
0.003
2994
19
6.5
3.9
10.1
2.6
10.4
1750
30%
5%
0.008
0.003
3327
22
6.5
4.1
9.9
2.8
10.2
1400
30%
5%
0.008
0.005
2662
19
7.1
4.2
11.1
2.8
11.4
1575
30%
5%
0.008
0.005
2994
21
7.1
4.4
10.8
3.1
11.1
1750
30%
5%
0.008
0.005
3327
24
7.1
4.5
10.6
3.3
10.9
1400
30%
5%
0.01
0.003
2662
21
7.9
4.9
12.1
3.4
12.4
1575
30%
5%
0.01
0.003
2994
24
7.9
5.0
11.8
3.6
12.2
1750
30%
5%
0.01
0.003
3327
26
7.9
5.2
11.5
3.8
12.0
1400
30%
5%
0.01
0.005
2662
23
8.5
5.4
12.8
3.8
13.2
1575
30%
5%
0.01
0.005
2994
25
8.5
5.5
12.5
4.1
12.9
1750
30%
5%
0.01
0.005
3327
28
8.5
5.7
12.3
4.3
12.7
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Coh
ort 
size
Proportion of 
adults
Percent
age 
followed 
during 
two 
years
Dropout rate (over 
2 years)
Incidence rates
Person-
years
Number of 
cases
Overall incidence 
(1000 p-y)
Confidence intervals (1000 p-y)
Children 
(p-y)*
Adults 
(p-y)
Exact 
(Poisson)
Normal approx + 
DE**
Lower
Upper
Lower
Upper
2400
30%
75%
5%
0.008
0.003
4006
26
6.5
4.2
9.5
3.1
9.8
2700
30%
75%
5%
0.008
0.003
4507
29
6.4
4.3
9.2
3.3
9.6
3000
30%
75%
5%
0.008
0.003
5008
32
6.4
4.4
9.0
3.4
9.4
2400
30%
75%
5%
0.008
0.005
4006
28
7.0
4.6
10.1
3.5
10.5
2700
30%
75%
5%
0.008
0.005
4507
32
7.1
4.9
10.0
3.8
10.4
3000
30%
75%
5%
0.008
0.005
5008
35
7.0
4.9
9.7
3.9
10.1
2400
30%
75%
5%
0.01
0.003
4006
31
7.7
5.3
11.0
4.1
11.4
2700
30%
75%
5%
0.01
0.003
4507
35
7.8
5.4
10.8
4.3
11.2
3000
30%
75%
5%
0.01
0.003
5008
39
7.8
5.5
10.6
4.5
11.1
2400
30%
75%
5%
0.01
0.005
4006
34
8.5
5.9
11.8
4.7
12.3
2700
30%
75%
5%
0.01
0.005
4507
38
8.4
6.0
11.6
4.8
12.0
3000
30%
75%
5%
0.01
0.005
5008
42
8.4
6.1
11.3
5.0
11.8
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Section 12 16 Country Specific Requirements
The duration of individual follow-up is 12 month for 2 sites (one site out of 2 in Mexico, 
one site in Indonesia) and 24 months for all the other sites.
Section 16.1
Thailand specific information and requirements
Section 16.1.1 List name, affiliation and contact details of all investigators
Name and Address of Principal Investigator
Principal Investigator
Assoc. Prof. Dr. Pornthep Chanthavanich
Department of Tropical Pediatrics,
Faculty of Tropical Medicine, Mahidol University
, 
,
, 
, Thailand 10400
Tel: 
, 
, Mobile: 
Fax: 
Email: 
Consultant
Dr. 
, .FRCST
Deputy  Nakhonpathom Provincial  Chief  Medical  Officer,
Expert Public Health Physician (Preventive Medicine)
Nakhonpathom Provincial Health Office
, 
 
, Nakhon Pathom 73000
Tel: 
, Mobile: 
, 
Fax: 
Email: 
Sub-Investigator
Assoc. Prof. Dr. Chukiat Sirivichayakul
Department of Tropical Pediatrics,
Faculty of Tropical Medicine, Mahidol University
, 
,
, 
, Thailand 10400
Tel: 
, 
, Mobile: 
Fax: 
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Email: 
Assist. Prof. Dr. Kriengsak Limkittikul
Department of Tropical Pediatrics,
Faculty of Tropical Medicine, Mahidol University
, 
,
, 
, Thailand 10400
Tel: 
, 
, Mobile: 
Fax: 
Email: 
Assist. Prof. Watcharee Chokejindachai
Department of Tropical Pediatrics,
Faculty of Tropical Medicine, Mahidol University
, 
,
, 
, Thailand 10400
Tel: 
, 
, Mobile: 
Fax: 
Email: 
Dr. Weerawan Hattasingh
Department of Tropical Pediatrics,
Faculty of Tropical Medicine, Mahidol University
, 
,
, 
, Thailand 10400
Tel: 
, 
, Mobile: 
Fax: 
Email: 
Assist. Prof. Wijitr Fungladda (Retired)
Department of Social and Environmental Medicine,
Faculty of Tropical Medicine, Mahidol University
, 
,
, 
, Thailand 10400
Tel: 
 Mobile: 
Fax: 
Email: 
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Dr. Supawat Chatchen
Department of Tropical Pediatrics,
Faculty of Tropical Medicine, Mahidol University
, 
,
, 
, Thailand 10400
Tel: 
, 
, Mobile: 
Fax: 
Email: 
Section 16.1.2 Definition of burden on the study protocol
Burden on the study protocol is means to incidence and severity of Dengue infection 
and admission in hospital of volunteer/subjects.
Section 16.1.3 Duration of the study, site address and number of participating subjects 
in Thailand
Trial Site in Thailand
There is 1 trial site participating in Thailand at Sam Kwai Phuek Health Promoting 
Hospital, Muang District, Nakhon Pathom Province. The site is planning to enrol 500 
subjects in this study. Study team members are from Department of Pediatric Tropical 
Medicine, Faculty of Tropical Medicine, Mahidol University, and
Deputy  
Nakhonpathom Provincial Chief Medical Officer, Nakornpathom provincial Health 
Office, consultant of the study.
This study is multicentre (International) that will be conducted in 4 countries. As 
listing below:
!
Thailand is planning to enrol 500 subjects
!
Malaysia is planning to enrol 400 subjects
!
Philippines is planning to enrol 500 subjects
!
Mexico is planning to enrol 350 subjects
Duration of the study in Thailand
The study duration for Thailand is 2.0 years (24 months).
Planning of recruitment period is June – December 2017.
Follow up visit period of the study is June 2017 – June 2020.
Planning of the study closure is June 2020.
For each subject, the study duration is approximately 2 years (24 months)
Section 16.1.4 Study population groups and Calculation of study population groups in 
Thailand
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The size of Thailand study population is derived from the average yearly incidence of 
dengue fever in Sam Khwai Phuak Sub-district from 2009 to 2013 of 283 per 100,000 
people. If it is assumed that this incidence is lower than actual incidence, according to 
population data of Sam Khwai Phuak Sub-District of approximately 10,900 people, a 
sample size of 500 people will be able to detect the incidence of dengue fever in Sam 
Khwai Phuak Sub-district of close to 600 per 100,000 people, with a confidence level of 
80 percent and an estimated data deficiency of no more than 15 percent.
A total of approximately 500 subjects will be recruited from Thailand. 
In Thailand, a total of approximately 500 subjects will be recruited and there will be a 
total of 1 study site participating in this study, which is Sam Khwai Phuek Health 
Promotion Hospital, Muang District, Nakhon Pathom Province, which will recruit 
approximately 500 subjects.
Department of Tropical Paediatrics, Faculty of Tropical Medicine, Mahidol University, 
is the main site for administration with no recruitment. Investigator and study team are 
affiliated with Department of Tropical Paediatrics, Faculty of Tropical Medicine, 
Mahidol University.
Deputy Nakhonpathom Provincial  Chief  Medical  Officer [Expert Public Health 
Physician (Preventive Medicine)] from Nakhon Pathom Province, a local person, is the 
project consultant.
The roles of study team at the Sam Kwai Phuek Health Promotion Hospital is:
To be the local person coordinating public utility payments at the study site for which 
the research project is the party responsible for, and coordinating with community 
leaders and the community in the area. 
Section 16.1.5 Overview of the Subject Recruitment Plan for Thailand
The method of finding/recruiting appropriate subjects will be selected by each research 
site participating in the study. One of the following 2 methods may be selected:

School recruitment method

Community recruitment method (without schools being involved)
For Thailand, however, subjects will be recruited using the community recruitment 
method (without schools being involved).
Section 16.1.6
Adding Secondary Objective Explanations
To describe the clinical symptoms of patients infected with the dengue virus (which 
have been confirmed and which are within the scope of suspected infection).*
*This includes studying clinical symptoms and the severity of the dengue virus 
infection. The severity of the dengue virus infection in this study will be monitored with 
the subject by taking their physical examination history, and tracking the progress of
the disease and hospitalization during the follow-up contact after 21 days of the first 
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Section 16.1.7 Informed Consent and Consent for Thailand
Obtaining consent from subjects in this study means requesting consent from 
individual subjects.
Subjects/guardians of subjects will be given an explanation of various details, 
including both the natures and risks involved with the study. The above process will be 
carried out by the head of protocol or protocol participants prior to obtaining 
signatures of consent from the subjects/imprinting the fingerprints of the subjects 
and/or the father/mother/legally acceptable representative of the subjects prior to their 
participation in the study, including the signature of the person providing protocol 
details (investigator) and relevant witnesses. The person providing protocol details 
must allow sufficient time for the subjects to decide whether or not to participate in the 
study, with the signing of the Subject Information Sheet and Informed Consent Form 
for adults, and Subject Information Sheet and Informed Assent Form for children, or 
Subject Information Sheet and Informed Consent Form for the father/mother/legally 
acceptable representative of the subjects to the amount of 2 copies of each. The first 
copy will be kept at the study site and the second copy will be kept by the subject. In 
addition, if the subjects or the father/mother/legally acceptable representative of the 
subjects would like to appoint a designee* to help with certain parts of the study 
procedures, including to help in monitoring and checking the body temperature of the 
subjects in the event they have a fever, completing daily records, answering calls from 
study staff who will ask whether or not the participant has a fever, and reminding the 
subjects of the study procedure, and help planning study visits, this designee is not able 
to make any decision on behalf of the subjects, or the father/mother/legally acceptable 
representative of the subjects. Moreover, the above designee must sign their name to 
receive consent from the subjects or the father/mother/legally acceptable representative 
of the subjects, or the father/mother/legally acceptable representative of the subjects 
together with the signature of the person providing protocol details (investigator) and 
the witnesses in the designee information document and letter of consent to participate 
in the study, to the amount of 2 copies. The first copy will be kept at the research site 
and the second copy will be kept by the subject.
The Subject Information Sheet and Informed Consent Form for this study are as 
follows:

Subject Information Sheet for research subject that are between 20 – 50 years old

Informed Consent Form for subjects that are between 20 – 50 years old

Subject Information Sheet for the father/mother/legally acceptable representative 
of subject from 6 months old to less than 20 years old

Informed Consent Form for the father/mother/legally acceptable representative of 
subject from 6 months old to less than 20 years old

Subject Information Sheet for subject from 7 years to less than 13 years old

Assent Form for subject from 7 years to less than 13 years old.

Subject Information Sheet for subject from 13 years to less than 20 years old. 

Assent Form for subject from 13 years to less than 20 years old. 

Subject Information Sheet for designees.

Informed Consent Form for designees.
 
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Section 16.1.8 Compiling Areal Coordinates of Households in Thailand
This study involves the compilation of the geographical areal coordinates of 
households. The compilation of the areal coordinates of subjects participating in the 
protocol with a Global Positioning System (GPS) device will deliver only subject 
number data and their areal coordinates. It will not specify the name, surname, home 
address, or any data that could affect the privacy of the subjects. 
Section 16.1.9
Managing Biological Samples in Thailand
For Thailand, samples of subjects will be stored for a period of 5 years after the study 
is completed (counting from the date of the last subject's last contact in the study). In 
the event that investigator needs to store blood samples for longer than 5 years, the 
investigator and the study team will request approval from the Ethical Review 
Committee for Research in Human Subjects for a further period of 5 years, and the 
samples remaining after the period approved will be destroyed. 
Subject samples will only be used for tests in this study.
Section 16.1.10
Potential Risks and Inconveniences, and Prevention Measures 
Blood drawing may cause slight pain in the area blood is drawn from. There may be 
swelling or bruising, or dizziness. After blood is drawn some people may feel faint, or 
nauseous, and in very rare cases infection may arise in the area blood is drawn. 
Investigator will use sterile blood drawing techniques to prevent infection from blood 
drawing. We will use hospitals with experience in blood drawing, especially for small 
children, and will use a scalp vein of appropriate size. For any side effects that may be 
caused by blood drawing, investigator will offer treatment until full recovery free of 
charge. 
Section 16.1.11
Benefits, Compensation, Reimbursement, Treatment and 
Solution in the Event of Complications for the Investigator
Benefits, Compensation, Reimbursement, Treatment and Solution in the Event of 
Complications for the Investigator
Subjects may not receive direct benefits from this study. However, knowledge regarding 
dengue fever derived from this study will be of great benefit in the prevention and 
management of dengue fever and administration of vaccines to prevent dengue fever in 
the future. 
Subjects will receive treatment for any fever symptoms from a doctor or healthcare 
professional at the Sam Khwai Phuak Sub-District Health Promotion Hospital in 
accordance with standard medical practice guidelines.
Subjects will be tested for dengue virus diagnoses (NS1, IgG and IgM) as specified in 
the protocol, and will know the results within 15 minutes. This will benefit subject 
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Section 16.1.12
Compensation and Reimbursement
Compensation
None
Reimbursement
Subjects will receive the amount of 1,000 baht per person per visit as specified in the 
protocol as a reimbursement for time and travel costs, except the case where you have a 
fever when attending a scheduled visit, but the person providing treatment suspects that 
you may have dengue fever, in which case a return visit appointment will be made on 
the following day.
Section 16.1.13
Other Ethical Issues 
Confidentiality
Investigator will keep personal information, study documents, and study results related 
to this study confidential, without disclosing them publicly. The investigator certifies 
that the personnel working in this project and the study sponsor will act in the same 
way. This study will keep subject information confidential and will disclose it in the 
form of overall study results that does not include any names or other data which may 
be able to identify the subjects, by using a numerical code instead of a name. This 
information, including medical records, may be examined by authorized personnel 
from Ministry of Public Health of Thailand or the Ethical Review Committee for 
Research in Human Subjects, only for the benefit of oversight, monitoring, and 
research evaluation. Regardless, the study sponsor and representatives, including those 
involved in all parts, must act in accordance with good clinical practices, and must, to 
the best of their ability, endeavour to keep personal information of subjects 
confidential. 
Budget Details and Funding Sources
Study sponsor GlaxoSmithKline Biologicals
Budget Details: As per documents showing research budget details.
 
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Section 16.1.14
Signature of all investigators
Principal Investigator
____________________________________
________________
(Assoc. Prof. Dr. Pornthep Chanthavanich)
Day / Month/ Year
Consultant
____________________________________
________________
(Dr. 
)
Day / Month/ Year
Sub-Investigator
____________________________________
________________
      (Assoc. Prof. Dr. Chukiat Sirivichayakul)
Day / Month/ Year
____________________________________
________________
           (Assist. Dr. Kriengsak Limkittikul)
Day / Month/ Year
____________________________________
________________
      (Assist. Prof. Dr. Watcharee Chokejindachai)
Day / Month/ Year
____________________________________
________________
                (Dr. Weerawan Hattasingh)
Day / Month/ Year
____________________________________
________________
          (Assoc. Prof. Dr. Wijitr Fungladda)
Day / Month/ Year
___________________________________
________________
                (Dr. Supawat Chatchen)
Day / Month/ Year
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Appendix B Clinical laboratories
Table 16
Outsourced laboratories
Laboratory
Address
Laboratório de Tecnologia Virológica, Bio-
Manguinhos, Fiocruz
Rio de Janeiro, Brazil
Q2 Solutions
26081 Avenue Hall, Suite 150, Valencia Ca, 
91355-1241, United States
Eijkman Institute for Molecular Biology
Jalan Diponegoro 69, Jakarta 10430, 
Indonesia, (within the Cipto 
Mangunkusumo Hospital / RSCM)
 
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GlaxoSmithKline Biologicals
Vaccine Value & Health Science (VVHS)
Protocol Amendment 3
eTrack study number and 
Abbreviated Title
200318 (EPI-DENGUE-007 BOD)
Amendment number:
Amendment 3
Amendment date:
24 July 2017
Co-ordinating author:
, freelance writer for GSK Biologicals
Rationale/background for changes: 
This amendment was developed to clarify that an additional blood sample will be 
collected for testing related to tertiary endpoints even when dengue is ruled out by 
the study physician during clinical examination.
Amended text has been included in bold italics and deleted text in strikethrough in 
the following sections:
Title page- Contributing authors
, 
, 
, Study Delivery LeadsSection 
7.5.1.9 Collect blood sample
At Visit 1, a blood sample of 3.5 mL-5 mL will be collected from subjects aged between 
6 months and 2 years (all sites), and a blood sample of 10-15 mL will be collected from 
subjects aged between 2 and 50 years (all sites).
Section 7.5.2 Procedures at Visit 2 and conclusion visit (Visit 3)
A blood sample of 3.5 - 5mL will be collected from subjects aged between 6 months and 
2 years (all sites), and a blood sample of 10-15 mL will be collected from subjects aged 
between 2 and 50 years (all sites).
Section 7.5.4 Procedures at unscheduled visits and management of SDC
!
All subjects with acute febrile illness should be seen at a designated study healthcare 
centre/hospital by a study physician. If dengue is ruled out by the study physician 
during clinical examination because of another obvious alternative diagnosis 
(identified focus of fever), the physician will document his alternative diagnosis, and 
collect a blood sample for testing related to tertiary endpoints no further study 
procedures will be conducted.
Section 7.6.2 Biological samples
Table 5 Volume of whole blood for study related testing
Age group
Type of Visit
Visits 1, 2 
and 3
SDC – first visit
Cases where dengue 
is ruled out
Early presenter
Late Presenter
6 months to <2 years
3.5 ml- 5ml
3.5 ml- 5ml
3.5 ml- 5ml
3.5 mL
2-50 years
10 – 15 ml
10 ml
10 ml
9 mL
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If blood collection is not feasible in a young child with SDC, a finger prick test may 
alternatively be performed for testing with a IgM/IgG/NS1 ICT.
Section 7.6.3 Laboratory assays to be performed for primary and secondary 
endpoints
Table 7 Humoral immunity (DENV antibody determination) for primary and 
secondary endpoints
System
Component
Method
Kit / Manufacturer
Laboratory
Serum
IgM/IgG to 
DENV
Rapid ICT assay or 
Dengue NS1 Ag + IgM/IgG
Standard Diagnostics 
(ICT) or equivalent/ SD 
BIOLINE Dengue 
Duo(Dengue NS1 Ag 
+IgG/IgM)
or equivalent
Local
IgG to DENV
(for scheduled 
visit 1, 2 and 3*)
ELISA
Indirect IgG Panbio or 
equivalent
Central or local GSK 
designated laboratory 
DENV types
1-4 neutralising 
antibodies
Dengue neutralisation
assay
In-house
GSK designated 
laboratory
*Blood sample will not be collected at Visit 2 and Visit 3 for subjects in Mexico.
Section 7.6.4 Laboratory assays to be performed for tertiary endpoints
Table 9 Possible laboratory assays on study blood samples for tertiary endpoints
Time point
Possible parameters/assays measured
Scheduled visit 1, 2 and 3*
- DENV neutralising antibody assay
- JEV neutralising antibody assay
- WNV neutralising antibody assay
- Tertiary exploratory assays to characterize DENV immune 
status (e.g. antibody avidity assay, etc.)
- Chikungunya serology (IgM/IgG detection) and neutralizing 
antibodies
- Zika serology (IgM/IgG detection) and neutralizing antibodies
Unscheduled Visit: SDC, early presenter (≤ 5 
days following onset of symptoms)
- - PCR/serology for other infectious agents causing febrile 
illness (chikungunya, Zika, influenza, leptospirosis)
Unscheduled Visit: SDC, late presenter (> 6 
days following onset of symptoms)
- DENV NS1 rapid test 
- IgM/IgG rapid test or IgM/IgG capture ELISA
- PCR/serology for other infectious agents causing febrile 
illness (chikungunya, Zika, influenza, leptospirosis)
Unscheduled Visit: Cases where dengue is 
ruled out
-PCR/serology for other infectious agents causing febrile 
illness (chikungunya, Zika, influenza, leptospirosis)
Section 8 Safety
In this prospective cohort study no test product/vaccine will be given. However, blood 
samples will be collected at the enrolment visit (Visit 1), Visit 2, conclusion visit (Visit 
3), and for suspected dengue and for subjects where dengue is ruled out. SAEs related to 
any study procedure will be recorded.
 
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Section 12.1.3 Duration of the study, site address and number of participating 
subjects in Thailand
The study duration for Thailand is 2.0 years (24 months).
Planning of recruitment period is June – December 2017October 2017-June 2018.
Follow up visit period of the study is June 2017 – June 2020October 2017- October 
2020.
Planning of the study closure is OctoberJune 2020.
Section 12.1.2 Definition of burden on the study protocol
Burden on in the study protocol is  means to incidence and severity of Dengue infection 
and hospital admission in hospital of volunteer/subjects.
Section 12.1.3 Duration of the study, site address and number of participating 
subjects in Thailand
This study is an international multicentre (International) cohort study that will be 
conducted in 4 countries. As Sample sizes are listeding below:
Section 12.1.4 Study population groups and calculation of study population groups 
in Thailand
The size of Thailand study population is derived from the average yearly incidence of 
dengue fever in Sam Khwai Phuak Sub-district from 2009 to 2013 of 283 per 100,000 
people. If a lower than actual incidence is assumed, it is assumed that this incidence is 
lower than actual incidence, according to population data of Sam Khwai Phuak Sub-
District of approximately 10,900 people, a sample size of 500 people will be able to 
detect the incidence of dengue fever in Sam Khwai Phuak Sub-district of close to 600 per 
100,000 people, with a confidence level of 80 percent and an estimated data deficiency of 
no more than 15 percent.
A total of approximately 500 subjects will be recruited from Thailand. 
In Thailand, a total of approximately 500 subjects will be recruited and there will be a 
total of 1 study site participating in this study, which is Sam Khwai Phuek Health 
Promotion Hospital, Muang District, Nakhon Pathom Province, which will recruit 
approximately 500 subjects.
Section 12.1.12 Compensation and Reimbursement
Subjects will receive the amount of 1,000 baht per person per visit as specified in the 
protocol as a reimbursement for time and travel costs, except the case where you have a 
fever when attending a scheduled visit, but the person providing treatment suspects that 
you he/she may have dengue fever, in which case a return visit appointment will be made 
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