A Cohort Study to Determine the Incidence of Dengue Fever and to Build Capacity for Dengue Vaccine Trials in Dengue-endemic Regions of South Asia
Phase: NA
Conditions: Dengue
Sponsor: GlaxoSmithKline

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Study Protocol
Sponsor:
GlaxoSmithKline Biologicals
Rue de l’Institut, 89
B-1330 Rixensart – Belgium
eTrack study number and 
Abbreviated Title
200274 (DPIV-021 EXPLO)
Date of protocol
Final Version 2: 31 July 2014
Date of protocol amendment
Amendment 1 Final: 16 December 2014
Amendment 2 Final: 02 April 2015
Amendment 3 Final: 01 March 2017
Title
A cohort study to determine the incidence of dengue 
fever and to build capacity for dengue vaccine trials in 
South Asia. (Amended 01 March 2017)
Detailed Title
A prospective, cohort study to determine the incidence 
of acute febrile dengue illness and to build capacity for 
dengue vaccine clinical endpoint trials in South Asian 
communities. (Amended 01 March 2017)
Co-ordinating authors
(4Clinics Belgium for GSK Biologicals)/ 
 / 
/
 
(XPE Belgium for GSK ) (Amended 01 March 2017)
Contributing authors
!
, Clinical Research and 
Development Lead
!
, Clinical and Epidemiol. Project 
Leader 
!
, Senior Epidemiologist
!
, Epidemiologist
!
, Project Statistician
!
, Project Statistician
!
, Project Statistician
!
, Lead Statistician
!
, 
, Study 
Delivery Lead
!
, Study Delivery Manager 
!
, Study Delivery Manager 
!
, 
, Clinical Safety 
Physician
!
, 
, Study Data 
Manager
!
, Clinical Laboratory Science 
Clinical Readout Team Leader

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eTrack study number and 
Abbreviated Title
200274 (DPIV-021 EXPLO)
Detailed Title
A prospective, cohort study to determine the incidence 
of acute febrile dengue illness and to build capacity for 
dengue vaccine clinical endpoint trials in South Asian 
communities. (Amended 01 March 2017)
Contributing authors
!
, Clinical Laboratories 
Representative
!
, Senior Manager, Global 
Regulatory Affairs
!
, Global Regulatory Leader
!
, Clinical Laboratory Sciences 
(CLS) Study Manager
!
, CLS Study Manager
!
, Vaccine Supply 
Coordinator
!
, R&D and Medical Affairs
!
, Oversight Data Manager
(Amended 01 March 2017)
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
Copyright 2014-2017 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
200274 (DPIV-021 EXPLO)
Date of protocol amendment
Amendment 3 Final: 01 March 2017
Detailed Title
A prospective, cohort study to determine the incidence 
of acute febrile dengue illness and to build capacity 
for dengue vaccine clinical endpoint trials in South 
Asian communities. (Amended 01 March 2017)
Sponsor signatory
Robert Paris, Clinical and Epidemiology Project Lead
Signature
Date
 
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Protocol Amendment 3 Rationale
Amendment number:
Amendment 3
Rationale/background for changes: 
After study enrolment was initiated, the 3 sites in India were still delayed due to the 
inability to export the blood samples for dengue testing. Following Sponsor review, the 
India portion of the study was cancelled due to logistical difficulties. Therefore, sites in 
India will no longer be included this study protocol.  Among the 5000 subjects planned 
in the study, 3000 were to come from India. The planned number of study subjects 
changed from 5000 to 2000 and all subjects will be enrolled from Sri Lanka.
Sample size calculations were adjusted accordingly.
Added text to clarify that an unscheduled visit (either for first, return, or follow-up 
dengue visit) will be considered as the weekly contact for the week in which it occurs.
 
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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) 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
200274 (DPIV-021 EXPLO)
Date of protocol amendment
Amendment 3 Final: 01 March 2017
Detailed Title
A prospective, cohort study to determine the incidence 
of acute febrile dengue illness and to build capacity 
for dengue vaccine clinical endpoint trials in South 
Asian communities (Amended 01 March 2017)
Investigator name
Signature
Date
 
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Sponsor Information
1.
Sponsor
GlaxoSmithKline Biologicals
Rue de 1’Institut, 89
B-1330 Rixensart - Belgium
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 Report of a Serious Adverse Event (SAE) related to study 
participation
GSK Biologicals Central Back-up Study Contact for Reporting SAEs realted to study 
participation: refer to protocol Section7.3.2
 
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SYNOPSIS
Detailed Title
A prospective, cohort study to determine the incidence of 
acute febrile dengue illness and to build capacity for dengue 
vaccine clinical endpoint trials in South Asian communities.
(Amended 01 March 2017):
Rationale for the 
study 
This study aims to estimate the burden of dengue illness in 
selected sites in South Asia and to prepare sites for the 
conduct of future vaccine efficacy trials.
Operational goals include:

Build long-term collaboration with sites in dengue-
endemic regions of South Asia where the incidence of 
clinical dengue illness can be studied.

Establish dengue surveillance cohorts that can be 
followed long-term.

Establish operational feasibility of future Phase III studies 
with regard to recruitment, case capture and sampling 
procedures.

Prepare sites for participation in Phase III clinical 
endpoint studies.
Scientific Objectives
Primary

To determine the incidence of acute febrile illness (AFI) 
due to Laboratory Confirmed Dengue (LCD) in the study 
population.
Secondary

To determine the incidence of AFI due to non-LCD in the 
study population.

To describe the signs and symptoms of AFI due to LCD 
and of AFI due to non-LCD. 

To estimate the incidence of AFI due to LCD by Dengue 
Virus (DENV) type, study site, and age group. 
 
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Tertiary (Research)

To describe DENVand Japanese Encephalitis Virus (JEV) 
antibody profiles in subsets of subjects with AFI.

To assess the concordance between dengue Reverse 
transcriptase-quantitative Polymerase Chain Reaction 
(RT-qPCR) and Non-Structural protein 1 (NS1)-antigen 
ImmunoChromaTographic (ICT) test and/or Enzyme-
Linked Immunosorbent Assay (ELISA) in the assessment 
of dengue infection.

To gather clinical data that will support the definition of 
clinical endpoints for future efficacy trials.

To describe spatio-temporal clustering of LCD cases.

To describe entomological characteristics at selected 
site(s).

To estimate the prevalence and incidence of tuberculosis 
diagnosis in the study population.

To describe the incidence of hospitalisation and discharge 
diagnosis in the study population.

To isolate and characterise infectious agents from a 
subset of subjects with AFI.

To evaluate the antibody response to infectious agents in 
a subset of subjects with AFI.To isolate and characterise 
infectious agents from a subset of subjects with AFI.

To evaluate the antibody response to infectious agents in 
a subset of subjects with AFI.
Study design

Type of design (Amended 01 March 2017):
Prospective, community-based, cohort study, household-
sampling, single country

Study population (Amended 01 March 2017):
approximately 2,000 subjects aged between 6 months and 
50 years at the time of enrolment living in randomly 
selected households in geographically-defined 
communities. Households including at least one member 
aged less than 18 years will be considered eligible if at 
least one adult (aged no more than 50 years) and one 
child (aged less than 18 years) consent (and assent if
applicable) to participate in the study.

Study visits The study will consist of scheduled visits 
(the enrolment visit [Visit 1], weekly contacts, and the 
close-out visit [Visit 2]) and, in case of AFI*, 
unscheduled Suspected Dengue Visits:
 
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
Visit 1 and Visit 2 will take place at home or at 
the hospital/clinic, in accordance with local ethics 
committee requirements and/or national laws or 
regulations

Weekly contacts will be home visits (at least 
every other week) or telephone contacts (TCs)

Unscheduled Suspected Dengue Visits will take 
place in case of AFI*
Note that an unscheduled visit (either for first, return, 
or follow-up dengue visits) will be considered as the 
weekly contact for the week in which it occurs. 
(Amended 01 March 2017)
*AFI is defined as fever (body temperature 38°C/100.4°F) on 
2 consecutive calendar days, measured at least twice, at least 8 hours 
apart.

Biological samples: Blood samples will be collected at 
each Suspected Dengue First Visit.

Case definitions
AFI due to LCD: ALL of the following findings must be 
met for an AFI due to LCD:

Fever (body temperature 38°C/100.4°F) on 
2 consecutive calendar days, measured at least 
twice, at least 8 hours apart, and

Laboratory confirmation of dengue through 
dengue RT-qPCR on the acute serum sample 
taken during the 7-day period (Days 2-7) from the 
onset of fever (body temperature 
38°C/100.4°F)
AFI due to non-LCD: ALL of the following findings 
must be met for an AFI due to non-LCD:

Fever (body temperature 38°C/100.4°F) on 
2 consecutive calendar days, measured at least 
twice, at least 8 hours apart and

Dengue RT-qPCR result on the acute serum 
sample taken during the 7-day period (Days 2-7) 
from the onset of fever (body temperature 
38°C/100.4°F) is (valid but) negative for 
dengue
 
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Undetermined case: at least ONE of the following 
findings must be met for an undetermined case:

Fever (body temperature 38°C/100.4°F) on 
2 consecutive calendar days, measured at least 
twice, at least 8 hours apart in a subject who has 
not been evaluated (at a Suspected Dengue First 
Visit or directly in a hospital/clinic) within the 7-
day period (Days 2-7) from the onset of fever or 
who had an obvious alternative diagnosis other 
than dengue (i.e., an identified focus of fever), and 
therefore dengue RT-qPCR was not requested, or

Dengue RT-qPCR result on the acute serum 
sample taken during the 7-day period (Days 2-7) 
from the onset of fever (body temperature 
38°C/100.4°F) is invalid or missing
Note: Two consecutive calendar days without fever (body 
temperature 38°C/100.4°F), in the absence of antipyretic 
medication, are required to separate two episodes of fever.
Note: Subjects with fever (body temperature 
38°C/100.4°F) on 2 consecutive calendar days,
measured at least twice, at least 8 hours apart who fail to 
attend a Suspected Dengue First Visit as they were directly 
hospitalised will be recorded as LCD, non-LCD or 
undetermined cases depending on the availability and result of 
the dengue RT-qPCR test.

Type of study: self-contained.

Data collection: Electronic Case Report Form (eCRF).

Duration of the study: approximately 2 years per 
subject:
Epoch 001: prospective data collection starting at Visit 1 
(Month 0) and ending at Visit 2 (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
≈2000
6 months to 50 years
x
(Amended 01 March 2017)
 
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Discussion of study 
design
This prospective cohort study including children aged less 
than 18 years and adults aged up to 50 years is designed to 
support the selection and definition of clinical and laboratory 
endpoints for future dengue vaccine efficacy trials and to 
provide incidence data for sample size calculation. In 
addition, the study aims to train investigators and their teams 
on protocol specific processes and procedures, in preparation 
for the conduct of anticipated future vaccine efficacy studies.
(Amended 01 March 2017)
Number of subjects
Approximately 2000 (Amended 01 March 2017)
Endpoints
Primary

Occurrence of AFI due to LCD. 
Secondary

Occurrence of AFI due to non-LCD.

Occurrence and intensity of signs and symptoms of 
interest, during the 7-day period following the onset of 
each episode of AFI due to LCD and due to non-LCD.

Occurrence of AFI due to LCD by DENV type, study 
site, and age group.
Tertiary (Research) (Amended 01 March 2017)
During the Suspected Dengue First Visit, additional blood 
volumes will be collected for tertiary study endpoint assays. 
These tertiary study endpoints include, but are not limited to, 
determination of neutralising antibody titres against dengue 
virus (DENV), Japanese encephalitis virus (JEV), and 
chikungunya virus, tissue culture to isolate infectious agents, 
and sequencing to characterise infectious agents.
Tertiary study endpoint assays other than dengue serology 
may be performed for a subset of samples (for example, JEV 
assays will only be conducted if additional studies indicate 
that JEV serology is needed to interpret the DENV antibody 
response), i.e., not all of these endpoint assays will be 
performed on all of the sera collected from adult study 
subjects. 
Research objectives may be assessed based on, but not limited 
to, the following endpoints:

Neutralising antibody titres against DENV 1-4.

Neutralising antibody titres against JEV.

Neutralising antibody titres against other viruses, 
including but not limited to, Chikungunya virus. 
 
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
Incidence of NS1-antigen (ICT test and/or ELISA)-
positive AFI by DENV type.

Concordance between dengue RT-qPCR and NS1-antigen 
(ICT and/or ELISA) assays.)

Occurrence of AFI due to LCD and due to non-LCD 
having ≥3 days of fever (body temperature 
≥38°C/≥100.4°F).

Occurrence of AFI due to LCD and due to non-LCD 
resulting in hospitalisation.

Occurrence of combinations of signs and symptoms 
during the 7-day period following the onset of each 
episode of AFI due to LCD and due to non-LCD.

Occurrence of AFI due to LCD within 2 weeks from an 
index case, in the same household or within 50 metres of 
the household of the index case.

Entomological characteristics for LCD cases (e.g., vector 
species, density and number of breeding sites) at selected 
site(s).

Occurrence (by medical history only) of a diagnosis of 
tuberculosis, from birth up to study conclusion, in the 
study population.

Occurrence of hospitalisation and discharge diagnosis.

Identification and characterisation of infectious agents 
isolated from blood (e.g., DENV, influenza viruses,
chikungunya viruses), in subjects with AFI.
 
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TABLE OF CONTENTS
PAGE
SPONSOR INFORMATION ............................................................................................7
SYNOPSIS......................................................................................................................8
LIST OF ABBREVIATIONS...........................................................................................21
GLOSSARY OF TERMS ...............................................................................................23
1. INTRODUCTION .....................................................................................................26
1.1.
Background ................................................................................................26
1.2.
Rationale for the study................................................................................27
2. OBJECTIVES ..........................................................................................................27
2.1.
Primary objective ........................................................................................27
2.2.
Secondary objectives..................................................................................27
2.3.
Tertiary (research) objectives......................................................................28
3. STUDY DESIGN OVERVIEW..................................................................................29
3.1.
Discussion of study design .........................................................................30
4. CASE DEFINITION..................................................................................................31
4.1.
Acute febrile illness due to LCD..................................................................31
4.2.
Acute febrile illness due to non-LCD...........................................................31
4.3.
Undetermined cases...................................................................................31
5. STUDY POPULATION.............................................................................................33
5.1.
Number of subjects / centres ......................................................................33
5.1.1.
Selection of communities and households ...................................33
5.1.2.
Overview of the recruitment plan .................................................34
5.2.
Inclusion criteria for enrolment....................................................................34
5.3.
Exclusion criteria for enrolment...................................................................35
6. CONDUCT OF THE STUDY....................................................................................35
6.1.
Regulatory and ethical considerations, including the informed 
consent process (and assent process if applicable)....................................35
6.2.
Subject identification...................................................................................37
6.3.
General study aspects ................................................................................37
6.4.
Outline of study procedures ........................................................................38
6.5.
Detailed description of study procedures ....................................................40
6.5.1.
Procedures at scheduled Visit 1 prior to enrolment......................40
6.5.1.1.
Informed consent (and assent if applicable) ...............40
6.5.1.2.
Check inclusion and exclusion criteria ........................40
6.5.2.
Procedures at scheduled Visit 1, after enrolment.........................41
6.5.2.1.
Subject and household number attribution..................41
6.5.2.2.
Record socio-demographic data.................................41
6.5.2.3.
Record medical history (including history of 
dengue illness, diagnosis of tuberculosis and 
JEV vaccination history) .............................................41
 
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6.5.2.4.
Subject card distribution .............................................41
6.5.2.5.
Distribution of dengue kits and instruction of 
subjects/subject’s parent(s)/LAR(s) to contact 
study staff in case of acute febrile illness....................41
6.5.2.6.
Recording of serious adverse events related to 
study participation ......................................................42
6.5.3.
Procedures during scheduled weekly contacts ............................43
6.5.3.1.
Distribution of dengue kits and instruction of 
subjects/subject’s parent(s)/LAR(s) to contact 
study staff in case of acute febrile illness....................43
6.5.3.2.
Collection and verification of diary cards, and 
recording of diary cards data from diary cards 
to the eCRF................................................................43
6.5.3.3.
Recording of serious adverse events related to 
study participation ......................................................44
6.5.4.
Procedures at scheduled Visit 2...................................................44
6.5.4.1.
Update medical and JEV vaccination history ..............44
6.5.4.2.
Collection and verification of diary cards, and 
recording of diary cards data from diary cards 
to the eCRF................................................................44
6.5.4.3.
Recording of serious adverse events related to 
study participation ......................................................44
6.5.5.
Procedures at Suspected Dengue Visits......................................44
6.5.5.1.
Procedures at Suspected Dengue First Visit...............44
6.5.5.1.1.
Important general remarks......................44
6.5.5.1.2.
Physical examination..............................45
6.5.5.1.3.
Record medical history ...........................46
6.5.5.1.4.
Blood sampling.......................................46
6.5.5.1.5.
Collection and recording of 
solicited symptoms from diary 
card ........................................................47
6.5.5.1.6.
Recording of serious adverse 
events related to study 
participation............................................47
6.5.5.2.
Procedures at Return Visit (optional) ..........................47
6.5.5.3.
Procedures at Suspected Dengue Follow-up 
Visit ............................................................................48
6.5.6.
Study conclusion..........................................................................48
6.6.
Solicited symptoms in case of acute febrile illness (suspected 
dengue infection) ........................................................................................48
6.6.1.
Assessment of the intensity of solicited symptoms.......................49
6.7.
Biological sample handling and analysis.....................................................50
6.7.1.
Use of specified study materials ..................................................51
6.7.2.
Biological samples.......................................................................52
6.8.
Laboratory assays ......................................................................................53
7. SAFETY ..................................................................................................................54
7.1.
Safety definitions ........................................................................................54
7.1.1.
Definition of an adverse event......................................................54
7.1.2.
Definition of a serious adverse event ...........................................54
7.1.3.
Clinical laboratory parameters and other abnormal 
assessments qualifying as SAEs .................................................55
 
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7.2.
Detecting and recording SAEs related to study participation.......................56
7.2.1.
Time periods for detecting and recording SAEs ...........................56
7.2.2.
Evaluation of SAEs related to study participation.........................56
7.2.2.1.
Active questioning to detect SAEs..............................56
7.2.2.2.
Assessment of the intensity SAEs ..............................57
7.2.2.3.
Assessment of causality .............................................57
7.2.2.4.
Assessment of outcomes............................................58
7.3.
Reporting of SAEs related to study participation .........................................58
7.3.1.
Prompt reporting of SAEs related to study participation to 
GSK.............................................................................................58
7.3.2.
Contact information for reporting SAEs related to study 
participation to GSK.....................................................................59
7.3.3.
Completion and transmission of reports of SAEs related to 
study participation to GSK ...........................................................59
7.3.3.1.
Back-up system in case the electronic 
reporting system does not work..................................59
7.3.4.
Updating of SAE information after freezing of the subject’s 
eCRF...........................................................................................59
7.3.5.
Regulatory reporting requirements for SAEs................................60
7.4.
Follow-up of SAEs related to study participation .........................................60
7.4.1.
Follow-up of SAEs related to study participation ..........................60
7.4.1.1.
Follow-up during the study..........................................60
7.4.1.2.
Follow-up after the subject is discharged from 
the study.....................................................................60
8. SUBJECT COMPLETION AND WITHDRAWAL ......................................................60
8.1.
Subject completion .....................................................................................60
8.2.
Subject withdrawal......................................................................................61
8.3.
Extension study ..........................................................................................62
9. STATISTICAL METHODS .......................................................................................62
9.1.
Endpoints....................................................................................................62
9.1.1.
Primary endpoint..........................................................................62
9.1.2.
Secondary endpoints ...................................................................62
9.1.3.
Tertiary (research) endpoints.......................................................63
9.2.
Sample size considerations ........................................................................64
9.3.
Cohorts for analyses...................................................................................65
9.4.
Derived and transformed data.....................................................................65
9.5.
Analysis of demographics / baseline characteristics ...................................67
9.6.
Analysis of primary and secondary objectives.............................................67
9.7.
Analysis of tertiary (research) objectives.....................................................68
9.8.
Analysis of SAEs related to study participation ...........................................69
9.9.
Interpretation of analyses............................................................................69
9.10.
Conduct of analyses ...................................................................................69
9.10.1.
Sequence of analyses..................................................................69
10. ADMINISTRATIVE MATTERS.................................................................................69
10.1.
Electronic Case Report Form instructions...................................................69
10.2.
Study monitoring by GSK Biologicals..........................................................70
10.3.
Record retention .........................................................................................71
10.4.
Quality assurance.......................................................................................71
 
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10.5.
Posting of information on publicly available registers and publication 
policy ..........................................................................................................71
10.6.
Provision of study results to investigators ...................................................72
11. COUNTRY SPECIFIC REQUIREMENTS ................................................................72
12. REFERENCES ........................................................................................................73
 
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LIST OF TABLES
PAGE
Table 1
Study groups and epochs foreseen in the study.....................................30
Table 2
List of study procedures for scheduled visits and contacts .....................38
Table 3
List of study procedures for unscheduled Suspected Dengue 
Visits (Amended 01 March 2017) ...........................................................39
Table 4
Intervals between study visits/contacts...................................................40
Table 5
Physical examination during Suspected Dengue First Visit ....................46
Table 6
Solicited symptoms in case of suspected dengue infection ....................49
Table 7
Intensity scales for solicited symptoms (excluding bleeding and 
reduced fluid intake, and body temperature) ..........................................49
Table 8
Intensity scales for bleeding ...................................................................49
Table 9
Intensity scales for reduced fluid intake..................................................50
Table 10
Grading scale for body temperature .......................................................50
Table 11
Biological samples (Amended 01 March 2017) ......................................52
Table 12
Laboratory confirmation of dengue infection (Amended 01 March 
2017)......................................................................................................53
Table 13
Haematology..........................................................................................53
Table 14
Other exploratory (tertiary, research) assays..........................................53
Table 15
Reporting periods for SAEs related to study participation.......................56
Table 16
Timeframes for submitting SAEs related to study participation to 
GSK .......................................................................................................58
Table 17
95% CI with design effect of 2.6.............................................................64
Table 18
GSK Biologicals’ laboratories .................................................................75
Table 19
Outsourced laboratories.........................................................................75
 
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LIST OF FIGURES
PAGE
Figure 1
Study design overview ...........................................................................29
Figure 2
Schematic overview of different cases ...................................................32
 
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LIST OF APPENDICES
PAGE
APPENDIX A LABORATORY ASSAYS .......................................................................74
APPENDIX B CLINICAL LABORATORIES ..................................................................75
APPENDIX C AMENDMENTS AND ADMINISTRATIVE CHANGES TO THE 
PROTOCOL...........................................................................................76
 
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LIST OF ABBREVIATIONS
µPRN
Micro-Plaque-Reduction Neutralisation
AE
Adverse Event
AFI
Acute Febrile Illness
ATP
According-To-Protocol
CBC
Complete Blood Count
CI
Confidence Interval
DENV
Dengue Virus
DSS
Dengue Shock Syndrome
eCRF
electronic Case Report Form
ELISA
Enzyme-linked Immunosorbent Assay
GCP
Good Clinical Practice
GIS
Geographic Information System
GMT
Geometric Mean Titre
GSK
GlaxoSmithKline
IAF
Informed Assent Form
ICF
Informed Consent Form
ICH
International Conference on Harmonisation
ICT
ImmunoChromaTographic
IEC
Independent Ethics Committee
IRB
Institutional Review Board
JEV
Japanese Encephalitis Virus
LAR
Legally Acceptable Representative
LCD
Laboratory Confirmed Dengue
MedDRA
Medical Dictionary for Regulatory Activities
NS1
Non-Structural protein 1
PII
Personal Identifiable Information
PRN
Plaque-Reduction Neutralisation
PT
Preferred Term
RBC
Red Blood Cells
RT-qPCR
Reverse Transcriptase quantitative Polymerase Chain Reaction
SAE
Serious Adverse Event
SBIR
GSK Biologicals’ randomisation system on internet
 
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SDV
Source Document Verification
SPM
Study Procedures Manual
TC
Telephone Contact
WBC
White Blood Cells
WHO
World Health Organisation
 
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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
Assent:
When a subject is below the legal age of consent (i.e., a minor) and 
is able to give assent to decisions about his/her participation in a 
clinical study, the investigator is encouraged to obtain it, in addition 
to obtaining the consent from the subject’s parent(s)/ Legally 
Acceptable Representative(s) (LAR[s]). The study investigator is 
accountable for determining a child's capacity to assent to 
participation in a research study, taking into consideration any 
standards set by the responsible Institutional Review Board (IRB) / 
Independent Ethics Committee (IEC) (or local requirements).
Assent refers to the child’s affirmative agreement to participate in 
the research.
Child in care:
A child who has been placed under the control or protection of an 
agency, organisation, institution or entity by the courts, the 
government or a government body, acting in accordance with powers 
conferred on them by law or regulation. The definition of a child in 
care can include a child cared for by foster parents or living in a care 
home or institution, provided that the arrangement falls within the 
definition above. The definition of a child in care does not include a 
child who is adopted or has an appointed legal guardian.
Coded:
Data from which personal identifier information has been removed 
and replaced by a key. These data are not anonymised since a decode 
listing exists and it is therefore possible to identify the patient under 
certain circumstances by an authorised or legally appointed third 
party data custodian, or by the original holder of the data.
 
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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). 
Community health 
worker:
A member of the local community who is selected, supported, and 
trained to work by the health system as a health aide in the 
community from which they come.
Eligible:
Qualified for enrolment into the study based upon strict adherence to 
inclusion/exclusion criteria.
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 9.3 for details on criteria for 
evaluability).
Impartial witness: A person, who is independent of the trial, who cannot be unfairly 
influenced by people involved with the trial, who attends the 
informed consent process (and assent process if applicable) if the 
subject of the subject’s legally acceptable representative cannot read, 
and who reads the informed consent form (and assent form if 
applicable) and any other written information supplied to the subject.
Interventional 
Human Subject 
Research:
Studies in which participants are administered medical care, 
medicinal products and/or medical/scientific procedures as described 
in a research protocol.
Legally 
Acceptable 
Representative 
(LAR):
An individual or juridical or other body authorised under applicable 
law to consent, on behalf of a prospective subject, to the subject’s 
participation to in the clinical trial.
Primary 
completion date:
The date that the final subject was examined or received an 
intervention for the purpose of final collection of data for the 
primary outcome, whether the clinical trial/pharmaco-
epidemiological study concluded according to the pre-specified 
protocol or was terminated prematurely for any reason.
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.
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.
 
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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, subjects files, and 
records kept at the pharmacy at the laboratories and at medico-
technical departments involved in the clinical trial).
Study population: Sample of population of interest.
Study staff:
All study personnel, including study coordinators, field workers, 
nurses, investigators, technicians and other study functions.
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 (or assenting when applicable) 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, the most common arthropod-borne viral disease worldwide, is caused by four 
types of dengue viruses (DENV 1-4) and transmitted primarily by Aedes aegypti, a 
mosquito that is highly adapted to urban environments. Dengue infection can cause a 
range of clinical manifestations from febrile illness to potentially fatal dengue shock 
syndrome (DSS).
It is estimated that between 2.5 to 3.5 billion people, or 40 to 55 % of the world’s 
population, live in areas where there is a risk of transmission of dengue fever [Kyle, 
2008; WHO, 2009; Guzman, 2010. Dengue is endemic in at least 100 countries in Asia, 
the Pacific, the Americas, Africa, and the Caribbean. New estimates indicate 
approximately 390 million (284-528) dengue infections occur per year, of which 96 
million (67-136) manifest apparently (any level of disease severity) [Bhatt, 2013]. The 
global dengue burden is believed to increase due to increasing mobility, increasing 
urbanisation, ecological changes, and the inability to sustain effective vector control.
Globally, 70% of the estimated 96 million apparent dengue infections are thought to 
occur in Asia.
Sero-surveillance data from Colombo, Sri Lanka, indicate that over 70% of children 
experienced at least 1 dengue infection by the age of 12 years, and the median age at 
infection there is 4.7 years. The risk of primary infection in children below 12 years of 
age was 14% per year [Tam, 2013. In 2012, the reported dengue fever incidence cases 
per 100,000 people were 200 for Sri Lanka [NVBDCP, 2017; Epidemiology Unit, 2017
and reports of dengue outbreaks and fatal cases frequently made headline news in South 
Asia.
In Sri Lanka, dengue surveillance and training of doctors in the management of dengue 
illness is a top priority of the Ministry of Health, especially because the rate of severe 
disease in Sri Lanka is one of the highest national rates reported. DENV infection and 
disease incidence in children in Colombo, Sri Lanka, have been reported to exceed 8% 
and 3% per year, respectively [Tissera, 2014].
(Amended 01 March 2017)
There are no licensed antivirals or vaccines to treat or prevent dengue. The development 
and widespread use of a safe and efficacious dengue vaccine is required to significantly 
reduce the global dengue burden. Estimating the incidence of clinically significant 
dengue fever is critical to the design of a dengue vaccine efficacy trial. In a given city or 
region, dengue incidence is highly variable from one year to another and cannot be 
reliably predicted. In a dengue vaccine efficacy trial, volunteers would be recruited from 
geographically distinct sites. Some of these sites would experience high dengue 
transmission during the trial duration while some other may have limited transmission. 
Therefore, it is important to make a valid assumption regarding the average incidence 
 
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across many sites in order to accurately calculate the sample size for a vaccine efficacy 
trial. In addition, a critical factor for the success of efficacy trials is the training of the 
various sites to perform febrile illness detection, dengue laboratory diagnosis and data 
collection in a cohort of subjects.
1.2.
Rationale for the study
This study aims to estimate the burden of dengue illness in selected sites in South Asia 
and to prepare sites for the conduct of future vaccine efficacy trials.
Operational goals include:

Build long-term collaboration with sites in dengue-endemic regions of South Asia
where the incidence of clinical dengue illness can be studied.

Establish dengue surveillance cohorts that can be followed long-term.

Establish operational feasibility of future Phase III studies with regard to 
recruitment, case capture and sampling procedures.

Prepare sites for participation in Phase III clinical endpoint studies
2.
OBJECTIVES
2.1.
Primary objective

To determine the incidence of acute febrile illness (AFI) due to Laboratory 
Confirmed Dengue (LCD) in the study population. 
Refer to Section 9.1.1 for the definition of the primary endpoint.
2.2.
Secondary objectives

To determine the incidence of AFI due to non-LCD in the study population. 

To describe the signs and symptoms of AFI due to LCD and of AFI due to non-LCD. 

To estimate the incidence of AFI due to LCD by Dengue Virus (DENV) type, study 
site, and age group. 
Refer to Section 9.1.2 for the definition of the secondary endpoints.
 
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2.3.
Tertiary (research) objectives
(Amended 01 March 2017)

To describe DENVand Japanese Encephalitis Virus (JEV) antibody profiles in 
subsets of subjects with AFI.

To assess the concordance between dengue Reverse transcriptase-quantitative 
Polymerase Chain Reaction (RT-qPCR) and Non-Structural protein 1 (NS1)-antigen 
ImmunoChromaTographic (ICT) test and/or Enzyme-Linked Immunosorbent Assay 
(ELISA) in the assessment of dengue infection.

To gather clinical data that will support the definition of clinical endpoints for future 
efficacy trials. 

To describe spatio-temporal clustering of LCD cases. 

To describe entomological characteristics at selected site(s). 

To estimate the prevalence and incidence of tuberculosis diagnosis in the study 
population. 

To describe the incidence of hospitalisation and discharge diagnosis in the study 
population. 

To isolate and characterise infectious agents from a subset of subjects with AFI. 

To evaluate the antibody response to infectious agents in a subset of subjects with 
AFI. 
Refer to Section 9.1.3 for the definition of the tertiary (research) endpoints.
 
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3.
STUDY DESIGN OVERVIEW
Protocol waivers or exemptions are not allowed. Therefore, adherence to the study design 
requirements, including those specified in the outline of study procedures (Section 6.4), 
are essential and required for study conduct.
Figure 1
Study design overview
Scheduled Visits
- Visit 1 and Visit 2 will take place at 
home or at the hospital/clinic 
- Weekly contacts will be home 
visits (at least every other week) or
telephone contacts (TCs)
Unscheduled Visits
- Suspected Dengue First Visit 
(hospital/clinic)
- Optional Return Visit 
(hospital/clinic)
- Suspected Dengue Follow-up Visit 
(hospital/clinic or home)
Notes: (Amended 01 March 2017)
The weekly contact may be a face to face visit outside the home in certain cases (i.e. at 
hospital during suspected dengue visit)
An unscheduled visit (either for first, return, or follow-up dengue visit) will be 
considered as the weekly contact for the week in which it occurs.
!
Type of design: Prospective, community-based, cohort study, household-sampling, 
single country. (Amended 01 March 2017)
!
Study population: Approximately 2000 subjects aged between 6 months and 
50 years at the time of enrolment living in randomly selected households in 
geographically-defined communities. Households including at least one member 
aged less than 18 years will be considered eligible if at least one adult (aged no more 
than 50 years) and one child (aged less than 18 years) consent (and assent if 
applicable) to participate in the study. (Amended 01 March 2017)
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
Study visits: The study will consist of scheduled visits (the enrolment visit [Visit 1], 
weekly contacts, and the close-out visit [Visit 2]) and, in case of AFI*, unscheduled 
Suspected Dengue Visits:

Visit 1 and Visit 2 will take place at home or at the hospital/clinic, in 
accordance with local ethics committee requirements and/or national laws or 
regulations

Weekly contacts will be home visits (at least every other week) or telephone 
contacts (TCs). The weekly contact may be a face to face visit outside the 
home in certain cases (i.e. at hospital during suspected dengue visit)
(Amended 01 March 2017)

Unscheduled Suspected Dengue Visits will take place in case of AFI*
*AFI is defined as fever (body temperature 38°C/100.4°F) on 2 consecutive calendar days,
measured at least twice, at least 8 hours apart.

Biological samples: Blood samples will be collected at each Suspected Dengue First 
Visit.

Type of study: self-contained.

Data collection: Electronic Case Report Form (eCRF).

Duration of the study: approximately 2 years per subject: 

Epoch 001: prospective data collection starting at Visit 1 (Month 0) and ending at 
Visit 2 (Month 24).
Table 1
Study groups and epochs foreseen in the study
Study Groups Number of subjects
Age (Min/Max)
Epoch 001
Prospective
≈2000
6 months to 50 years
x
(Amended 01 March 2017)
3.1.
Discussion of study design
This prospective cohort study including children aged less than 18 years and adults aged 
up to 50 years is designed to support the selection and definition of clinical and 
laboratory endpoints for future dengue vaccine efficacy trials and to provide incidence 
data for sample size calculation. In addition, the study aims to train investigators and their 
teams on protocol specific processes and procedures, in preparation for the conduct of 
anticipated future vaccine efficacy studies. (Amended 01 March 2017)
 
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4.
CASE DEFINITION
4.1.
Acute febrile illness due to LCD
ALL of the following findings must be met for an AFI due to LCD (see Figure 2): 

Fever (body temperature 38°C/100.4°F) on 2 consecutive calendar days,
measured at least twice, at least 8 hours apart, and

Laboratory confirmation of dengue through dengue RT-qPCR on the acute serum 
sample taken during the 7-day period (Days 2-7) from the onset of fever (body 
temperature 38°C/100.4°F)
4.2.
Acute febrile illness due to non-LCD
ALL of the following findings must be met for an AFI due to non-LCD (see Figure 2): 

Fever (body temperature 38°C/100.4°F) on 2 consecutive calendar days,
measured at least twice, at least 8 hours apart, and

Dengue RT-qPCR result on the acute serum sample taken during the 7-day period 
(Days 2-7) from the onset of fever (body temperature 38°C/100.4°F) is (valid 
but) negative for dengue
4.3.
Undetermined cases
At least ONE of the following findings must be met for an undetermined case (see Figure 
2)

Fever (body temperature 38°C/100.4°F) on 2 consecutive calendar days,
measured at least twice, at least 8 hours apart in a subject who has not been evaluated 
(at a Suspected Dengue First Visit or directly in a hospital/clinic) within the 7-day 
period (Days 2-7) from the onset of fever or who had an obvious alternative 
diagnosis other than dengue (i.e., an identified focus of fever), and therefore dengue 
RT-qPCR was not requested, or

Dengue RT-qPCR result on the acute serum sample taken during the 7-day period 
(Days 2-7) from the onset of fever (body temperature 38°C/100.4°F) is invalid or 
missing
 
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Figure 2
Schematic overview of different cases
AFI: Acute febrile illness; LCD: Laboratory confirmed dengue; RT-qPCR: Reverse transcriptase quantitative 
polymerase chain reaction (Amended 01 March 2017)
Note: Two consecutive calendar days without fever (body temperature 
# 38°C/# 100.4°F), in the absence of antipyretic medication, are required to separate two 
episodes of fever.
Note: Subjects with fever (body temperature # 38°C/# 100.4°F) on # 2 consecutive 
calendar days, measured at least twice, at least 8 hours who fail to attend a Suspected 
Dengue First Visit as they were directly hospitalised will be recorded as LCD, non-LCD 
or undetermined cases depending on the availability and result of the dengue RT-qPCR 
test. Refer to the Study Procedures Manual (SPM) for more information.
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5.
STUDY POPULATION
5.1.
Number of subjects / centres
This study will be a multi-centre, household-based study conducted in dengue-endemic 
regions of South Asia.
The target is to enrol approximately 2,000 eligible subjects in order to reach 
approximately 3300 person-years evaluable overall at the time of final analysis. Refer to 
Section 9.2 for a detailed description of the criteria used in the estimation of sample size.
(Amended 01 March 2017)
One or more members of each household (preferably all eligible household members) 
will be enrolled.
5.1.1.
Selection of communities and households
This study will enrol subjects in selected communities based on the following non-
exclusive characteristics: 

preferably in areas where access to the community is already established (previous 
community-based surveillance studies)

in areas with a high population density

in areas with low out-migration rate

in areas with preferably a single point-of-care for the community

preferably in areas where there is a confirmation of dengue endemicity

in areas where children are likely to constitute at least 30% of the study population 
per site

in households with at least one child (less than 18 years of age)

in households with at least one adult (aged no more than 50 years) and one child 
(aged less than 18 years) consenting (and assenting if applicable) to participate in the 
study
Refer to Section 5.1.2 for an overview of the recruitment plan.
(Amended 01 March 2017).
A list of all households in the sampling area will be generated by each site. Simple 
random sampling will be performed to select households for enrolment. If the sampling 
area is too large to compile a list of all households, a least eight clusters of similar 
population size (blocks or streets) will be selected by simple random sampling. 
Households in the selected clusters will then be listed and selected by simple random 
sampling.
 
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5.1.2.
Overview of the recruitment plan
Enrolment will continue until a total of approximately 2,000 subjects are recruited. 
Subjects lost to follow-up (i.e., with no contact for 4 consecutive weeks) during the 
enrolment period will be replaced (Refer to Section 8.2). (Amended 01 March 2017)
SBIR (GSK Biologicals’ randomisation system on internet) will be used for this study. 
All details pertaining to user access and specific procedures can be found in the SBIR 
version 8 User Guide and the SPM.
Recruitment will be organised by study staff at participating sites. Community health 
workers or equivalent will serve to the liaison, and may accompany study staff during the 
visits. New target households will be visited until the enrolment target for the study area 
is reached. The study will be explained to the individuals living in the household, and if 
any eligible individual is interested in study participation, informed consent (and assent if
applicable) will be obtained at the hospital/clinic or during a home visit, where eligibility 
criteria will be checked and subjects will be enrolled and interviewed for potential study 
participation.
Refusal (including reasons for refusal) of a household to participate will be documented. 
Refusal (including reasons for refusal) of individual household members will be 
documented as well. Household visits may be scheduled to occur during weekends if 
necessary. 
Possession of a phone/cell phone will be documented.

If a site decides to perform home visits only (i.e., no TC contacts), the possession of 
a phone/cell phone is not mandatory.

If a site decides to perform both home visits and TC contacts, the access to a 
phone/cell phone will be ensured.
Infants who become eligible (i.e., reach the age of 6 months) will be offered enrolment in 
the study. These children, if enrolled, will be followed from that time until the last adult 
household member has completed the study.
5.2.
Inclusion criteria for 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:

Subject and/or subject’s parent(s)/legally acceptable representative(s) (LAR[s) who, 
in the opinion of the investigator, can and will comply with the requirements of the 
protocol (e.g., willingness to go to the hospital/clinic for visit[sin case of AFI, able 
to observe the signs of dengue and to understand how to take and report body 
temperature, etc.).
 
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
Signed/thumb-printed (and video recorded if required by law) informed consent (and
assent if applicable) must be obtained from the subject/subject’s parent(s)/LAR(s) at 
the hospital/clinic or during a home visit. If the subject/subject’s parent(s)/LAR(s) 
are illiterate, the informed consent form (ICF) (or informed assent form [IAF] when 
applicable) will be countersigned by an impartial witness.

Subject is part of a household with at least one child (aged less than 18 years) and in 
which informed consent (and assent if applicable) to study participation was obtained 
from at least one adult and one child.

Male or female aged between and including 6 months and 50 years at the time of 
enrolment.

Subject who plans, at the time of enrolment, to remain at same residence/study area 
during the two-year study period.
5.3.
Exclusion criteria for 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 based on investigator's judgement.

Mental incapacity based on investigator’s judgement.
6.
CONDUCT OF THE STUDY
6.1.
Regulatory and ethical considerations, including the 
informed consent process (and assent process if
applicable)
The study will be conducted in accordance with all applicable regulatory requirements. 
GSK will obtain the favourable approvals from Ethics committees and other appropriate 
authorities prior to initiating a site.
The study will be conducted in accordance with the International Conference on 
Harmonisation (ICH) Guideline for Good Clinical Practice (GCP) and other applicable 
local guidelines, all applicable subject privacy requirements and the guiding principles of 
the Declaration of Helsinki.
 
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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.
Conduct of the study includes, but is not limited to, the following:

Institutional Review Board (IRB)/Independent Ethics Committee (IEC) review and 
favourable opinion/approval of study protocol and any subsequent amendments.

Subject/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, at the hospital/clinic or during a home visit prior to participation in the study.
GSK Biologicals will prepare a model ICF which will embody the applicable ICH GCP 
and other applicable local guidelines, 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 Good Clinical Practice, 
a subject who can only be enrolled in the study with the consent of his/her 
parent(s)/LAR(s) (e.g., a minor below the age of consent) but at the age for assent, should 
be informed about the study to the extent compatible with his/her understanding and, if 
capable, the subject should sign or thumb-print and personally date a written IAF (while 
being video recorded if required by law). 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(s)/LAR(s). It should be assessed whether an assent is required depending on the 
age of the study population and the local requirements.
The consenting process will be video recorded if required by law. If the subject/subject’s 
parent(s)/LAR(s) are illiterate, the ICF (and IAF if applicable) will be countersigned by 
an impartial witness. Informed consent/assent can be obtained at the study clinic or 
during a home visit. In either case, medically and GCP trained study staff will follow an 
SOP for the consenting/assenting process.
 
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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.
6.2.
Subject identification
Subject numbers will be assigned sequentially to subjects consenting (or assenting when
applicable) to participate/to be included in the study, according to the range of subject 
numbers allocated to each study centre.
Recruitment in this study will be monitored using SBIR.
6.3.
General study aspects
Supplementary study conduct information not mandated to be present in this protocol is 
provided in the accompanying Study Procedures Manual (SPM and central laboratory 
manual). 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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6.4.
Outline of study procedures
Table 2
List of study procedures for scheduled visits and contacts
Epoch
Epoch 001
Visit/contact
Visit 1
Home visits or 
TCs
Visit 2
Time point
Month 0
Weekly
between visits 1
Month 24
Informed consent (and assent if applicable)
●
Check inclusion/exclusion criteria
●
Subject number attribution
O
Household number attribution
●
Record socio-demographic data
●
Record medical history (including history of dengue illness, diagnosis of 
tuberculosis and JEV vaccination history)
●
Update medical history
●
Subject card distribution
O
Distribute dengue kits to subjects/subject’s parent(s)/LAR(s), including diary 
cards to be filled out in case of fever 2 
O
O
Instruct/remind subjects/subject’s parent(s)/LAR(s) to contact study staff and to 
report symptoms in diary card in case of fever 3
O
O
Contact subjects regarding any AFI and/or hospitalisation 4
●
Collect and verify diary cards (if applicable) 5
O 6
O
Record data (solicited symptoms, medication) from diary cards (if applicable)
●6
●
Record SAEs related to study participation
●
●
●
Study conclusion 7
●
● is used to indicate a study procedure that requires documentation in the individual eCRF.
is used to indicate a study procedure that does not require documentation in the individual eCRF.
AFI: acute febrile illness
eCRF: electronic Case Report Form
JEV: Japanese encephalitis virus
LAR: legally acceptable representative
SAE: serious adverse event
TC: telephone contact
1) Weekly contacts will be home visits (at least every other week) or TCs. The weekly contact may be a face to face 
visit outside the home in certain cases (i.e. at hospital during suspected dengue visit) (Amended 01 March
2017)
2) Diary cards will be distributed at the first scheduled visit. A new diary card will be issued by the study staff whenever 
necessary (i.e., if the subject no longer has one, e.g., the previous one was lost or used).
3) The subject/subject’s parent(s)/LAR(s) will be instructed to start completing a diary card in the event of fever (i.e.,
body temperature 38°C/100.4°F). Body temperature and the intensity of each of the solicited symptoms should 
be recorded on the diary cards for the next 6 subsequent days. After 7 days, only the end date for any ongoing 
solicited symptoms will be recorded on the diary cards. Please refer to Table 6 for the list of solicited symptoms to 
be recorded. Any medications (e.g., antipyretics) taken during this 7-day period will also be recorded. Two 
consecutive calendar days without fever, in the absence of antipyretic medication, are required to separate two 
episodes of fever. Signs and symptoms prior to the onset of fever will not be recorded.
4) Including recording of discharge diagnosis for hospitalised subjects.
5) The subject/subject’s parent(s)/LAR(s) will bring the diary card with him/her to each Suspected Dengue Visit (see 
Table 3). The investigator or the appointed study personnel will record the diary card information in the eCRF if a 
temperature of 38°C (100.4°F) was recorded on 2 consecutive calendar days, measured at least twice, at 
least 8 hours apart. The subject/subject’s parent(s)/LAR(s) will return the diary card when completed at the next visit 
or contact (i.e., at the next home visit or at a Suspected Dengue Visit). 
6) Only applicable for home visits. 
7) Only one study conclusion will be conducted, either at Visit 2, or if a dengue case is ongoing at Visit 2, at the last 
Suspected Dengue Follow-up Visit for the dengue case.
 
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Table 3
List of study procedures for unscheduled Suspected Dengue Visits
(Amended 01 March 2017)
Epoch
Epoch 001
Visit
Suspected Dengue 
First Visit
Return Visit 1
Suspected Dengue 
Follow-up Visit 2
Time point
Day 2 to Day 7, ideally 
on Day 2 (1 day after 
the onset of fever)
1 to 6 days 
after 
Suspected 
Dengue First 
Visit
7 to13 days after 
Suspected Dengue 
First Visit
Specific physical examination
●
●
Update medical history 
●
●
●
Blood sampling*
   Dengue NS1-antigen ICT test and/or ELISA 
(0.5 mL)
●
   Dengue RT-qPCR (2.5 mL)
●
   for haematology (2 mL) 3
●
   for other tertiary (research) endpoints related tests 
(3.5 mL) 4
●
Safety follow-up:
Collect and verify diary cards (if applicable) 5
O
O
O
Record data (solicited symptoms, medication) from 
diary cards (if applicable) 5
●
●
●
Issue diary cards to subjects/subject’s 
parent(s)/LAR(s) to be filled out in the event of fever 6
O
O
O
Record SAEs related to study participation
●
●
●
Study conclusion 7
●
● is used to indicate a study procedure that requires documentation in the individual eCRF.
is used to indicate a study procedure that does not require documentation in the individual eCRF.
µPRN: micro-plaque-reduction neutralisation; eCRF: electronic Case Report Form; ELISA: enzyme-linked 
immunosorbent assay; ICT: immunochromatographic; JEV: Japanese encephalitis virus; LAR: legally acceptable 
representative; NS1: non-structural protein 1; RT-qPCR: reverse transcriptase quantitative polymerase chain reaction; 
SAE: serious adverse event. (Amended 01 March 2017
* Blood samples should only be drawn in case of AFI (i.e., fever [body temperature 38°C/100.4°F] on 
2 consecutive calendar days, measured at least twice, at least 8 hours apart)) during the Suspected Dengue First 
Visit from the second day of fever (i.e., from Day 2, the second calendar day with fever) and onwards, up to Day 7. 
1) A Return Visit is a visit linked to the Suspected Dengue First Visit and should take place 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.
2) A Suspected Dengue Follow-up Visit may occur during a weekly home visit. This visit should be performed by a 
medically trained site staff member. Note that when a subject comes in for a suspected dengue follow-up 
visit, this will replace the regularly scheduled weekly home visit. (Amended 01 March 2017)
3) For all subjects ≥5 years of age. For subjects < 5 years of age only if clinically indicated, as per investigator 
judgement. Complete blood count (White blood cells [WBC], red blood cells [RBC], platelets and haemoglobin).
4) For subjects ≥18 years of age only. Including, but not limited to, neutralising antibody against JEV and 
Chikungunya virus. A subset of samples will be tested. 
5) The subject/subject’s parent(s)/LAR(s) will bring the diary card with him/her to each Suspected Dengue Visit. The 
investigator or appointed study personnel will record the diary card information in the eCRF if a temperature of 
38°C (100.4°F) was recorded on 2 consecutive calendar days, measured at least twice, at least 8 hours 
apart. The subject/subject’s parent(s)/LAR(s) will return the diary card when completed at the next scheduled visit 
or contact (i.e., at the next home visit or at a Suspected Dengue Visit).
6) A new diary card will be issued by the study staff whenever necessary (i.e., if the subject no longer has one, e.g., 
the previous one was lost or used).
7) Only one study conclusion will be conducted, either at Visit 2, or if a suspected dengue case is ongoing at Visit 2, at 
the last Suspected Dengue Follow-up Visit for the suspected dengue case.
 
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Table 4
Intervals between study visits/contacts
Interval
Optimal length of interval
Allowed interval *
Scheduled Visits:
Visit 1 (Month 0) Visit 2 (Month 24)
24 months
23 months - 25 months
Weekly contacts 
7 days
6 – 8 days
Unscheduled Visits:
Onset of fever (body temperature 38°C/100.4°F) 
(Day 1) Suspected Dengue First Visit
1 day (on Day 2)
up to 6 days (Day 2 to 
Day 7) **
Suspected Dengue First Visit (optional) Return Visit 
(as per investigator judgement)
1 – 6 days
1 – 6 days
Suspected Dengue First Visit Suspected Dengue 
Follow-up Visit
7 – 8 days (may occur during a 
weekly home visit)
7 – 13 days
*Whenever possible the investigator should arrange study visits/contacts within this interval.
** If the subject has not been evaluated within the 7-day period (Days 2-7) from the onset of fever, the Suspected 
Dengue First Visit is no longer required and the case will be considered as an undetermined case.
6.5.
Detailed description of study procedures
6.5.1.
Procedures at scheduled Visit 1 prior to enrolment
The enrolment visit (Visit 1) will take place at home or at the hospital/clinic. 
6.5.1.1.
Informed consent (and assent if applicable)
The signed/thumb printed (and video recorded, if required by law) informed consent of 
the subject/subject’s parent(s)/LAR(s) must be obtained before study participation at the 
hospital/clinic or during a home visit. The signed/thumb printed informed assent of a 
subject below the age of consent (i.e., minor) but at the age for assent should also be 
obtained in addition to the signed/thumb printed informed consent by his/her 
parent(s)/LAR(s) according to local rules and regulations. If the subject/subject’s 
parent(s)/LAR(s) are illiterate, the ICF (and IAF if applicable) will be countersigned by 
an impartial witness. Refer to Section 6.1 for the requirements on how to obtain informed 
consent and assent, as appropriate.
The initial contact and consenting process (and assenting process if applicable) will take 
place at the hospital/clinic or during a home visit.
6.5.1.2.
Check inclusion and exclusion criteria 
Check all applicable inclusion and exclusion criteria as described in Sections 5.2 and 5.3
before enrolment.
 
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6.5.2.
Procedures at scheduled Visit 1, after enrolment
6.5.2.1.
Subject and household number attribution
A subject number and household number will be attributed. The household number will 
be allocated by each site, according to their local practice. Existing system of house 
numbering or census method will be used. The household number will be recorded in the 
eCRF.
6.5.2.2.
Record socio-demographic data
Socio-demographic data such as date of birth, gender, education, occupation and socio-
economic status will be recorded in the eCRF.
A Geographic Information System (GIS) will be used to map the location of each study 
household. Based on these data, a map of study households without GIS coordinates will 
be generated and transferred to GSK Biologicals for analysis of spatio-temporal 
clustering of cases. The individual GIS coordinates, which are considered as personal 
identifiable information (PII), will be maintained at local sites only. They will not be 
recorded in the eCRF and will not be transferred to GSK Biologicals.
The location of each study hospital/clinic will also be mapped.
Refer to the SPM for more information.
6.5.2.3.
Record medical history (including history of dengue illness, diagnosis 
of tuberculosis and JEV vaccination history)
Medical history (e.g., diabetes, cardiovascular diseases, asthma, cancers, haematologic 
diseases, genetic disorders), including history of dengue illness, tuberculosis, and JEV 
vaccination will be taken and results will be recorded in the eCRF.
A JEV vaccination record is preferable but self-reported history will be allowed if no 
vaccination record is available. 
6.5.2.4.
Subject card distribution
All subjects/subjects’ parent(s)/LAR(s) will be provided with the address and telephone 
number of the main contact for information about the study.
6.5.2.5.
Distribution of dengue kits and instruction of subjects/subject’s 
parent(s)/LAR(s) to contact study staff in case of acute febrile illness
Each household will be given at least one dengue kit, which includes one thermometer 
per subject, study contact information (phone numbers), and diary cards. 
Study personnel will instruct subject/subject’s parent(s)/LAR(s) to contact the study 
personnel or come to a designated study hospital/clinic for medical evaluation at the 
occurrence of fever (body temperature 38°C/100.4°F) on 2 consecutive calendar 
days, measured at least twice, at least 8 hours apart.
 
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Diary cards
Diary cards will be given to each of the subjects in the household to be used in the event 
of fever (body temperature 38°C/100.4°F). Diary cards will be distributed at the first 
scheduled visit. New diary cards will be issued by the study staff whenever necessary 
(i.e., if the subject no longer has one, e.g., the previous one was lost or used). 
The subject/subject’s parent(s)/LAR(s) will be instructed to start completing a diary card 
in the event of fever (body temperature 38°C/100.4°F). If necessary, assistance to 
complete the diary cards will be provided to the subject/subject’s parent(s)/LAR(s).
Body temperature and the intensity of each of the solicited symptoms should be recorded 
on the diary cards for the next 6 subsequent days (with Day 1 the day of the onset of 
fever and Day 7 the last day of recording solicited symptoms). After 7 days, only the end 
date for any ongoing solicited symptoms will be recorded on the diary cards. Please refer 
to Table 6 for the list of solicited symptoms to be recorded. Any medications (e.g., 
antipyretics) taken during this 7-day period will also be recorded. Two consecutive 
calendar days without fever, in the absence of antipyretic medication, are required to 
separate two episodes of fever. Symptoms prior to the onset of fever will not be recorded 
on the diary card.
The subject/subject’s parent(s)/LAR(s) will bring the diary card with him/her to each 
Suspected Dengue Visit. The investigator or appointed study personnel will review the 
diary card information with the subject and will record the information in the eCRF if a 
temperature of 38°C (100.4°F) was recorded on 2 consecutive calendar days, 
measured at least twice, at least 8 hours apart. The subject/subject’s parent(s)/LAR(s) will 
return the diary card when completed at the next visit or contact (i.e., at the next home 
visit or at a Suspected Dengue Visit).
Note: Diary cards can be filled in by a minor subject under the supervision of the 
subject’s parent(s)/LAR(s) provided that the minor has the competence to assess and 
report the information to be provided on the diary card. The ultimate accountability for 
the completion of the diary cards remains with the subject’s parent(s)/LAR(s). The 
investigator should discuss this accountability with the subject’s parent(s)/LAR(s).
Note: If the diary card has been filled in by a minor subject, the investigator or delegate 
should verify the reported information during a discussion with the minor subject 
preferably in the presence of his/her parent(s)/LAR(s).
6.5.2.6.
Recording of serious adverse events related to study participation

Refer to Section 7.2 for procedures for the investigator to record SAEs related to 
study participation. Refer to Section 7.3 for guidelines on how to submit SAE reports 
to GSK Biologicals.

The subjects/subjects’ parent(s)/LAR(s) will be instructed to contact the investigator 
immediately should the subject manifest any signs or symptoms they perceive as 
serious.
 
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6.5.3.
Procedures during scheduled weekly contacts
Weekly contacts will be home visits (at least every other week) or telephone contacts 
(TCs). The weekly contact may also be a face to face visit outside the home in certain 
cases (i.e. at hospital during suspected dengue visit) (Amended 01 March 2017)
During the weekly contacts, an adult household member enrolled in the study may 
respond for all enrolled members of the household. If no adult household member can be 
reached during the weekly contact (home visit or TC), 3 additional attempts to contact the 
household will be made on the 3 days following the initial attempted contact (see Section 
8.2).
6.5.3.1.
Distribution of dengue kits and instruction of subjects/subject’s 
parent(s)/LAR(s) to contact study staff in case of acute febrile illness
Each household will be given at least one dengue kit, which includes one thermometer 
per subject, study contact information (phone numbers), and diary cards. 

During the weekly contact (home visit or TC), study personnel will ask about AFI 
(fever [body temperature 38°C/100.4°F] on 2 consecutive calendar days,
measured at least twice, at least 8 hours apart) in the past week and will 
instruct/remind subject/subject’s parent(s)/LAR(s) to contact the study personnel or 
come to a designated study hospital/clinic for medical evaluation at the occurrence of 
AFI and solicited symptoms.
6.5.3.2.
Collection and verification of diary cards, and recording of diary cards 
data from diary cards to the eCRF

During the weekly contact (home visit or TC), a structured check list will be used to 
inquire about any AFI since the last contact (home visit or TC). Refer to the SPM for 
the structured check list. This will include inquiring about any hospitalisation that 
may have occurred and hospitalisation discharge diagnosis for all cases of 
hospitalisation. If necessary, assistance to complete the diary cards will be provided 
to the subject/subject’s parent(s)/LAR(s). If the subject is hospitalised at the study 
health care facility, information will be available via the hospital record. Information 
from both the diary card and hospital/clinic records can be used to update clinical 
data in the eCRF.

If a subject or subject’s parent/LAR reports fever (body temperature 
38°C/100.4°F) during any home visit or TC, the local study coordinator will 
arrange for a Suspected Dengue First Visit at the designated study hospital/clinic for 
medical evaluation and blood sampling (refer to Section 6.5.5.1). The Suspected 
Dengue First Visit should be scheduled within 7 days from the onset of fever 
(Days 2-7) and should ideally take place on the second day of fever (Day 2). If the 
visit has not taken place within the 7-day period (Days 2-7) from the onset of fever,
it is no longer required and the case will be considered as an undetermined case.

Collection and distribution of diary cards as needed (only applicable for home visits).

Recording of diary cards information by the investigator.
 
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6.5.3.3.
Recording of serious adverse events related to study participation

Refer to Section 7.2 for procedures for the investigator to record SAEs related to 
study participation. Refer to Section 7.3 for guidelines on how to submit SAE reports 
to GSK Biologicals.

The subjects/subjects’ parent(s)/LAR(s) will be instructed to contact the investigator 
immediately should the subject manifest any signs or symptoms they perceive as 
serious.
6.5.4.
Procedures at scheduled Visit 2
The close-out visit (Visit 2) will take place at home or at the hospital/clinic.
6.5.4.1.
Update medical and JEV vaccination history
Update medical history, including tuberculosis diagnosis and JEV vaccination.
6.5.4.2.
Collection and verification of diary cards, and recording of diary cards 
data from diary cards to the eCRF

Collection and recording of diary card if needed.

Recording of SAEs related to study participation.
6.5.4.3.
Recording of serious adverse events related to study participation
Refer to Section 6.5.2.6.
6.5.5.
Procedures at Suspected Dengue Visits
6.5.5.1.
Procedures at Suspected Dengue First Visit
6.5.5.1.1.
Important general remarks
All study subjects with AFI (fever [body temperature 38°C/100.4°F] on 
2 consecutive calendar days, measured at least twice, at least 8 hours apart) should be 
seen at a designated study hospital/clinic by the study physician. The Suspected Dengue 
First Visit should be scheduled within 7 days from the onset of fever (Days 2-7) and 
should ideally take place on the second day of fever (Day 2). 
 
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If a subject with fever (body temperature 38°C/100.4°F) on 2 consecutive calendar 
days, measured at least twice, at least 8 hours apart has not been evaluated at a Suspected 
Dengue First Visit (or directly in a hospital/clinic) within the 7-day period (Days 2-7) 
from the onset of fever, the Suspected Dengue First Visit is no longer required and the 
case will be considered as an undetermined case. If a diary card has been filled, the data 
needs to be recorded in the eCRF.
Blood samples for study procedures should not be drawn from subjects who attend a 
study healthcare facility on the first day of fever (body temperature 38°C/100.4°F), 
i.e., on Day 1. Medical assistance can be provided to these subjects but they will be asked 
to come back the following day should the fever persist. Blood samples for study 
procedures should only be drawn from the second day of fever (i.e., from Day 2, the 
second calendar day with fever) and onwards, up to Day 7.
Although subjects will be instructed to contact the study staff in the event of AFI (fever 
[body temperature 38°C/100.4°F] on 2 consecutive calendar days, measured at least 
twice, at least 8 hours apart), there may be cases where the subject is taken directly to a
hospital/clinic (study or non-study health care facility). If this occurs, a family member 
should inform the study staff as early as possible, and the study staff should contact the 
hospital/clinic where the subject is hospitalised. In addition, weekly contacts will also be 
used to identify subjects who have been hospitalised with AFI where study staff has not 
been informed. In such case, clinical data will be retrospectively collected in the eCRF. 
The study physician will be responsible for collecting the retrospective data and 
informing the local study coordinator for appropriate follow-up.
All subjects with AFI who fail to attend a Suspected Dengue Visit as they were directly 
hospitalised will be recorded as LCD, non-LCD, or undetermined cases depending on the 
availability and result of the dengue RT-qPCR test. Refer to the SPM for more 
information.
6.5.5.1.2.
Physical examination
A detailed physical examination to assess the subject’s general condition and any dengue 
associated clinical signs/symptoms as well as relevant clinical signs/symptoms will be 
performed (see Table 5). Collected information needs to be recorded in the “Suspected 
Dengue First Visit” section of the eCRF.
 
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Table 5
Physical examination during Suspected Dengue First Visit
Physical examination
(all subjects)

body temperature and route, 

height and weight, 

cardiac and respiratory rates, 

blood pressure,

dengue-like rash, 

abdominal tenderness

liver enlargement > 2 cm,

tourniquet test (positive/negative), only pertains to subjects 
≥5 years of age (Amended 01 March 2017)

haemorrhagic manifestations; petechiae, bleeding from mucosa, 
gastrointestinal tract, injection sites or other locations,

Injected pharynx,

clinical signs of plasma leakage: oedema, pleural effusion, ascites
Additional information
(subjects with severe disease 
and suspected Dengue Shock 
Syndrome [DSS] - according to 
investigator’s judgement)

narrow pulse pressure (< 30 mm Hg)

hypotension 

cold, clammy skin 

restlessness
Treatment of any condition observed during the physical examination will be performed 
according to local medical practice.
6.5.5.1.3.
Record medical history
Update medical history in the eCRF (in relation to what was encoded during the 
Suspected Dengue First Visit).
6.5.5.1.4.
Blood sampling
Blood samples should only be drawn in case of AFI (i.e., fever [body temperature 
38°C/100.4°F] on 2 consecutive calendar days, measured at least twice, at least 
8 hours apart) during the Suspected Dengue First Visit from the second day of fever (i.e., 
from Day 2, the second calendar day with fever) and onwards, up to Day 7.
The following blood samples should be collected unless an obvious alternative diagnosis, 
i.e., an obvious cause of fever other than dengue, has been identified and is documented
(Amended 01 March 2017):

Whole blood for laboratory confirmation of dengue by NS1-antigen (ICT test and/or 
ELISA): 0.5 mL, from all subjects (any age). The results of this/these test(s) should 
be recorded in the eCRF.

Whole blood for laboratory confirmation of dengue infection by (generic and/or
serotype-specific) RT-qPCR: 2.5 mL, from all subjects (any age).
 
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
Whole blood for haematology assessment (complete blood count): 2 mL, from all 
subjects ≥5 years of age and from subjects < 5 years of age only if clinically 
indicated, as per investigator judgement or local recommendations. The results of 
these tests should be recorded in the eCRF.

Whole blood for other tertiary (research) endpoints related tests (including, but not 
limited to, neutralising antibody against JEV and Chikungunya virus, tissue culture 
to isolate infectious agents, and sequencing to characterise infectious agents): 3.5 
mL, from all subjects ≥18 years of age. A subset of these samples will be tested.
6.5.5.1.5.
Collection and recording of solicited symptoms from diary card

During the Suspected Dengue First Visit, the diary card (partially completed) is 
reviewed by the physician and data recorded in the “solicited symptoms” section of 
the eCRF if the subject meets AFI criteria. Refer to the Section 6.6 for a description 
of the solicited symptoms to be recorded.

After the Suspected Dengue First Visit, the subject/subject’s parent(s)/LAR(s)
continues to complete the diary card until resolution of all symptoms. If necessary, 
assistance to complete the diary cards will be provided to the subject/subject’s 
parent(s)/LAR(s).

Once completed, the diary card is collected and the data are reviewed with the 
subject and encoded in the eCRF, either by the physician during a Return or 
Suspected Dengue Follow-up Visit at hospital/clinic or by the study staff during a 
home visit.
6.5.5.1.6.
Recording of serious adverse events related to study participation
Refer to Section 6.5.3.3.
6.5.5.2.
Procedures at Return Visit (optional)
Based on the physician’s assessment during the Suspected Dengue First Visit, a Return 
Visit may be needed and will be conducted as directed as per local medical practices. 
A Return Visit is a visit linked to the Suspected Dengue First Visit and should take place 
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. 

Update physical examination: medical data related to any suspected dengue case will 
be collected for hospitalised study subjects and for those followed in the outpatient 
setting at the Return Visit. Diagnostic and treatment intervention will be conducted 
as per local medical practices.

Verify diary cards and record any new data in the eCRF.

Record any SAEs related to study participation. Refer to Section 6.5.3.3.
 
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6.5.5.3.
Procedures at Suspected Dengue Follow-up Visit
All AFI cases need to be followed up until resolution of fever (body temperature 
38°C/100.4°F) and/or resolution of symptoms and signs are reported. The signs and 
symptoms should be recorded on the Suspected Dengue Follow-up (convalescent) Visit
page of the eCRF or on the weekly contact page (in case of home visit). This information 
may be collected during a home visit by medically trained member of the study
personnel.

Verify diary cards and record any new data in the eCRF. 

Record any SAEs related to study participation. Refer to Section 6.5.3.3.
6.5.6.
Study conclusion
The Study Conclusion screen in the eCRF will be completed at the last study contact. 
This last contact could occur at Visit 2 or at the last Suspected Dengue Follow-up Visit if 
dengue is suspected at Visit 2. 
The study staff will review data collected to ensure accuracy and completeness and will 
complete the Study Conclusion screen in the eCRF.
The sponsor may decide to continue to follow up subjects for a specified time period. 
This would be detailed in a protocol amendment and subject will be asked to sign a new 
ICF (and IAF if applicable).
If so, at the end of the study (study conclusion visit/contact), the investigator will ask 
each subject/subject’s parent(s)/LAR(s) if they are interested in participating/allowing the 
subject to participate in a long-term (follow-up) study. If a subject/subject’s 
parent(s)/LAR(s) is/are not interested in participating in the long-term study the reason 
for refusal will be documented in the subject’s eCRF.
6.6.
Solicited symptoms in case of acute febrile illness 
(suspected dengue infection)
Solicited symptoms will be collected in case of fever (body temperature 
38°C/100.4°F). The Diary card should be completed from the first day of fever.
 
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The following solicited symptoms will be recorded on the diary cards in case of fever:
Table 6
Solicited symptoms in case of suspected dengue infection
Signs and symptoms
body temperature and route * _ (minimum of two measurements)
headache / irritability **
eye pain ***
myalgia (muscle pain) ***
arthralgia (joint pain) ***
abdominal pain ***
nausea ***
vomiting 
rash
any bleeding (skin, mouth, anus)
loss of appetite 
Fatigue/decrease in normal activity **
reduced fluid intake **
* Refer to Table 10
** Wording intended for infants and toddlers.
*** When possible (depending on age in young children).
6.6.1.
Assessment of the intensity of solicited symptoms
The intensity of the following solicited symptoms will be assessed as described 
below:
Table 7
Intensity scales for solicited symptoms (excluding bleeding and 
reduced fluid intake, and body temperature)
Grade
0 (none)
=
No discomfort
1 (mild)
=
Any solicited symptom which is easily tolerated by the subject, causing minimal 
discomfort and not interfering with everyday activities.
2 (moderate) =
Any solicited symptom which is sufficiently discomforting to interfere with normal 
everyday activities.
3 (severe)
=
Any solicited symptom which prevents normal, everyday activities. (in a young child, 
such a solicited symptom would, for example, prevent attendance at 
school/kindergarten/day-care centre and would cause the parent(s)/LAR(s) to seek 
medical advice. In adults/adolescents, such a solicited symptom would, for example, 
prevent attendance at work/school and would necessitate the administration of 
corrective therapy.)
Table 8
Intensity scales for bleeding
Grade
0 (none)
=
No bleeding or bruising
1 (mild)
=
Small skin bleed or easy bruising
2 (moderate) =
Nose bleed or gum bleed not requiring medical attention
3 (severe)
=
Bleeding that requires medical attention
 
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Table 9
Intensity scales for reduced fluid intake
Grade
*
0 (none)
=
Normal feeding
1 (mild)
=
Drinks a little less than usual
2 (moderate) =
Does not drink well
3 (severe)
=
Lethargic, does not drink
* Wording intended for infants.
Table 10
Grading scale for body temperature
Grade
Temperature (°C)
Temperature (°F)
0
=
< 38.0
< 100.4
1
=
[38.0 - 38.4]
[100.4 – 101.2]
2
=
[38.5 - 38.9]
[101.3 – 102.0]
3
=
> 38.9
> 102.0
The preferred route for recording temperature in this study will be axillary for infants and 
younger children, and oral for older children and adults. For all age groups, tympanic 
measurements are not recommended, although they may be used for screening when 
followed up by another route of measurement.
Note: Ideally, body temperature should be measured twice a day. 
Any symptom that is assessed as Grade 3 (severe) should not be confused with an SAE. 
Grade 3 is a category used for rating the intensity of an event; and both symptoms and 
SAEs can be assessed as Grade 3.
6.7.
Biological sample handling and analysis
Biological samples will only be taken in case of AFI (i.e., fever [body temperature 
38°C/100.4°F] on 2 consecutive calendar days, measured at least twice, at least 
8 hours apart), during the Suspected Dengue First Visit.
Please refer to the SPM for details of biospecimen management (handling, storage and 
shipment).
Samples will not be labelled with information that directly identifies the subjects but will 
be coded with the identification number of the subject (subject number and Visit 
number). 

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.
 
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
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 (and assent if applicable) 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 except those described in the protocol or its 
amendment(s). 
Collected samples will be stored for a maximum of 20 years (counting from when the last 
subject performed the last study visit/ contact), unless local rules, regulations or 
guidelines require different timeframes or different procedures, which will then be in line 
with the subject consent (or assent when applicable). These extra requirements need to be 
communicated formally to and discussed and agreed with GSK Biologicals.
For routine sample collection, when a coded sample/aliquot is shipped to GSK 
Biologicals and needs to be linked to coded subject data, (re-) consent (and assent if 
applicable) from the subject/subject's parent(s)/LAR(s) and/or IRB/IEC approval will be 
sought.
6.7.1.
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 according-to-protocol (ATP) analysis (See Section 9.3 for the definition 
of study cohorts 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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6.7.2.
Biological samples
Table 11
Biological samples (Amended 01 March 2017)
Purpose
Collected 
sample 
type
QuantityUnit
Tested sample type
Sub-cohort
Time point
Haematology 
assessment
Whole 
blood
2
mL Whole blood
Suspected dengue 
infection
– all subjects 
≥5 years of age
– subjects 
< 5 years of age 
only if clinically 
indicated, as per 
investigator 
judgement
At each 
occurrence of 
AFI, i.e., each 
Suspected 
Dengue First 
Visit
Laboratory 
confirmation of 
dengue by RT-qPCR 
Whole 
blood
2.5
mL Serum*
Suspected 
dengue infection
– all subjects (any 
age)
Tertiary endpoint 
laboratory 
confirmation of 
dengue by NS1-
antigen ICT test 
and/or ELISA
Whole 
blood
0.5
mL Whole Blood/Serum*/Plasma*
Other tertiary 
(research) endpoint 
related tests (including, 
but not limited to, 
neutralising antibody 
against JEV)
Whole 
blood
3.5
mL Serum*
Suspected dengue 
infection
– all subjects 
≥18 years of age
At each 
occurrence of 
AFI, i.e., each 
Suspected 
Dengue First Visit
ELISA: enzyme-linked immunosorbent assay
ICT: immunochromatographic
JEV: Japanese encephalitis virus
NS1: non-structural protein 1
RT-qPCR: reverse transcriptase quantitative polymerase chain reaction
* Assumption: 1 mL of whole blood usually allows a yield of between 30% and 40% of serum/plasma.
 
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6.8.
Laboratory assays
Please refer to APPENDIX A for a detailed description of the assays performed in the 
study. Please refer to APPENDIX B for the address of the clinical laboratories used for 
sample analysis.
Table 12
Laboratory confirmation of dengue infection (Amended 01 March
2017)
System
Component *
Method
Kit/Manufacturer
Laboratory†
Whole Blood/Serum/
Plasma
NS1-antigen
ICT test
SD Bioline Dengue Duo
(or other manufacturer) **
Local
Serum
DENV RNA
RT-qPCR
Dengue Simplexa kit or 
comparable assay
Q²Solutions -Focus Diagnostics
or validated laboratory designated
by GSK Biologicals††
ELISA: enzyme-linked immunosorbent assay
ICT: immunochromatographic
NS1: non-structural protein 1
RT-qPCR: reverse transcriptase quantitative polymerase chain reaction
* Refer to the SPM and the central laboratory manual for more information. 
** GSK Biologicals recommends SD Bioline Dengue Duo.
† Refer to the APPENDIX B for the laboratory addresses.
†† GSK Biologicals laboratory refers to the Clinical Laboratory Sciences (CLS) in Rixensart, Belgium, Wavre, 
Belgium, or validated laboratory designated by GSK Biologicals.
Table 13
Haematology
System
Component
Method
Laboratory
Whole blood
Complete blood count (CBC)*
Per local standard practice
Local
* CBC: Complete blood count: white blood cells (WBC), red blood cells (RBC), platelets and haemoglobin.
Table 14
Other exploratory (tertiary, research) assays
System
Component *
Method
Kit/ManufacturerLaboratory
Serum Viral serology (e.g., JEV, Chikungunya virus)TBD
TBD
TBD **
Serum Isolation of infectious agents †
Tissue cultureNA
TBD **
Characterisation of infectious agents †
Sequencing
TBD
TBD **
AFI: acute febrile illness
JEV: Japanese encephalitis virus
TBD: to be determined
* A subset of samples will be tested.
† Additional potential exploratory tests (tertiary endpoints related tests) for the diagnosis of other common causes of 
AFI, such as malaria, typhoid, scrub typhus, influenza, viral hepatitis, etc. (Amended 01 March 2017)
** The testing laboratory(ies) that will perform the additional potential exploratory (tertiary, research) tests have not 
been identified yet.
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. The local clinical laboratories must also have a Quality 
System established and supported by procedures.
 
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7.
SAFETY
In this prospective cohort study no test product/vaccine will be given. Blood samples will 
be collected during each Suspected Dengue First Visit.
SAEs related to study participation will be recorded throughout the study. 
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) will be instructed to contact the investigator 
immediately should they/the subject manifest any signs or symptoms they perceive as 
serious.
7.1.
Safety definitions
7.1.1.
Definition of an adverse event
An AE is 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. 
7.1.2.
Definition of a serious adverse event
Only SAEs related to study participation will be recorded during this study.
An SAE is any untoward medical occurrence that:
a.
Results in death,
b.
Is life-threatening,
Note: 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,
Note: 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.
 
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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, 
Note: 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.
7.1.3.
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 related to study 
participation if they meet the definition of an SAE (refer to Section 7.1) and if deemed by 
the investigator to be causally related to study participation. 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, if 
deemed by the investigator to be causally related to study participation. However, 
clinically significant abnormal laboratory findings or other abnormal assessments that are 
associated with dengue will not be reported as SAEs but will be reported in Medical 
history section during Suspected Dengue Visits.
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.
 
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7.2.
Detecting and recording SAEs related to study participation
7.2.1.
Time periods for detecting and recording SAEs
The time period for collecting and recording SAEs related to study participation will 
begin at the time the subject consents (or assents when applicable) to participate in the 
study until he/she is discharged from the study. See Section 7.3 for instructions on 
reporting of SAEs related to study participation.
An overview of the protocol-required reporting periods for SAEs related to study 
participation is given in Table 15.
Table 15
Reporting periods for SAEs related to study participation
Study activity
Visit 1 *
(Month 0) 
Home visits/TCs/
any Suspected Dengue Visits
Visit 2
(Month 24)**
SAEs related to study participation
SAE: serious adverse event
TC: telephone contact
* i.e., at the time consent (or assent when applicable) is obtained. 
** Only one study conclusion will be conducted, either at Visit 2, or if a dengue case is ongoing at Visit 2, at the last 
Suspected Dengue Follow-up Visit for the dengue case.
7.2.2.
Evaluation of SAEs related to study participation
7.2.2.1.
Active questioning to detect SAEs
Each subject/subject’s parents/LAR(s) will be instructed to contact the investigator 
immediately should the subject manifest any signs and symptoms he/she 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) 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 related to study participation 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.
 
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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.
7.2.2.2.
Assessment of the intensity SAEs
The investigator will assess the maximum intensity that occurred over the duration of the 
event for all SAEs related to study participation 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/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.)
A symptom that is assessed as Grade 3 (severe) should not be confused with an SAE. 
Grade 3 is a category utilised for rating the intensity of an event; and both symptoms and 
SAEs can be assessed as Grade 3. An event is defined as 'serious' when it meets one of 
the pre-defined outcomes as described in Section 7.1.2.
7.2.2.3.
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 an SAE related to study participation 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. 
If an event meets the criteria to be considered as ‘serious’ (see Section 7.1), additional 
examinations/tests will be performed by the investigator in order to determine ALL 
possibly contributing factors to each SAE related to study participation.
 
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Possibly contributing factors include:

Medical history.

Concurrent medication.

Protocol required procedure.

Other procedure not required by the protocol.

Other cause (specify).
7.2.2.4.
Assessment of outcomes
The investigator will assess the outcome of all SAEs related to study participation 
recorded during the study as:

Recovered/resolved.

Recovering/resolving.

Not recovered/not resolved.

Recovered with sequelae/resolved with sequelae.

Fatal.
7.3.
Reporting of SAEs related to study participation
7.3.1.
Prompt reporting of SAEs related to study participation to GSK
SAEs related to study participation that occur in the time period defined in Section 7.2.1
will be reported promptly to GSK within the timeframes described in Table 16 once the 
investigator determines that the event meets the protocol definition of an SAE.
Table 16
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*
electronic Expedited 
Adverse Event Report
24 hours*
electronic Expedited Adverse 
Event Report
SAE: serious adverse event
* Timeframe allowed after receipt or awareness of the information.
** Timeframe allowed after the diagnosis is established and known to the investigator.
 
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7.3.2.
Contact information for reporting SAEs related to study 
participation to GSK 
Back-up Study Contact for Reporting SAEs related to study participation
24/24 hour and 7/7 day availability:
GSK Biologicals Clinical Safety & Pharmacovigilance
Fax: +
 or +
7.3.3.
Completion and transmission of reports of SAEs related to study 
participation to GSK
Once an investigator becomes aware that an SAE related to study participation 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.
7.3.3.1.
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. 
7.3.4.
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 16.
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7.3.5.
Regulatory reporting requirements for SAEs
The investigator will promptly report SAEs related to study participation to GSK 
Biologicals in accordance with the procedures detailed in Section 7.3.1. GSK Biologicals 
has a legal responsibility to promptly notify, as appropriate, both the local regulatory 
authority and other regulatory agencies. Prompt notification of SAEs related to study 
participation 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.
7.4.
Follow-up of SAEs related to study participation
7.4.1.
Follow-up of SAEs related to study participation
7.4.1.1.
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 16).
All SAEs related to study participation 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.
7.4.1.2.
Follow-up after the subject is discharged from the study
The investigator will follow-up subjects with SAEs related to study participation, 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.
8.
SUBJECT COMPLETION AND WITHDRAWAL
8.1.
Subject completion
A subject who returns for the concluding visit/is available for the concluding contact 
foreseen in the protocol is considered to have completed the study.
 
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8.2.
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 related to study 
participation until resolution of the event (see Section 7.4.1.2).
Withdrawals will not be replaced.
From an analysis perspective, a ‘withdrawal’ from the study refers to any subject who did 
not come back for the concluding visit/was not available for the concluding contact 
foreseen in the protocol.
All data collected until the date of withdrawal/last contact 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.
If no adult household member can be reached during a weekly contact (home visit or 
TC), 3 additional attempts to contact the household should be made on the 3 days 
following the initial attempted contact. If after a period of 4 weeks, the household can 
still not be contacted, its members will be considered as a lost to follow-up (see below). 
Each attempt to contact a household should be documented.
Investigators will make an attempt to contact those subjects who do not return for Visit 2 
or for Suspected Dengue Follow-up Visits.
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)/LAR(s), or by the investigator, as 
well as which of the following possible reasons was responsible for withdrawal:

SAE*

Protocol violation (specify)

Consent (or assent when applicable) withdrawal, not due to an SAE**

Moved from the study area

Lost to follow-up***

Other (specify)
* Subjects who are withdrawn from the study because of SAEs related to study 
participation 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 related to study participation until resolution of the event (see Section 7.4.1.2).
 
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**In case a subject is withdrawn from the study because he/she/the subject’s 
parent(s)/LAR(s) has withdrawn consent (or assent when applicable), the investigator 
will document the reason for withdrawal of consent (or assent when applicable), if 
specified by the subject/the subject’s parent(s)/LAR(s), in the eCRF.
*** Subjects who cannot be contacted (via home visits/TCs or visits at study 
hospital/clinic) during 4 consecutive weeks will be considered as lost to follow-up. 
Subjects lost to follow-up will only be replaced during the enrolment period.
8.3.
Extension study
The sponsor may decide to extend the study for a specified time period. This would be 
detailed in a protocol amendment and the subject will be asked to sign a new ICF (or IAF 
when applicable).
If so, at the end of the study (study conclusion visit/contact), the investigator will ask 
each subject/subject’s parent(s)/LAR(s) if they are interested in participating/allowing the 
subject to participate in future studies. If a subject/subject’s parent(s)/LAR(s) is/are not 
interested in participating in future studies the reason for refusal will be documented in 
the subject’s eCRF.
9.
STATISTICAL METHODS
9.1.
Endpoints
9.1.1.
Primary endpoint

Occurrence of AFI due to LCD. 
Please refer to Section 4 for the case definitions of AFI due to LCD.
9.1.2.
Secondary endpoints

Occurrence of AFI due to non-LCD. 

Occurrence and intensity of signs and symptoms of interest, during the 7-day period 
following the onset of each episode of AFI due to LCD and due to non-LCD. 

Occurrence of AFI due to LCD by DENV type, study site, and age group. 
Please refer to Section 4 for the case definitions of AFI due to LCD and due to non-LCD.
 
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9.1.3.
Tertiary (research) endpoints
During the Suspected Dengue First Visit, additional blood volumes will be collected for 
tertiary study endpoint assays. These tertiary study endpoints include, but are not limited 
to, determination of neutralising antibody titres against dengue virus (DENV), Japanese 
encephalitis virus (JEV), and chikungunya virus, tissue culture to isolate infectious 
agents, and sequencing to characterise infectious agents. (Amended 01 March 2017)
Tertiary study endpoint assays may be performed for a subset of samples (for example, 
JEV assays will only be conducted if additional studies indicate that JEV serology is 
needed to interpret the DENV antibody response), i.e., not all of these endpoint assays 
will be performed on all of the sera collected from adult study subjects. (Amended 01 
March 2017)
Research objectives may be assessed based on, but not limited to, the following 
endpoints: (Amended 01 March 2017)

Neutralising antibody titres against JEV. 

Neutralising antibody titres against other viruses, including but not limited to, 
Chikungunya virus. 

Incidence of NS1-antigen (ICT test and/or ELISA)-positive AFI by DENV type.

Concordance between dengue RT-qPCR and NS1-antigen (ICT and/or ELISA) 
assays. 

Occurrence of AFI due to LCD and due to non-LCD having (≥3 days of fever (body 
temperature (≥38°C/≥100.4°F). 

Occurrence of AFI due to LCD and due to non-LCD resulting in hospitalisation. 

Occurrence of combinations of signs and symptoms during the 7-day period 
following the onset of each episode of AFI due to LCD and due to non-LCD. 

Occurrence of AFI due to LCD within 2 weeks from an index case, in the same 
household or within 50 metres of the household of the index case. 

Entomological characteristics for LCD cases (e.g., vector species, density and 
number of breeding sites) at selected site(s). 

Occurrence (by medical history only) of a diagnosis of tuberculosis, from birth up to 
study conclusion, in the study population.

Occurrence of hospitalisation and discharge diagnosis. 

Identification and characterisation of infectious agents isolated from blood (e.g., 
DENV, influenza viruses, chikungunya viruses), in subjects with AFI. 
Please refer to Section 4 for the case definitions of AFI due to LCD and due to non-LCD.
 
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9.2.
Sample size considerations
A maximum of 2,000 subjects will be enrolled for a 2-year follow-up period. Assuming a 
drop-out rate of 10% the first year and a drop-out rate of 20% the second year, the final 
analysis will be done on approximately 3300 person-years overall.
In order to evaluate the precision with a sample size of 3,000 subjects, the 95% 
confidence interval (CI) (Poisson exact distribution and normal approximation of the 
Poisson distribution accounting for a design effect) for a range of expected incidences 
rates of AFI due to LCD were computed. The 95% CI for a range of expected incidences 
rates for DENV specific LCD were also presented (see Table 17).
(Amended 01 March 2017)
Estimating design effect and precision
The normal approximation of the Poisson distribution was used for calculating 95% CI 
for this cluster design. The variance was adjusted for a design effect of 2.6 to account for 
the between-cluster variability. The design effect measures the increase in the standard 
error of the estimate due to the sampling design used and is given by: D = 1 + (b – 1) rho, 
where rho is the rate of homogeneity (a measure of variability and equivalent to the 
“intra-cluster correlation”) and b is the average number of subjects sampled per 
household. Here we assumed b to be 5. Although, in theory rho can have a value up to 1, 
in practice values higher than 0.4 are uncommon [Bennett, 1991]. We used a conservative 
estimate of 0.4 for this study. The design effect is then estimated to 2.6.
Table 17
95% CI with design effect of 2.6
Number 
of cases
Number of 
person-
years
Incidence 
of LCD 
(per 100 
person-
years)
95% CI Poisson 
exact
95% CI Normal 
approx with design 
effect of 2.6
LL
UL
LL
UL
Overall
10
3300
0.30
[
0.15
;
0.56
]
[
0.00
;
0.61
]
20
3300
0.61
[
0.37
;
0.94
]
[
0.18
;
1.03
]
30
3300
0.91
[
0.61
;
1.30
]
[
0.38
;
1.43
]
40
3300
1.21
[
0.87
;
1.65
]
[
0.61
;
1.82
]
50
3300
1.52
[
1.12
;
2.00
]
[
0.84
;
2.19
]
60
3300
1.82
[
1.39
;
2.34
]
[
1.08
;
2.56
]
DENV 
specific
4
3300
0.12
[
0.03
;
0.31
]
[
0.00
;
0.31
]
8
3300
0.24
[
0.10
;
0.48
]
[
0.00
;
0.51
]
12
3300
0.36
[
0.19
;
0.64
]
[
0.03
;
0.70
]
16
3300
0.48
[
0.28
;
0.79
]
[
0.10
;
0.87
]
(Amended 01 March 2017)
 
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CI: confidence interval
DENV: dengue virus
LCD: laboratory Confirmed Dengue
LL lower limit; 
UL: upper limit
At time of analysis, the design effect of this study will be estimated using the following 
formula [Bennett, 1991]:
With
Where, c is the number of clusters, 
and 
are the number of person-years and the 
number of cases, respectively in the 
cluster, n is the total number of person-years and 
p is the overall proportion
.
The intra-cluster correlation ρ may then be estimated as:
9.3.
Cohorts for analyses
All the analyses of objectives and of demographics will be performed on the total cohort. 
The Total cohort will include all subjects enrolled in the study. 
9.4.
Derived and transformed data

An AFI case will be defined as:

LCD, if fever (body temperature 38°C/100.4°F) on 2 consecutive 
calendar days is measured at least twice, at least 8 hours apart and if dengue 
RT-qPCR result on a blood sample taken during the 7-day period (Days 2-7) 
from the onset of fever is positive.

Non-LCD, if fever (body temperature 38°C/100.4°F) on 2 consecutive 
calendar days is measured at least twice, at least 8 hours apart and if dengue 
RT-qPCR result on a blood sample taken during the 7-day period (Days 2-7) 
from the onset of fever is negative.

Undetermined, if fever (body temperature 38°C/100.4°F) on 
2 consecutive calendar days is measured at least twice, at least 8 hours 
apart but is not evaluated within the 7-day period (Days 2-7) from the onset
 
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of fever and/or if dengue RT-qPCR result on a blood sample taken during the 
7-day period (Days 2-7) from the onset of fever is invalid or missing.

DENV-1 LCD, if fever (body temperature 38°C/100.4°F) on 
2 consecutive calendar days is measured at least twice, at least 8 hours 
apart in diary card and if positive result for DENV-1 by serotype-specific 
RT-qPCR on a blood sample taken within the 7-day period (Days 2-7) from 
the onset of fever is positive.

DENV-2 LCD, if fever (body temperature 38°C/100.4°F) on 
2 consecutive calendar days is measured at least twice, at least 8 hours 
apart in diary card and if positive result for DENV-2 by serotype-specific 
RT-qPCR on a blood sample taken within the 7-day period (Days 2-7) from 
the onset of fever is positive.

DENV-3 LCD, if fever (body temperature 38°C/100.4°F) on 
2 consecutive calendar days is measured at least twice, at least 8 hours 
apart in diary card and if positive result for DENV-3 by serotype-specific 
RT-qPCR on a blood sample taken within the 7-day period (Days 2-7) from 
the onset of fever is positive.

DENV-4 LCD, if fever (body temperature 38°C/100.4°F) on 
2 consecutive calendar days is measured at least twice, at least 8 hours 
apart in diary card and if positive result for DENV-4 by serotype-specific 
RT-qPCR on a blood sample taken within the 7-day period (Days 2-7) from 
the onset of fever is positive.

Age in the study will be computed as the difference between the date of enrolment 
(i.e., date of ICF [and IAF if applicable]) and the date of birth. The age will be 
expressed in years.

The subject age at time of AFI will be computed as the difference between the date 
of the onset of fever (body temperature 38°C/100.4°F) completed on diary card 
and the date of birth.

The follow-up time for estimation of incidence of AFI due to LCD, will be 
computed, for each subject, as the difference between the date of first occurrence of 
AFI due to LCD (by serotype) or the date of last contact if no LCD occurred (i.e., 
last weekly Suspected Dengue Follow-up or last Visit) and the date of enrolment.

The follow-up time for estimation of incidence of AFI due to non-LCD, will be 
computed, for each subject, as the difference between the date of first occurrence of 
AFI due to non-LCD or the date of last contact if any (i.e., last weekly Suspected 
Dengue Follow-up or last Visit) and the date of enrolment.

The follow-up time for estimation of incidence of AFI due to LCD by DENV-1 type, 
will be computed, for each subject, as the difference between the date of first 
occurrence of AFI due to DENV-1 LCD or date of last contact if any (i.e., last 
weekly Suspected Dengue Follow-up or last Visit) and the date of enrolment. The 
same computations will be done for DENV-2-3 and 4 specific cases.
 
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
The total person-years at risk will be computed as the sum of the follow-up times in 
year for each individual.

The number of consecutive calendar days with fever (body temperature 
38°C/100.4°F) used for definition of cases will be computed as the number of 
consecutive calendar days with fever during the 7-day period following the onset of 
fever.

The duration of symptoms will be computed as the number of days with grade 1, 2 or 
3 symptoms during the 7 days following the onset of fever.

For the analysis of solicited symptom, missing or non-evaluable measurements will 
not be replaced. Therefore the analysis of the solicited symptoms will include only 
episodes of AFI with documented safety data (i.e., diary card completed).

The geometric mean titre (GMT) calculations are performed by taking the anti-log of 
the mean of the log10 titre transformations. Antibody titres below the cut-off of the
assay will be given an arbitrary value of half the cut-off for the purpose of GMT 
calculation. For a given subject and a given immunogenicity measurement, missing 
or non-evaluable measurements will not be replaced. Therefore, an analysis will 
exclude subjects with missing or non-evaluable measurements.
9.5.
Analysis of demographics / baseline characteristics
Socio-demographic and patient characteristics (e.g., age at study enrolment, gender, 
household conditions, medical history and vaccination history) will be summarised 
overall and for LCD cases and by site using descriptive statistics.
9.6.
Analysis of primary and secondary objectives
The following analyses will be performed:

Computation of incidence rate of AFI due to LCD and due to non-LCD with 95% CI: 
the numerator will be the number of subjects with AFI due to LCD and due to non-
LCD during the study period. The denominator will be the total person-years at risk.

Description of signs and symptoms of AFI due to LCD and due to non-LCD will 
include the percentage of AFI presenting each sign or symptom (any intensity and 
grade 3) during the 7-day period from the onset of fever (body temperature 
38°C/100.4°F). Duration of signs and symptoms will also be described for LCD 
and non-LCD episodes. Analysis will be done overall and then separately for 
episodes of AFI occurring in subjects < 5 years of age, in subjects 5 to 11 years of 
age, and in subjects 12 years of age.

Computation of incidence rates of AFI due to LCD with 95% CI by type (DENV 1-
4), study site and age group (6 months to 4 years, 5 years-11 years, 12 years to 
17 years, 18 years): the numerator will be the number of subjects with AFI due to 
LCD in the category. The denominator will be the total person-years at risk in the 
category.
 
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9.7.
Analysis of tertiary (research) objectives
Some tertiary (research) analyses may be done including:
(Amended 01 March 2017)

Neutralising antibody titres against JEV: the seropositivity rates to JEV with exact 
95% CI and GMTs with 95% CI and range of antibody titres will be tabulated.

A concordance analysis between dengue virus detection by RT-qPCR and by NS1-
antigen (ICT test) may be performed. This analysis may be stratified by day of 
sampling if sufficient data are available. Sensitivity, specificity, positive predictive 
value and negative predictive value of NS1-antigen (ICT test) in prediction of RT-
qPCR test results will be computed. (Amended 01 March 2017)

Computation of incidence rate of AFI due to dengue confirmed by NS1-antigen (ICT 
test and/or ELISA) with 95% CI: the numerator will be the number of subjects with 
AFI due to dengue confirmed by NS1-antigen (ICT test and/or ELISA) during the 
study period. The denominator will be the total person-years at risk.

Evaluation of different case definitions by:

Computation of incidence rates of AFI due to LCD having 3 consecutive calendar 
days of fever (body temperature 38°C/100.4°F) or incidence of AFI due to LCD 
resulting in hospitalisation with 95% CI.

Descriptive analysis of the signs and symptoms associated with AFI due to LCD 
having 3 consecutive calendar days of fever (body temperature 38°C/100.4°F)
or resulting in hospitalisation.

Multivariate analyses could be done to identify categories of medically relevant signs 
and symptoms associated with high probability of LCD (any severity, having 
3 consecutive calendar days of fever (body temperature 38°C/100.4°F) and 
resulting in hospitalisation) versus non-LCD episodes using logistic regressions 
and/or classification and regression tree analysis to provide support for the definition 
of a moderate-to-severe dengue case for future clinical endpoint trials. Same 
analyses may be done to identify category of signs and symptoms associated with 
high probability of LCD having 3 consecutive calendar days of fever (body 
temperature 38°C/100.4°F) or resulting to hospitalisation.

Values of signs and symptoms in predicting LCD in AFI will be computed 
(sensitivity, specificity, positive predictive value and negative predictive value). 
Same computations may be done in predicting LCD having 3 consecutive calendar 
days of fever (body temperature 38°C/100.4°F) or resulting to hospitalisation.

Descriptive analysis of the spatio-temporal clustering of cases using a map of study 
households. Occurrence of AFI due to LCD within 2 weeks from an index case, in 
the same household or within 50 metres of the household of the index case will be 
analysed.

Descriptive analysis of incidence and reason for hospitalisation in the study 
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
Description of entomological characteristics such as vector species, vector density 
and breeding sites at selected site(s).
9.8.
Analysis of SAEs related to study participation
The verbatim reports of SAEs related to study participation will be reviewed by a 
physician and the signs and symptoms will be coded according to the Medical Dictionary 
for Regulatory Activities (MedDRA). Every verbatim term will be matched with the 
appropriate PT. SAEs related to study participation reported during the entire study 
period will be described in individual data listings.
9.9.
Interpretation of analyses
There are no confirmatory objectives with a pre-defined success criterion.
The exploratory (tertiary, research) analyses are not controlled for Type 1 error.
9.10.
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.
9.10.1.
Sequence of analyses
The final analysis will be performed when all prospective data have been collected and 
cleaned. A final study report containing data from the entire study will be written at this 
time.
(Amended 01 March 2017)
10.
ADMINISTRATIVE MATTERS
To comply with ICH GCP or other applicable guidelines administrative obligations 
relating to data collection, monitoring, archiving data, audits, confidentiality, ownership 
and publications must be met.
10.1.
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.
10.2.
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 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.
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.
In accordance with applicable regulations, GCP and GSK procedures, GSK monitors will 
contact the site prior to the start of the study to review with the site staff the protocol, 
study requirements, and their responsibilities to satisfy regulatory, ethical, and GSK 
requirements. When reviewing data collection procedures, the discussion will also 
include identification, agreement and documentation of data items for which the eCRF 
entries will serve as the source document.
 
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10.3.
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. 
10.4.
Quality assurance
To ensure compliance with GCP and all applicable regulatory requirements, GSK may 
conduct a quality assurance audit. Regulatory agencies may also conduct a regulatory 
inspection of this study. Such audits/inspections can occur at any time during or after 
completion of the study. If an audit or inspection occurs, the investigator and institution 
agree to allow the auditor/inspector direct access to all relevant documents and to allocate 
his/her time and the time of his/her staff to the auditor/inspector to discuss findings and 
any relevant issues.
10.5.
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.
Interventional 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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10.6.
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.
COUNTRY SPECIFIC REQUIREMENTS
Not applicable.
 
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12.
REFERENCES
(Amended 01 March 2017)
Bennett S, Woods T, Liyanage WM, et al. A simplified general method for cluster-
sample surveys of health in developing countries. World Health Stat Q. 1991;44:98-106.
Bhatt S, Gething P, Brady O, et al. The global distribution and burden of dengue. Nature. 
2013;496:504-7.
Epidemiology Unit, Ministry of Health. Distribution of Notification (H399) Dengue 
Cases by Month;
http://www.epid.gov.lk/web/index.php?option=com_casesanddeaths&Itemid=448&lang=
en# (last accessed 28 February 2017).
Guzman MG, Halstead SB, Artsob H, et al. Dengue: a continuing global threat. Nat Rev 
Microbiol. 2010;8:S7-S16.
Kyle JL, Harris E. Global spread and persistence of dengue. Annu Rev Microbiol.
2008;62:71-92.
Government of India. National Vector Borne Disease Control Programme (NVBDCP). 
Directorate General of Health Services, Ministry of Health and Family Welfare. Dengue 
Cases and Deaths in the Country since 2007; http://nvbdcp.gov.in/den-cd.html (last 
accessed 18 February 2014).
Sasmono RT, Aryati A, Wardhani P, et al. Performance of Simplexa dengue molecular 
assay compared to conventional and SYBR Green RT-PCR for detection of dengue 
infection in Indonesia. PLoS One. 2014;9:e103815 
Tam CC, Tissera H, de Silva AM, et al. Estimates of dengue force of infection in children 
in Colombo, Sri Lanka. PLoS Negl Trop Dis. 2013;7:e2259.
Tissera H, Amarasinghe A, De Silva AD, et al. Burden of dengue infection and disease in 
a pediatric cohort in urban Sri Lanka. Am J Trop Med Hyg. 2014;91:132-7. 
WHO (World Health Organization). Dengue: Guidelines for Diagnosis, Treatment, 
Prevention and Control -- New Edition. A joint publication of the World Health 
Organization (WHO) and the Special Programme for Research and Training in Tropical 
Diseases (TDR) WHO/HTM/NTD/DEN/2009.1 Geneva:2009.
 
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APPENDIX A
LABORATORY ASSAYS
NS1-antigen ICT test 
The Dengue NS1 ICT test will be a commercial individual test for the qualitative 
detection of Dengue virus NS1 antigen in human serum, plasma or on whole blood. 
Performance characteristics of the assay are described in the manufacturer package insert.
NS1-antigen ELISA
The NS1 antigen in serum or plasma samples will be detected by using a commercial 
assay. Performance characteristics of the assay are described in the manufacturer package 
insert.
Dengue RT-qPCR assay 
The detection of the dengue virus in human serum is done by reverse transcription of 
viral RNA, followed by amplification and quantification of the resultant cDNA using a 
real-time RT-qPCR assay, such as the Focus Diagnostics Simplexa™ Dengue RT-PCR 
assay. 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. Performance 
characteristics of the assay are described in the manufacturer package insert and in a 
recent publication [Sasmono, 2014].
(Amended 01 March 2017)
Neutralising antibody against JEV and Chikungunya virus 
To be determined. A testing laboratory has not been identified yet. 
Virus isolation and characterisation
To be determined. A testing laboratory has not been identified yet. 
 
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APPENDIX B
CLINICAL LABORATORIES
Table 18
GSK Biologicals’ laboratories
Laboratory
Address
GSK Biologicals Global Vaccine Clinical Laboratory, 
Rixensart
Biospecimen Reception - B7/44
Rue de l'Institut, 89 - B-1330 Rixensart – Belgium
GSK Biologicals Global Vaccine Clinical Laboratory, 
Wavre-Nord Noir Epine
Avenue Fleming, 20 - B-1300 Wavre – Belgium
Table 19
Outsourced laboratories
Laboratory
Address
Focus (US)
Q²Solutions -Focus Diagnostics, Inc.
(d/b/a Quest Diagnostics)
33608 Ortega Highway 
San Juan Capistrano,
CA 92675-2042
USA
(Amended 01 March 2017)
 
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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
200274 (DPIV-021 EXPLO)
Amendment number:
Amendment 1
Amendment date:
16 December 2014
Co-ordinating author:
, 4Clinics Belgium, contractor for GSK 
Biologicals
Rationale/background for changes: 
Dengue virus RNA and NS1 detection assays will be performed for all suspected 
dengue cases. During the same study visit and the same procedure, additional blood 
volumes will be collected for tertiary study endpoints: 
!
for dengue serology (sites in India only since seroprevalence is well defined for 
sites in Sri Lanka), at the first Suspected Dengue First Visit only, from all subjects 
≥5 years of age, and
!
for tertiary study endpoint assays other than dengue serology* (in both India and 
Sri Lanka), at each Suspected Dengue First Visit, from adult study subjects only.
* including, but not limited to, determination of neutralising antibody titres against 
Japanese encephalitis virus (JEV), and chikungunya virus, tissue culture to isolate 
infectious agents, and sequencing to characterise infectious agents.
All of the dengue serology specimens collected from children aged 5 to 17 years will 
be tested for dengue neutralising antibody titres. In addition, sera collected from adult 
study subjects who tested positive for dengue by RT-qPCR, and up to three matched 
controls, will be tested for dengue serology. For tertiary study endpoints other than 
dengue serology, only a subset of samples will be tested. The current amendment 
clarifies that all of the endpoint assays will be performed on all of the sera collected 
from children while not all of the endpoint assays will be performed on all of the sera 
collected from adult study subjects.
Clarification was added regarding laboratory assay methodologies and responsible 
laboratories.
The listing of contributing authors has been updated and typographical errors have 
been corrected.
Amended text has been included in bold italics and deleted text in strikethrough in 
the following sections:
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Cover page
Contributing authors 
!
, Project Statistician
!
, Study Delivery Manager
!
, Clinical Laboratories 
Representative
Synopsis
Rationale for 
the study
This study aims to estimate the burden of dengue illness in selected 
sites in India and Sri Lanka South Asia and to prepare sites for the 
conduct of future vaccine efficacy trials. 
Scientific 
Objectives
Tertiary (Research)
!
To describe DENV and Japanese Encephalitis Virus (JEV) antibody 
profiles in a subset of subjects with AFI.
!
To assess the concordance between dengue Reverse transcriptase-
quantitative Polymerase Chain Reaction (RT-qPCR) and Non-
Structural protein 1 (NS1)-antigen Enzyme-Linked Immunosorbent 
Assay (ELISA) and/or ImmunoChromaTographic (ICT) test in the 
diagnosis assessment of dengue infection.
!
To isolate and characterise infectious agents from a subset of 
subjects with AFI.
!
To evaluate the antibody response to infectious agents in a subset 
of subjects with AFI.
Endpoints
Tertiary (Research)
During the Suspected Dengue First Visit, additional blood volumes
will be collected for tertiary study endpoint assays. These tertiary 
study endpoints include, but are not limited to, determination of
neutralising antibody titres against dengue virus (DENV), 
Japanese encephalitis virus (JEV), and chikungunya virus, tissue 
culture to isolate infectious agents, and sequencing to characterise 
infectious agents.
Dengue serology (sites in India only since seroprevalence is well 
defined for sites in Sri Lanka) will be performed on samples 
collected at the first Suspected Dengue First Visit only, from all 
children aged 5 to 17 years, as well as from adult study subjects 
who tested positive for Dengue RT-qPCR and up to three matched 
controls. Matching will be done by study site, age and gender.
Tertiary study endpoint assays other than dengue serology will be 
performed for a subset of samples (for example, JEV assays will 
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only be conducted if additional studies indicate that JEV serology 
is needed to interpret the DENV antibody response), i.e., not all of 
these endpoint assays will be performed on all of the sera collected 
from adult study subjects.
Research objectives may be assessed based on, but not limited to, 
the following endpoints:

Neutralising antibody titres against other viruses, including 
but not limited to, Chikungunya virus.
Section 1.1 Background
In 2012, the Government of India convened a meeting of dengue experts to address the 
growing burden of disease and to form a task force on dengue, indicating a growing sense 
of urgency and increasing awareness of the importance of this disease. In Sri Lanka, 
dengue surveillance and training of doctors in the management of dengue illness is a top 
priority of the Ministry of Health, especially because the rate of severe disease in 
Sri Lanka is one of the highest national rates reported. DENV infection and disease 
incidence in children in Colombo, Sri Lanka, have been reported to exceed 8% and 3% 
per year, respectively [Tissera, 2014].
Section 1.2 Rationale for the study
This study aims to estimate the burden of dengue illness in selected sites in India and 
Sri Lanka South Asia and to prepare sites for the conduct of future vaccine efficacy trials. 
Section 2.3 Tertiary (research) objectives

To describe DENV and Japanese Encephalitis Virus (JEV) antibody profiles in a 
subset of subjects with AFI.

To assess the concordance between dengue Reverse transcriptase-quantitative 
Polymerase Chain Reaction (RT-qPCR) and Non-Structural protein 1 (NS1)-antigen 
Enzyme-Linked Immunosorbent Assay (ELISA) and/or ImmunoChromaTographic 
(ICT) test in the diagnosis assessment of dengue infection.

To isolate and characterise infectious agents from a subset of subjects with AFI.

To evaluate the antibody response to infectious agents in a subset of subjects with 
AFI.
Section 5.1 Number of subjects/centres
One or more (preferably all eligible) members of each household (preferably all eligible 
household members) will be enrolled.
 
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Section 6.3 General study aspects
Supplementary study conduct information not mandated to be present in this protocol is 
provided in the accompanying Study Procedures Manual (SPM and central laboratory 
manual).
Section 6.4 Outline of study procedures
Table 3
List of study procedures for Suspected Dengue Visits
Blood sampling*
   for dDengue infection diagnosis by NS1-antigen ELISA and/or ICT test (0.5 mL)
   for dDengue infection diagnosis by (generic and/or serotype-specific) RT-qPCR (2.5 mL)
   for dengue infection diagnosis by anti-DENV 1-4 µPRN, microneutralisation or comparable assay serology (4 
mL) 3
   for other tertiary (research) endpoints related tests (4 mL) 5
* Blood samples should only be drawn in case of AFI (i.e., fever [body temperature 38°C/100.4°F] on 
2 consecutive calendar days measured at least 12 hours apart) during the Suspected Dengue First Visit from 
the second day of fever (i.e., from Day 2, the second calendar day with fever) and onwards, up to Day 7.
3) For subjects ≥5 years of age only at the first Suspected Dengue First Visit only and in India only.
5) For subjects ≥18 years of age only. Including, but not limited to, neutralising antibody against JEV and 
Chikungunya virus. A subset of samples will be tested.
Section 6.5.5.1.4 Blood sampling
Blood samples should only be drawn in case of AFI (i.e., fever [body temperature 
38°C/100.4°F] on 2 consecutive calendar days measured at least 12 hours apart) 
during the Suspected Dengue First Visit from the second day of fever (i.e., from Day 2, 
the second calendar day with fever) and onwards, up to Day 7.
The following blood samples should be collected unless an obvious alternative diagnosis, 
i.e., an obvious cause of fever other than dengue, has been identified and is documented:

Whole blood for laboratory confirmation of dengue infection diagnosis by NS1-
antigen (ELISA and/or ICT test): 0.5 mL, from all subjects (any age). The results of 
this/these test(s) should be recorded in the eCRF.

Whole blood for laboratory confirmation of dengue infection diagnosis by (generic 
and/or serotype-specific) RT-qPCR: 2.5 mL, from all subjects (any age).

Whole blood for haematology assessment (complete blood count): 2 mL, from all 
subjects ≥5 years of age and from subjects < 5 years of age only if clinically 
indicated, as per investigator judgement or local recommendations. The results of 
these tests should be recorded in the eCRF.

Whole blood for dengue serology infection by anti-DENV 1-4 µPRN, 
microneutralisation or comparable assay: (India only) 4 mL, from all subjects
children aged ≥5 to 17 years of age, as well as from adult study subjects who tested
positive for Dengue RT-qPCR and up to three matched controls.

Whole blood for other tertiary (research) endpoints related tests (including, but not 
limited to, neutralising antibody against JEV and Chikungunya virus, tissue culture 
to isolate infectious agents, and sequencing to characterise infectious agents): 
4 mL, from all subjects ≥18 years of age. A subset of these samples will be tested.
 
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Section 6.7.2 Biological samples
Table 11
Biological samples
Purpose
Time point
Laboratory confirmation of Ddengue infection diagnosis by 
NS1-antigen ELISA and/or ICT test
At each occurrence of AFI, i.e., each Suspected 
Dengue First Visit
Laboratory confirmation of Ddengue infection diagnosis by 
(generic and/or serotype-specific) RT-qPCR
At each occurrence of AFI, i.e., each Suspected 
Dengue First Visit
Haematology assessment
At each occurrence of AFI, i.e., each Suspected 
Dengue First Visit
Dengue serology infection diagnosis by anti-DENV 1-4 
µPRN, microneutralisation or comparable assay *
Only at the first occurrence of AFI, i.e., during 
the first Suspected Dengue First Visit only
Tertiary (research) endpoint related tests (including, but not 
limited to, neutralising antibody against JEV) **
At each occurrence of AFI, i.e., each Suspected 
Dengue First Visit
* For subjects ≥5 years of age only at the first Suspected Dengue First Visit only and in India only.
** For subjects ≥18 years of age only.
Section 6.8 Laboratory assays
Table 12
Diagnosis Laboratory confirmation of dengue infection
System
Component 1
Method
Kit/Manufacturer
Laboratory*
Whole Blood/Serum/PlasmaNS1-antigen
ICT test
SD Bioline Dengue 
Duo Bio-Rad (or other 
manufacturer) 2
Local
Serum
DENV RNA
Generic RT-qPCR 2 and/or 
serotype-specific RT-qPCR 3
Dengue Simplexa kit o
comparable assay In-
house
Fiocruz and/or GSK 
Biologicals** and/or 
Local Quest and/or 
Focus
Serum
Neutralising 
antibodies 
against DENV 
1-4
Micro-plaque-reduction 
neutralisation (µPRN), 
microneutralisation or 
comparable assay 3
In-house
Fiocruz and/or GSK 
Biologicals**
1) Refer to the SPM and the central laboratory manual for more information. Generic RT-qPCR will be run at 
Fiocruz and/or GSK Biologicals but may not be used in Local lab.
2) As per local practices. GSK Biologicals recommends SD Bioline Dengue DuoDENGUE NS1 AG STRIP, Bio-Rad.
3) If serotype-specific testing is done at Fiocruz and/or GSK Biologicals, only samples tested positive with dengue 
generic RT-qPCR will be tested on serotype-specific RT-qPCR. If testing done locally, serotype-specific RT-qPCR 
can be used only. Dengue serology (India only) will be performed on samples collected at the first 
Suspected Dengue First Visit only, from all children aged 5 to 17 years, as well as from adult study 
subjects who tested positive for Dengue RT-qPCR and up to three matched controls. Matching will be 
done by study site, age and gender.
** GSK Biologicals laboratory refers to the Global Vaccines Clinical Laboratories (GVCL) in Rixensart, Belgium; Wavre, 
Belgium; Laval, Canada, or validated laboratory designated by GSK Biologicals.
Table 14
Other exploratory (tertiary, research) assays
System
Component
Method
Kit/ManufacturerLaboratory
Serum Viral serology (e.g., Neutralising antibodies against JEV, 
Chikungunya virus)
TBD
TBD In-house
TBD **
Serum Characterisation of infectious agents †
Sequencing of PCR 
product
TBD
TBD **
* A subset of samples will be tested.
** The laboratory(ies) that will perform the additional potential exploratory (tertiary, research) tests is/are not yet 
identified and will be defined as soon as available.
 
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The GSK Biologicals’ and Fiocruz clinical laboratories have established a Quality 
System supported by procedures.
Section 9.1.3 Tertiary (research) endpoints
During the Suspected Dengue First Visit, additional blood volumes will be collected for 
tertiary study endpoint assays. These tertiary study endpoints include, but are not 
limited to, determination of neutralising antibody titres against dengue virus (DENV), 
Japanese encephalitis virus (JEV), and chikungunya virus, tissue culture to isolate 
infectious agents, and sequencing to characterise infectious agents.
Dengue serology (sites in India only since seroprevalence is well defined for sites in Sri 
Lanka) will be performed on samples collected at the first Suspected Dengue First Visit 
only, from all children aged 5 to 17 years, as well as from adult study subjects who 
tested positive for Dengue RT-qPCR and up to three matched controls. Matching will 
be done by study site, age and gender.
Tertiary study endpoint assays other than dengue serology will be performed for a 
subset of samples (for example, JEV assays will only be conducted if additional studies 
indicate that JEV serology is needed to interpret the DENV antibody response), i.e., not 
all of these endpoint assays will be performed on all of the sera collected from adult 
study subjects.
Research objectives may be assessed based on, but not limited to, the following 
endpoints:

Neutralising antibody titres against other viruses, including but not limited to, 
Chikungunya virus.
Section 9.10.2 Statistical considerations for interim analyses
At time of interim analysis, all primary and secondary endpoints will be assessed to 
monitor the quality of data collected. The statistical report will then include the incidence 
of AFI due to LCD and due to non-LCD overall, by DENV type, study site and age group 
as well as a description of signs and symptoms of AFI due to LCD and due to non-LCD 
cases.
Section 12 References
Tissera H, Amarasinghe A, De Silva AD, et al. Burden of dengue infection and disease 
in a pediatric cohort in urban Sri Lanka. Am J Trop Med Hyg. 2014;91:132-7.
Section APPENDIX A. LABORATORY ASSAYS
Dengue generic RT-qPCR
The detection of the dengue virus in human serum is done by the amplification and 
quantification of its genetic material using a real time RT-qPCR assay.
Viral RNA is extracted from serum samples and reverse-transcribed into cDNA. The 
resulting cDNA is detected amplified and quantified by real time qPCR in one 
amplification reaction using specific generic primers and fluorogenic probes selected 
from 3’ untranslated highly conserved region of targeting the dengue viral genome.
 
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Dengue serotype-specific RT-qPCR
The detection and typing of the dengue virus 1,2,3 and 4 in human serum is done by 
the amplification of each serotype RNA using a real time RT-qPCR assay. 
The dengue serotype-specific RT-qPCR assay has been developed at GSK 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 amplified
by real time qPCR using type-specific DNA primers and fluorogenic probes targeting
the dengue viral genome DENV capsid gene. PCR is performed as 2 duplex 
amplification (DENV-1/-3 and DENV-2/-4).
Neutralising antibody against JEV and Chikungunya virus
Section APPENDIX B. CLINICAL LABORATORIES
Table 19
Outsourced laboratories
Laboratory
Address
Fiocruz
Laboratório de Tecnologia Virológica, Bio-Manguinhos
Rio de Janeiro
QUEST (India)
Quest Diagnostics Private Limited
A-17 Info City, Sector 34
Gurgaon-122001 Haryana
INDIA
Focus (US)
Focus Diagnostics, Inc.
(d/b/a Quest Diagnostics)
33608 Ortega Highway 
San Juan Capistrano,
CA 92675-2042
USA
 
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GlaxoSmithKline Biologicals
Vaccine Value & Health Science (VVHS)
Protocol Amendment 2
eTrack study number 
and Abbreviated Title
200274 (DPIV-021 EXPLO)
Amendment number:
Amendment 2
Amendment date:
02 April 2015
Co-ordinating author:
, 4Clinics Belgium, contractor for GSK 
Biologicals
Rationale/background for changes: 
The location where the scheduled enrolment visit (Visit 1) and the scheduled close-out 
visit (Visit 2) will take place has been extended, i.e., both visits will take place either at 
home or at the hospital/clinic, in accordance with local ethics committee requirements 
and/or national laws or regulations.
The testing schedule of dengue serology in adults has been aligned to the one of 
children 5 to 17 years of age, i.e., all subjects 5 years of age and above will be tested 
for dengue serology (in India only) on samples collected at the first Suspected Dengue 
First Visit.
The definition of acute febrile illness has been further clarified.
Detailed description of the quantification process of the dengue viral genome has been 
provided.
Typographical errors have been corrected and table legends reformatted
Copyright 2014-2015 of the GlaxoSmithKline group of companies. All rights reserved.
Synopsis
Scientific 
Objectives
Tertiary (Research)
!
To describe DENV and Japanese Encephalitis Virus (JEV) antibody 
profiles in a subsets of subjects with AFI.
!
To assess the concordance between dengue Reverse transcriptase-
quantitative Polymerase Chain Reaction (RT-qPCR) and Non-
Structural protein 1 (NS1)-antigen ImmunoChromaTographic 
(ICT) test and/or Enzyme-Linked Immunosorbent Assay (ELISA) 
and/or ImmunoChromaTographic (ICT) test in the assessment of 
dengue infection.
All occurrences of "NS1-antigen ELISA and/or ICT test" have been updated to "NS1-
antigen ICT test and/or ELISA": as the ICT test is the preferred test, and the ELISA is an 
optional test. Changes have been implemented in the following sections: synopsis 
(scientific objectives, endpoints), 2.3, 6.4 (Table 3), 6.5.5.1.4, 6.7 (Table 11), 
6.8 (Table 12), 9.1.3, and 9.7. The order of presentation of both assays has also been 
modified in Appendix A.

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Study 
design

Study visits: The study will consist of 2 scheduled visits and (the 
enrolment visit [Visit 1], weekly contacts, and the close-out visit 
[Visit 2]) and, in case of AFI*, unscheduled Suspected Dengue 
Visits:

Visit 1 and Visit 2 will take place at home or at the 
hospital/clinic, in accordance with local ethics committee
requirements and/or national laws or regulations

Weekly contacts will be i.e., home visits (at least every other 
week) or telephone contacts (TCs) over a period of 
approximately two years per subject.

In addition, study subjects will be asked to attend
Unscheduled Suspected Dengue Visits will take place in 
case of AFI*
*AFI is defined as if fever (body temperature 38°C/100.4°F) is recorded or 
reported on 2 consecutive calendar days, and measured at least twice, at least 
812 hours apart.
The definition of AFI has been updated accordingly throughout the whole document. 
Changes have been implemented in the following sections: synopsis (study design/study 
visits, case definitions), 3, 4 (Figure 2), 4.1, 4.2, 4.3, 6.4 (Table 3), 6.5.2.5, 6.5.3.1, 
6.5.5.1.1, 6.5.5.1.4, 6.7, and 9.4, 
Scientific 
Objectives
Tertiary (Research)
Dengue serology (sites in India only since seroprevalence is well defined 
for sites in the Sri Lanka site) will be performed on samples collected at 
the first Suspected Dengue First Visit only, from all subjects children 
aged 5 to 17 years of age and above, as well as from adult study subjects 
who tested positive for Dengue RT-qPCR and up to three matched 
controls. Matching will be done by study site, age and gender. 
Endpoints
Tertiary (Research)

Occurrence (by medical history only) of a Confirmation of 
diagnosis of tuberculosis, from birth up to study conclusion, in the 
study population.
Section 2.3 Tertiary (research) objectives

To describe DENV and Japanese Encephalitis Virus (JEV) antibody profiles in a
subsets of subjects with AFI. 
 
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Section 3 Study design overview
Figure 1 Study design overview
Scheduled Visits 
- Visit 1 and Visit 2 will take place at 
home or at the hospital/clinic 
and
Weekly contacts: home 
visits/Telephone contacts 
(TCs)
- Weekly contacts will be home visits 
(at least every other week) or 
telephone contacts (TCs)
!
Study visits: The study will consist of 2 scheduled visits and (the enrolment visit 
[Visit 1], weekly contacts, and the close-out visit [Visit 2]) and, in case of AFI*, 
unscheduled Suspected Dengue Visits:
∀
Visit 1 and Visit 2 will take place at home or at the hospital/clinic, in 
accordance with local ethics committee requirements and/or national laws 
or regulations
∀
Weekly contacts will be i.e., home visits (at least every other week) or 
telephone contacts (TCs) over a period of approximately two years per 
subject.
∀
In addition, study subjects will be asked to attend Unscheduled Suspected 
Dengue Visits will take place in case of AFI*
*AFI is defined as if fever (body temperature # 38°C/# 100.4°F) is recorded or reported on 
# 2 consecutive calendar days, and measured at least twice, at least 128 hours apart.
Section 4 Case definition 
Section 4.1 Acute febrile illness due to LCD
ALL of the following findings must be met for an AFI due to LCD (see also Figure 2):
Section 4.2 Acute febrile illness due to non-LCD
ALL of the following findings must be met for an AFI due to non-LCD (see also
Figure 2)
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Figure 2 Schematic overview of different cases
AFI: Acute febrile illness; LCD: Laboratory Confirmed Dengue; NS1-antigen test: Non-
Structural protein 1 antigen test by ELISA or ICT; RT-qPCR: reverse transcriptase 
quantitative polymerase chain reaction
Section 6.1 Regulatory and ethical considerations, including the 
informed consent process (and assent process if applicable)
The consenting process will be video recorded if required by law. If the subject/subject’s 
parent(s)/LAR(s) are illiterate, the ICF (and IAF if applicable) will be countersigned by 
an impartial witness. Informed consent/assent can be obtained at the study clinic or 
during a home visit. In either case, medically trained and GCP trained study staff will 
follow an SOP for the consenting/assenting process.
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Section 6.4 Outline of study procedures 
Table 2 List of study procedures for scheduled visits and contacts
Epoch
Epoch 001
Visit/contact
Visit 1
Home visits or 
TCs
Visit 2
Time point
Month 0
Weekly
between visits 1
Month 24
Record data (solicited symptoms, medication) from diary cards (if applicable)
●6
●
5) The subject/subject’s parent(s)/LAR(s) will bring the diary card with him/her to each Suspected Dengue Visit (see 
Table 3). The investigator or the appointed study personnel will record the diary card information in the eCRF if a 
temperature of 38°C (100.4°F) was recorded on 2 consecutive calendar days, measured at least 
twice, at least 8 hours apart.. The subject/subject’s parent(s)/LAR(s) will return the diary card when completed 
at the next visit or contact (i.e., at the next home visit or at a Suspected Dengue Visit). 
Table 3 List of study procedures for unscheduled Suspected Dengue Visits
6) The subject/subject’s parent(s)/LAR(s) will bring the diary card with him/her to each Suspected Dengue Visit. The 
investigator or appointed study personnel will record the diary card information in the eCRF if a temperature of 
38°C (100.4°F) was recorded on 2 consecutive calendar days, measured at least twice, at least 
8 hours apart. The subject/subject’s parent(s)/LAR(s) will return the diary card when completed at the next 
scheduled visit or contact (i.e., at the next home visit or at a Suspected Dengue Visit).
Section 6.5.1 Procedures at scheduled Visit 1 prior to enrolment
The enrolment visit (Visit 1) will take place at home or at the hospital/clinic.
Section 6.5.2.2 Record socio-demographic data
Socio-demographic data such as date of birth, gender, education, occupation and socio-
economic status and household/living conditions will be recorded in the eCRF.
Section 6.5.2.5 Distribution of dengue kits and instruction of 
subjects/subject’s parent(s)/LAR(s) to contact study staff in case of 
acute febrile illness
Diary cards
The subject/subject’s parent(s)/LAR(s) will bring the diary card with him/her to each 
Suspected Dengue Visit. The investigator or appointed study personnel will review the 
diary card information with the subject and will record the information in the eCRF if a 
temperature of 38°C (100.4°F) was recorded on 2 consecutive calendar days, 
measured at least twice, at least 8 hours apart.
Section 6.5.3 Procedures at scheduled weekly contacts
Weekly contacts will be home visits (at least every other week) or telephone contacts 
(TCs).
Section 6.5.4 Procedures at scheduled Visit 2
The close-out visit (Visit 2) will take place at home or at the hospital/clinic.
 
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Section 6.5.5.1.1 Important gGeneral remarks
Blood samples for study procedures should not be drawn from subjects who attend a 
study healthcare facility on the first day of fever (body temperature 38°C/100.4°F), 
i.e., on Day 1. Medical assistance can be provided to these subjects but they will be asked 
to come back the following day should the fever persist.
Section 6.5.5.1.2 Physical examination
Table 5 Physical examination during Suspected Dengue First Visit
Additional information
(subjects with severe disease 
and suspected Dengue Shock 
Syndrome [DSS] - according to 
investigator’s judgement)

narrow pulse pressure (< 20 30 mm Hg)

hypotension 

cold, clammy skin 

restlessness
Section 6.5.5.1.4 Blood sampling

Whole blood for dengue serology by anti-DENV 1-4 µPRN, microneutralisation or 
comparable assay: (India only) 4 mL collected at the first Suspected Dengue First 
Visit only, from all subjects children aged 5 to 17 years of age and above, as well as 
from adult study subjects who tested positive for Dengue RT-qPCR and up to three 
matched controls. 
Section 6.5.5.1.5 Collection and recording of solicited symptoms from 
diary card
1.
During the Suspected Dengue First Visit, the diary card (partially completed) is 
reviewed by the physician and data recorded in the “solicited symptoms” section of 
the eCRF if the subject meets AFI criteria.
Section 6.6 Solicited symptoms in case of acute febrile illness (suspected 
dengue infection)
Table 6 Solicited symptoms in case of suspected dengue infection
Signs and symptoms
abdominal pain ***
*** When possible (depending on age in young children).
Section 6.6.1 Assessment of the intensity of solicited symptoms
Note: Ideally, body temperature should be measured twice a day. Should additional 
temperature measurements be performed at other times of day, the highest temperature 
will be recorded in the eCRF.
 
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Section 6.7.2 Biological samples 
Table 11 Biological samples
Purpose
Collected 
sample 
type
QuantityUnit
Tested sample type
Sub-cohort
Time point
Laboratory confirmation 
of dengue by NS1-
antigen ELISA and/or ICT 
test 
Whole 
blood
0.5
mL Whole Blood/Serum/Plasma
Suspected 
dengue infection
– all subjects 
(any age)
At each 
occurrence of 
AFI, i.e., each 
Suspected 
Dengue First Visit
Laboratory confirmation 
of dengue by RT-qPCR 
Whole 
blood
2.5
mL Serum
At each 
occurrence of 
AFI, i.e., each 
Suspected 
Dengue First Visit
Haematology 
assessment
Whole 
blood
2
mL Whole blood
Suspected 
dengue infection
– all subjects 
≥5 years of age
– subjects 
< 5 years of age 
only if clinically 
indicated, as per 
investigator 
judgement
At each 
occurrence of 
AFI, i.e., each 
Suspected 
Dengue First 
Visit
Laboratory 
confirmation of dengue 
by RT-qPCR 
Whole 
blood
2.5
mL Serum*
Suspected 
dengue 
infection
– all subjects 
(any age)
Tertiary endpoint 
Laboratory 
confirmation of dengue 
by NS1-antigen ICT test 
and/or ELISA 
Whole 
blood
0.5
mL Whole Blood/Serum*/Plasma*
Tertiary endpoint 
Dengue serology by anti-
DENV 1-4 µPRN, 
microneutralisation or 
comparable assay
Whole 
blood
4
mL Serum*
Suspected 
dengue infection
– all subjects 
≥5 years of age 
in India only
Only at the first 
occurrence of 
AFI, i.e., during 
the first 
Suspected 
Dengue First Visit 
only
Other tertiary (research) 
endpoint related tests 
(including, but not limited 
to, neutralising antibody 
against JEV)
Whole 
blood
4
mL Serum *
Suspected 
dengue infection
– all subjects 
≥18 years of age
At each 
occurrence of 
AFI, i.e., each 
Suspected 
Dengue First Visit
* For subjects ≥5 years of age only at the first Suspected Dengue First Visit only and in India only.
** For subjects ≥18 years of age only. 
* Assumption: 1 mL of whole blood usually allows a yield of between 30% and 40% of serum/plasma.
 
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Section 6.8 Laboratory assays 
Table 12 Laboratory confirmation of dengue infection
System
Component 1*
Method
Kit/Manufacturer
Laboratory*†
Whole Blood/Serum/PlasmaNS1-antigen
ICT test
SD Bioline Dengue 
Duo (or other 
manufacturer) 2**
Local
ELISA
PLATELIA™ Dengue 
NS1 Ag (or other 
manufacturer)
Local
Serum
DENV RNA
RT-qPCR
Dengue Simplexa kit o
comparable assay
Quest and/or Focus
or validated 
laboratory 
designated by GSK 
Biologicals
Serum
Neutralising 
antibodies 
against DENV 1-
4
Micro-plaque-reduction 
neutralisation (µPRN), 
microneutralisation or 
comparable assay 3***
In-house
GSK Biologicals **††
* 1) Refer to the SPM and the central laboratory manual for more information. 
** 2) GSK Biologicals recommends SD Bioline Dengue Duo. 
*** 3) Dengue serology (India only) will be performed on samples collected at the first Suspected Dengue First Visit 
only, from all subjects children aged 5 to 17 years of age and above, as well as from adult study subjects who 
tested positive for Dengue RT-qPCR and up to three matched controls. Matching will be done by study site, age 
and gender.
† * Refer to the APPENDIX B for the laboratory addresses.
†† ** GSK Biologicals laboratory refers to the Global Vaccines Clinical Laboratories (GVCL) in Rixensart, Belgium;,
Wavre, Belgium;, Laval, Canada, or validated laboratory designated by GSK Biologicals.
Table 14 Other exploratory (tertiary, research) assays
** The testing laboratory(ies) that will perform the additional potential exploratory (tertiary, research) tests is/ (are) not 
yet identified and will be defined as soon as available have not been identified yet.
Section 9.1.3 Tertiary (research) endpoints
Dengue serology (sites in India only since seroprevalence is well defined for sites in the
Sri Lanka site) will be performed on samples collected at the first Suspected Dengue First 
Visit only, from all subjects children aged 5 to 17 years of age and above, as well as 
from adult study subjects who tested positive for Dengue RT-qPCR and up to three 
matched controls. Matching will be done by study site, age and gender.

Occurrence (by medical history only) of a Confirmation of diagnosis of 
tuberculosis, from birth up to study conclusion, in the study population.
Section 12. REFERENCES
Sasmono RT, Aryati A, Wardhani P, et al. Performance of Simplexa dengue molecular 
assay compared to conventional and SYBR Green RT-PCR for detection of dengue 
infection in Indonesia. PLoS One. 2014;9:e103815
 
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Section APPENDIX A. LABORATORY ASSAYS
NS1-antigen ICT test
The Dengue NS1 ICT test will be a commercial individual test for the qualitative 
detection of Dengue virus NS1 antigen in human serum, plasma or on whole blood. 
Performance characteristics of the assay are described in the manufacturer package 
insert.
NS1-antigen ELISA
The NS1 antigen in serum or plasma samples will be detected by using a commercial CE 
labelled assay. Performance characteristics of the assay are described in the manufacturer 
package insert.
NS1-antigen ICT test
The Dengue NS1 ICT test will be a commercial CE labelled individual test for the 
qualitative detection of Dengue virus NS1 antigen in human serum, plasma or on whole 
blood. Performance characteristics of the assay are described in the manufacturer package 
insert.
Dengue generic RT-qPCR assay
The detection of the dengue virus in human serum is done by reverse transcription of 
viral RNA, followed by the amplification and quantification of its genetic material the 
resultant cDNA using a real-time RT-qPCR assay, such as the Focus Diagnostics 
Simplexa™ Dengue RT-PCR assay. Viral RNA is extracted from serum samples and 
reverse-transcribed into cDNA. The resulting cDNA is amplified and quantified by real 
time qPCR using generic primers, and fluorogenic probes targeting the dengue viral 
genome.
Dengue serotype-specific RT-qPCR 
The detection and typing of the dengue virus 1,2,3 and 4 in human serum is done by the 
amplification of each serotype RNA using a real time RT-qPCR assay. 
Viral RNA is extracted from serum samples and reverse-transcribed into cDNA. The 
resulting cDNA is amplified by real time qPCR using type-specific primers targeting and 
fluorogenic probes targeting the dengue viral genome.
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. 
Performance characteristics of the assay are described in the manufacturer package 
insert and in a recent publication [Sasmono, 2014].
 
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Neutralising antibody against JEV and Chikungunya virus 
To be determined. A testing laboratory has not been identified yet.
Virus isolation and characterisation
To be determined. A testing laboratory has not been identified yet.
Section APPENDIX B. CLINICAL LABORATORIES
Table 18 GSK Biologicals' laboratories
Laboratory
Address
GSK Biologicals Global Vaccine Clinical Laboratory, 
North America- Laval
Biospecimen Reception - Clinical Serology
525 Cartier blvd West - Laval - Quebec – Canada - H7V 3S8
 
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GlaxoSmithKline Biologicals
Vaccine Value & Health Science (VVHS)
Protocol Amendment 3
eTrack study number 
and Abbreviated Title
200274 (DPIV-021 EXPLO)
Amendment number:
Amendment 3
Amendment date:
01 March 2017
Co-ordinating author:
, contractor for GSK Biologicals
Rationale/background for changes: 
After study enrolment was initiated, the 3 sites in India were still delayed due to the 
inability to export the blood samples for dengue testing. Following Sponsor review, the 
India portion of the study was canceled due to logistical difficulties.  Therefore, sites in 
India will no longer be included this study protocol.  Among the 5000 subjects planned 
in the study, 3000 were to come from India. The planned number of study subjects 
changed from 5000 to 2000 and all subjects will be enrolled from Sri Lanka. 
Sample size calculations were adjusted accordingly.
Added text to clarify that an unscheduled visit (either for first, return, and follow-up 
dengue visits) will be considered as the weekly contact for the week in which it occurs. 
Title A cohort study to determine the incidence of dengue fever and to build capacity 
for dengue vaccine trials in dengue-endemic regions of South Asia.
Detailed Title
A prospective, multi-centre, cohort study to determine the incidence 
of acute febrile dengue illness and to build capacity for dengue vaccine clinical endpoint 
trials in South Asian communities.
Co-ordinating authors
!
 (4Clinics Belgium for GSK Biologicals) / 
 / 
 
/
 (XPE Belgium for GSK Biologicals
Contributing authors
!
, Clinical and Epidemiology Project Leader 
!
, Senior Epidemiologist
!
 Project Statistician
!
, 
, Study Delivery Lead
!
, 
, Clinical Safety Representative Physician
!
, 
, Study Data Manager
!
, Clinical Laboratories Representative Clinical Laboratory 
Science Clinical Readout Team Leader

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PPD
PPD
PPD
PPD
PPD
PPD
PPD
PPD
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, Global Regulatory Leader
!
, Global Vaccines Clinical Laboratories Clinical Laboratory 
Sciences (CLS) Study Manager
!
, CLS GVCL Study Manager
!
, Oversight Data Manager
Synopsis:
Detailed Title: A prospective, multi-centre, cohort study to determine the incidence of 
acute febrile dengue illness and to build capacity for dengue vaccine clinical endpoint 
trials in South Asian communities
Tertiary Research Objective: To describe neutralising antibody titre profiles in a subset 
of LCD and non-LCD subjects.
!
Study design: Type of design Prospective, multi-centre, community-based, cohort 
study, household-sampling, single country multi-country
!
Study population: approximately 2,000 5,000 subjects ...
!
Unscheduled Suspected Dengue Visits will take place in case of AFI*
Note that an unscheduled visit (either for first, return, or follow-up dengue visits) 
will be considered as the weekly contact for the week in which it occurs. 
Synopsis Table 1 Study groups and epochs foreseen in the study
Study Groups
Number of subjects
Prospective
~2000 ≈2000
Discussion of study design: This prospective multiple-site cohort study including 
children aged less than 18 years and adults aged up to 50 years...
Number of subjects: Approximately 5,000 2000 
Endpoint: Tertiary (Research) 
Dengue serology (sites in India only since seroprevalence is well defined for the Sri 
Lanka site) will be performed on samples collected at the first Suspected Dengue First 
Visit only, from all subjects 5 years of age and above. (Amended 01 March 2017)
Introduction, Background:
Deleted text regarding India
Globally, 70% of the estimated 96 million apparent dengue infections are thought to 
occur in Asia. India alone accounts for approximately 34% (33 million [22-44∃
infections) of the global total. Current national and global estimations of the disease 
burden of dengue in India are considered uncertain. Underreporting and misdiagnoses are 
major obstacles to understanding the full burden, and active surveillance studies in 
prospective cohorts are thought to be needed to estimate the burden of the disease more 
accurately [Gupta, 2006; Kakkar, 2012; Gupta, 2013∃. Dengue fever became a notifiable 
disease in 2005, and the Government of India has established a network of Sentinel 
Surveillance Hospitals linked to referral Laboratories. In 2006, India’s National Vector 
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Control Program reported an incidence of 1.2 cases/100,000 people per year and a case 
fatality rate of 1.5%. This incidence rate is much lower than the expected rate reported 
from other dengue endemic regions, and most reported cases in India lack laboratory 
confirmation [Kanungo, 2012, indicating that there is a pressing need for active 
surveillance studies. Even though solid incidence rate estimates for dengue fever are not 
available for India, surveillance data indicate that dengue is a substantial contributor to 
febrile illness and that all 4 dengue virus serotypes co-circulate in large parts of India 
[Bharaj, 2008; Chakravarti, 2012. A recent study in Kolkata found that 8% of all acute 
febrile illness (AFI) cases were due to dengue [Kanungo, 2012. The number of reported 
cases has increased considerably over the past decade and hyper-endemicity seems to 
contribute to increased disease severity. 
The reported age-distribution of dengue in India suggests that dengue illness is most 
common in young adults and teenagers [Chakravarti, 2012, with a tendency for more 
severe disease in younger people. In contrast, sero-surveillance data from Colombo, 
Sri Lanka, indicate that over 70% of children experienced at least 1 dengue infection by 
the age of 12 years, and the median age at infection there is 4.7 years. The risk of primary 
infection in children below 12 years of age was 14% per year [Tam, 2013. In 2012, the 
reported dengue fever incidence cases per 100,000 people were 200, 20 and 4 for 
Sri Lanka, the state of Tamil Nadu, and all of India, respectively [NVBDCP, 2014; 
Epidemiology Unit, 2014and reports of dengue outbreaks and fatal cases frequently 
made headline news in South Asia. 
In 2012, the Government of India convened a meeting of dengue experts to address the 
growing burden of disease and to form a task force on dengue, indicating a growing 
sense of urgency and increasing awareness of the importance of this disease.
2.3 Tertiary objectives

To describe neutralising antibody titre profiles in a subset of LCD and non-LCD 
subjects.
3 Study Design Overview
Figure1 Study design overview 
Added footnote: 
Notes: 
The weekly contact may be a face to face visit outside the home in certain cases (i.e. at 
hospital during suspected dengue visit)
An unscheduled visit (either for first, return, or follow-up dengue visit) will be 
considered as the weekly contact for the week in which it occurs.
Type of design: Prospective, multi-centre, community-based, cohort study, household-
sampling, single country.

Study population: approximately 5,000 2000 subjects aged 
 
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Study Visits 
Weekly contacts will be home visits (at least every other week) or telephone contacts 
(TCs) The weekly contact may be a face to face visit outside the home in certain cases 
(i.e. at hospital during suspected dengue visit)
Table1 Study groups and epochs foreseen in the study
Study Groups Number of subjects
Age (Min/Max)
Epoch 001
Prospective
≈2000 5000
6
6 months to 
50 years
x
3.1 Discussion of study design
This prospective, multi-center cohort study including children aged less than 18 years and 
adults aged up to 50 years is designed to support the selection and definition of 
clinical.....
5.1 Number of subjects / centres
The target is to enrol approximately 2,000 5,000 eligible subjects in order to reach 
approximately 3300 8,000 person-years evaluable overall at the time of final analysis. 
5.1.1. Selection of communities and households
A minimum of 500 subjects will be recruited per site.
5.1.2. Overview of the recruitment plan
Each site will have approximately 4 months from global study start to reach the 
minimum target of 500 subjects. Thereafter e Enrolment will be competitive continue 
until a total of approximately 2,000 5,000 subjects are recruited. 
6.4.
Outline of study procedures
Table 2
List of study procedures for scheduled visits and contacts 
Table footnote text added: 1) Weekly contacts will be home visits (at least every other week) or TCs. The weekly 
contact may be a face to face visit outside the home in certain cases (i.e. at hospital during suspected 
dengue visit) 
Deleted amendment reference after row entitled “record data” and moved notes regarding 
abbreviations to occur adjacent to other notes in order to have table appear on a single 
page
 
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Table 3 List of study procedures for unscheduled Suspected Dengue Visits 
Epoch
Epoch 001
Visit
Suspected Dengue 
First Visit
Return Visit 1
Suspected Dengue 
Follow-up Visit 2
Time point
Day 2 to Day 7, ideally 
on Day 2 (1 day after 
the onset of fever)
1 to 6 days 
after 
Suspected 
Dengue First 
Visit
7 to13 days after 
Suspected Dengue 
First Visit
Blood sampling*
   Dengue NS1-antigen ICT test and/or ELISA 
(0.5 mL) (Amended 01 March 2017)
●
   Dengue RT-qPCR (2.5 mL)
●
   DENV 1-4 serology (4 mL) 3
●
   for haematology (2 mL) 4 3
●
   for other tertiary (research) endpoints related tests 
(~4 3.5 mL) 5 4
●
Safety follow-up:
µPRN: micro-plaque-reduction neutralisation; DENV: dengue virus; eCRF: electronic Case Report Form; 
2) A Suspected Dengue Follow-up Visit may occur during a weekly home visit. This visit should be performed by a 
medically trained site staff member. Note that when a subject comes in for a suspected dengue follow-up 
visit, this will replace the regularly scheduled weekly home visit.
3) For subjects ≥5 years of age only at the first Suspected Dengue First Visit only and in India only. 
4 3) For all subjects ≥5 years of age. For subjects < 5 years of age only if clinically indicated, as per investigator 
judgement. Complete blood count (White blood cells [WBC], red blood cells [RBC], platelets and haemoglobin).
5 4) For subjects ≥18 years of age only. Including, but not limited to, neutralising antibody against JEV and ....
6 5) The subject/subject’s parent(s)/LAR(s) will bring the diary card with him/her to each Suspected Dengue ....
7 6) A new diary card will be issued by the study staff whenever necessary (i.e., if the subject no longer ... 
8 7) Only one study conclusion will be conducted, either at Visit 2, or if a suspected dengue case is....
6.5.3 Procedures during scheduled weekly contacts (Amended 01 March 2017)
Weekly contacts will be home visits (at least every other week) or telephone contacts 
(TCs). (Amended 02 April 2015). The weekly contact may also be a face to face visit
outside the home in certain cases (i.e. at hospital during suspected dengue visit)
Table 5 Physical examination during Suspected Dengue First Visit
Physical examination
(all subjects)

tourniquet test (positive/negative), only pertains to subjects 
≥5 years of age (Amended 01 March 2017)

haemorrhagic manifestations; petechiae, bleeding from mucosa, 
gastrointestinal tract, injection sites or other locations,

Injected pharynx,

clinical signs of plasma leakage: oedema, pleural effusion, ascites
 
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6.7.2  Biological samples
Table 11 Biological samples 
Purpose
Collected 
sample 
type
QuantityUnit
Tested sample type
Sub-cohort
Time point
Laboratory confirmation 
of dengue by RT-qPCR 
Whole 
blood
2.5
mL Serum*
Suspected 
dengue 
infection
– all subjects 
(any age)
Tertiary endpoint 
laboratory confirmation 
of dengue by NS1-
antigen ICT test and/or 
ELISA
Whole 
blood
0.5
mL Whole Blood/Serum*/Plasma*
Tertiary endpoint Dengue 
serology * by anti-DENV 1-
4 µPRN, 
microneutralisation or 
comparable assay 
Whole 
blood
4
mL Serum*
Suspected 
dengue 
infection
– all subjects 
≥5 years of 
age in India 
only
Only at the first 
occurrence of 
AFI, i.e., during 
the first 
Suspected 
Dengue First Visit 
only
Other tertiary (research) 
endpoint related tests 
(including, but not limited 
to, neutralising antibody 
against JEV) **
Whole 
blood
4  3.5
mL Serum*
Suspected 
dengue 
infection
– all subjects 
≥18 years of 
age
At each 
occurrence of 
AFI, i.e., each 
Suspected 
Dengue First Visit
µPRN: micro-plaque-reduction neutralisation
DENV: dengue virus
6.8 Laboratory Assays
Table 12 Laboratory confirmation of dengue infection 
System
Component *
Method
Kit/Manufacturer
Laboratory†
Whole Blood/Serum/PlasmaNS1-antigen
ICT test
SD Bioline Dengue 
Duo (or other 
manufacturer) **
Local
ELISA
PLATELIA™ Dengue 
NS1 Ag (or other 
manufacturer)
Local
Serum
DENV RNA
RT-qPCR
Dengue Simplexa kit o
comparable assay
Quest and/or Focus
Q²Solutions -Focus 
Diagnostics or 
validated laboratory 
designated by GSK 
Biologicals††
Serum
Neutralising 
antibodies 
against DENV 
1-4
Micro-plaque-reduction 
neutralisation (µPRN), 
microneutralisation or 
comparable assay ***
In-house
GSK Biologicals††
DENV: dengue virus
†† GSK Biologicals laboratory refers to the Global Vaccines Clinical Laboratories (GVCL) Clinical Laboratory 
Sciences (CLS) in Rixensart, Belgium, Wavre, Belgium, or validated laboratory designated by GSK Biologicals.
 
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Table 14
Other exploratory (tertiary, research) assays
System
Component *
Method
Kit/ManufacturerLaboratory
Serum Isolation of infectious agents †
Tissue cultureNA
TBD **
Characterisation of infectious agents †Sequencing
TBD
TBD **
† Additional potential exploratory tests (tertiary endpoints related tests) for the diagnosis of other common causes of 
AFI, such as malaria, typhoid, scrub typhus, flu influenza, viral hepatitis A , etc. 
9.1.3 Tertiary (research) endpoints
During the Suspected Dengue First Visit, additional blood volumes will be collected for 
tertiary study endpoint assays. These tertiary study endpoints include, but are not limited 
to, determination of neutralising antibody titres against dengue virus (DENV), Japanese 
encephalitis virus (JEV), and chikungunya virus, tissue culture to isolate infectious 
agents, and sequencing to characterise infectious agents.
Dengue serology (sites in India only since seroprevalence is well defined for the
Sri Lanka site) will be performed on samples collected at the first Suspected Dengue First 
Visit only, from all subjects 5 years of age and above. (Amended 01 March 2017)
Tertiary study endpoint assays may be performed for a subset of samples (for example, 
JEV assays will only be conducted if additional studies indicate that JEV serology is 
needed to interpret the DENV antibody response), i.e., not all of these endpoint assays 
will be performed on all of the sera collected from adult study subjects. 
Research objectives may be assessed based on, but not limited to, the following 
endpoints: 

Neutralising antibody titres against DENV 1-4. 
9.2 Sample size considerations
A maximum of 2,000 5,000 subjects will be enrolled for a 2-year follow-up period. 
Assuming a drop-out rate of 10% the first year and a drop-out rate of 20% the second 
year, the final analysis will be done on approximately 3300 8,000 person-years overall.
In order to evaluate the precision with a sample size of 3,000 5,000 subjects, the 95% 
confidence interval (CI) (Poisson exact distribution and normal approximation of the 
Poisson distribution accounting for a design effect) for a range of expected incidences 
rates of AFI due to LCD were computed. The 95% CI for a range of expected incidences 
rates for DENV specific LCD were also presented (see Table 17). 
 
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New table
Table 17 95% CI with design effect of 2.6
Number 
of cases
Number of 
person-
years
Incidence 
of LCD 
(per 100 
person-
years)
95% CI Poisson 
exact
95% CI Normal 
approx with design 
effect of 2.6
LL
UL
LL
UL
Overall
10
3300
0.30
[
0.15
;
0.56
]
[
0.00
;
0.61
]
20
3300
0.61
[
0.37
;
0.94
]
[
0.18
;
1.03
]
30
3300
0.91
[
0.61
;
1.30
]
[
0.38
;
1.43
]
40
3300
1.21
[
0.87
;
1.65
]
[
0.61
;
1.82
]
50
3300
1.52
[
1.12
;
2.00
]
[
0.84
;
2.19
]
60
3300
1.82
[
1.39
;
2.34
]
[
1.08
;
2.56
]
DENV 
specific
4
3300
0.12
[
0.03
;
0.31
]
[
0.00
;
0.31
]
8
3300
0.24
[
0.10
;
0.48
]
[
0.00
;
0.51
]
12
3300
0.36
[
0.19
;
0.64
]
[
0.03
;
0.70
]
16
3300
0.48
[
0.28
;
0.79
]
[
0.10
;
0.87
]
Deleted table: 
CI: confidence interval
DENV: dengue virus
LCD: laboratory Confirmed Dengue
LL lower limit; 
UL: upper limit
 
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9.7.
Analysis of tertiary (research) objectives
Some tertiary (research) analyses may be done including:
• Neutralising antibody titres against DENV 1-4 on serum: the seropositivity rates for 
each DENV serotype with exact 95% CI and GMTs with 95% CI and range of antibody 
titres for each DENV serotype will be tabulated.
• A concordance analysis between dengue virus detection by RT-qPCR and by NS1-
antigen (ICT test and/or ELISA) may be performed. This analysis may be stratified by 
day of sampling if sufficient data are available. Sensitivity, specificity, positive predictive 
value and negative predictive value of NS1-antigen (ICT test and/or ELISA) in prediction 
of RT-qPCR test results will be computed. 
9.10.1 Sequence of analyses

An interim analysis will be performed (on as clean as possible data) once 
1,500 subjects have been followed up for at least 1 year. If the observed incidence of 
cases is lower than expected, possible causes will be explored, including quality of 
surveillance (weekly contacts and suspected dengue visits, diary card completion), 
time from fever onset to blood draw, sample management and laboratory assay 
performance. The outcome of this analysis could lead to process clarification or even 
a protocol amendment to improve surveillance and or case confirmation.
The final analysis will be performed when all prospective data have been collected and 
cleaned. A final study report containing data from the entire study will be written at this 
time. Depending on whether or not some of the processes of surveillance before / after 
the interim analysis have occurred, the new data may be analysed separately.
9.10.2 Statistical considerations for interim analyses
At time of interim analysis, all primary and secondary endpoints will be assessed to 
monitor the quality of data collected. The statistical report will then include the incidence 
of AFI due to LCD and due to non-LCD overall, by study site and age group as well as a 
description of signs and symptoms of AFI due to LCD and due to non-LCD cases. 
Additionally, the number of subjects withdrawn from the study and the compliance in 
returning diary card in case of AFI will also be generated.
Section 12 References
Bharaj P, Chahar HS, Pandey A, et al. Concurrent infections by all four dengue virus 
serotypes during an outbreak of dengue in 2006 in Delhi, India. Virol J. 2008;5:1. 
Chakravarti A, Arora R and Luxemburger C. Fifty years of dengue in India. Trans R Soc 
Trop Med Hyg. 2012;106:273-82. 
Epidemiology Unit, Ministry of Health. Distribution of Notification (H399) Dengue 
Cases by Month; 
http://www.epid.gov.lk/web/index.php?option=com_casesanddeaths&Itemid=448&lang=
en# (last accessed 18 February 2014 27 February 2017).
 
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Gupta E, Dar L, Kapoor G, et al. The changing epidemiology of dengue in Delhi, India. 
Virol J. 2006;3:92.  
Gupta B and Reddy BP. Fight against dengue in India: progresses and challenges. 
Parasitol Res. 2013;112:1367-78. 
Kakkar M. Dengue fever is massively under-reported in India, hampering our response. 
BMJ. 2012;345:e8574. 
Kanungo R. Are clinical microbiology laboratories missing out quality control and 
quality assurance in laboratory management? Indian J Med Microbiol. [serial online] 
2012 [cited 2014 Feb 18]; 30: 1-2. Available from: 
http://www.ijmm.org/text.asp?2012/30/1/1/93012 (last accessed 18 February 2014). 
APPENDIX B
CLINICAL LABORATORIES
Table 19
Outsourced laboratories
Laboratory
Address
QUEST (India)
Quest Diagnostics Private Limited
A-17 Info City, Sector 34
Gurgaon-122001 Haryana
INDIA
Focus (US)
Q²Solutions -Focus Diagnostics
Focus Diagnostics, Inc.
(d/b/a Quest Diagnostics)
33608 Ortega Highway 
San Juan Capistrano,
CA 92675-2042
USA
 
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